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PCR efficiency or DNA yield with single primer

PCR efficiency or DNA yield with single primer


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How to calculate number of DNA molecules synthesized after 'n' no. cycles with single primer (either only forward primer or reverse primer)? Is there any formula to calculate the DNA yield after such PCR?

Supposing, I am setting a PCR reaction for 100 template DNA molecules for 30 cycles, with only one primer - forward primer for target gene. How many DNA molecules will be generated at the end of 30 cycles. All other conditions remain the same.

Thanks.


Technically, if you use only 1 primer, it will not be PCR anymore.

But that's what gonna happen if you have 100 template DNA molecules (made of strand A and complimentary strand B) and abundance of primer (say, 10x, or 1000 molecules):

  1. At first cycle primers will bind to template DNA, but only to one strand (for example, A)
  2. DNA polymerase will extend those primers, creating complimentary strands B'. Some strands B' will be shorter than template because DNA pol is not perfect
  3. DNA-primer complex will melt at the beginning of cycle 2
  4. At the beginning of cycle 2 primer molecules will again bind to template, again to strands A, and process continues

Since there is no step where strand A is being copied, this process will be linear, not a "chain reaction". At each cycle you will get the same amount of strands B'.

It is hard to say how much B' molecules you will get at each cycle, but at the end of 30 cycles, it will be at best 30x of the first cycle. Remember, though, that DNA polymerase degrades, and cycle 30 will be less efficient than cycle 1.

Assuming perfect polymerase, and that extension time is long enough, you will get 100 strands B' on cycle 1, 100 strands B' on cycle 2 and so on. At the end of 30 cycles you will have 30x100 strands B', and 100 strands A and 100 strands B.


Understanding qPCR efficiency and why it can exceed 100%

Quantitative polymerase chain reaction (or qPCR) is a well-established assay for nucleic acid quantification and is still regarded as the method of choice in most areas of molecular biology. Though different types of qPCR quantification exist (absolute and relative), determining the amplification efficiency should be among the first things to do when setting up a qPCR assay. Understanding efficiency and how to calculate it is crucial for accurate data interpretation.

Ideally, the number of molecules of the target sequence should double during each replication cycle, corresponding to a 100% amplification efficiency. Similarly, if the number of replicated molecules is less than double this is due to poor efficiency – below 100%. The most common reasons for lower efficiencies are bad primer design and non-optimal reagent concentrations or reaction conditions. Secondary structures like dimers and hairpins or inappropriate melting temperatures (Tm) can affect primer-template annealing which results in poor amplification. Since each additional dilution contains appropriately lower starting amounts of DNA, differences occur between Ct values in serially diluted samples (see below).

Differences between Ct values of known dilution steps are higher than predicted. 10-fold dilutions should be 3.3 cycles apart, but in this case, they are further apart.

One way of calculating the amplification efficiency is by making serial dilutions of your target. Once you obtain their Ct values, plot them on a logarithmic scale along with corresponding concentrations. Next, generate a linear regression curve through the data points and calculate the slope of the trend line. Finally, efficiency is calculated using the equation: E = -1+10 (-1/slope) . Or use this calculator which does the work for you. Be sure to understand what influences the slope of the amplification curve, as it can otherwise be misleading.

Typically, desired amplification efficiencies range from 90% to 110%. The theoretical maximum of 100% indicates that polymerase enzyme is working at maximum capacity. How are efficiencies over 100% even possible then? That would mean more than two copies of the sequence are generated in each qPCR cycle, right?

Pipetting 96- and 384-well plates can be a very frustrating and tedious task.

Using pipetting tools like the Pipetting Aid PlatR can drastically improve your precision while keeping you calm and relaxed.


Overview

The specific complementary association due to hydrogen bonding of single-stranded nucleic acids is referred to as "annealing": two complementary sequences will form hydrogen bonds between their complementary bases ( G to C, and A to T or U ) and form a stable double-stranded, anti-parallel "hybrid" molecule. One may make nucleic acid (NA) single-stranded for the purpose of annealing - if it is not single-stranded already, like most RNA viruses - by heating it to a point above the "melting temperature" of the double- or partially-double-stranded form, and then flash-cooling it: this ensures the "denatured" or separated strands do not re-anneal. Additionally, if the NA is heated in buffers of ionic strength lower than 150mM NaCl, the melting temperature is generally less than 100oC - which is why PCR works with denaturing temperatures of 91-97 o C.

A more detailed treatment of annealing / hybridisation is given in an accompanying page, together with explanations of calculations of complexity, conditions for annealing / hybridisation, etc.

Taq polymerase is given as having a half-life of 30 min at 95 o C, which is partly why one should not do more than about 30 amplification cycles: however, it is possible to reduce the denaturation temperature after about 10 rounds of amplification, as the mean length of target DNA is decreased : for templates of 300bp or less, denaturation temperature may be reduced to as low as 88 o C for 50% (G+C) templates (Yap and McGee, 1991), which means one may do as many as 40 cycles without much decrease in enzyme efficiency.

"Time at temperature" is the main reason for denaturation / loss of activity of Taq: thus, if one reduces this, one will increase the number of cycles that are possible , whether the temperature is reduced or not. Normally the denaturation time is 1 min at 94 o C: it is possible, for short template sequences, to reduce this to 30 sec or less. Increase in denaturation temperature and decrease in time may also work: Innis and Gelfand (1990) recommend 96 o C for 15 sec.

Annealing Temperature and Primer Design

Primer length and sequence are of critical importance in designing the parameters of a successful amplification: the melting temperature of a NA duplex increases both with its length, and with increasing (G+C) content: a simple formula for calculation of the Tm is

Tm = 4(G + C) + 2(A + T) o C.

Thus, the annealing temperature chosen for a PCR depends directly on length and composition of the primer(s). One should aim at using an annealing temperature (Ta) about 5 o C below the lowest Tm of ther pair of primers to be used (Innis and Gelfand, 1990). A more rigorous treatment of Ta is given by Rychlik et al. (1990): they maintain that if the Ta is increased by 1 o C every other cycle, specificity of amplification and yield of products <1kb in length are both increased. One consequence of having too low a Ta is that one or both primers will anneal to sequences other than the true target, as internal single-base mismatches or partial annealing may be tolerated: this is fine if one wishes to amplify similar or related targets however, it can lead to "non-specific" amplification and consequent reduction in yield of the desired product, if the 3'-most base is paired with a target.

A consequence of too high a Ta is that too little product will be made , as the likelihood of primer annealing is reduced another and important consideration is that a pair of primers with very different Tas may never give appreciable yields of a unique product, and may also result in inadvertent "asymmetric" or single-strand amplification of the most efficiently primed product strand.

Annealing does not take long: most primers will anneal efficiently in 30 sec or less, unless the Ta is too close to the Tm, or unless they are unusually long.

An illustration of the effect of annealing temperature on the specificity and on the yield of amplification of Human papillomavirus type 16 (HPV-16) is given below (Williamson and Rybicki, 1991: J Med Virol 33: 165-171).

Plasmid and biopsy sample DNA templates were amplified at different annealing temperatures as shown: note that while plasmid is amplified from 37 to 55 o C, HPV DNA is only specifically amplified at 50 o C.

Primer Length

The optimum length of a primer depends upon its (A+T) content, and the Tm of its partner if one runs the risk of having problems such as described above. Apart from the Tm, a prime consideration is that the primers should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low. (See hybridn doc).

For example, there is a ¼ chance (4 -1 ) of finding an A, G, C or T in any given DNA sequence there is a 1/16 chance (4 -2 ) of finding any dinucleotide sequence (eg. AG) a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every 4 16 bases (=4 294 967 296, or 4 billion): this is about the size of the human or maize genome, and 1000x greater than the genome size of E. coli . Thus, the association of a greater-than-17-base oligonucleotide with its target sequence is an extremely sequence-specific process, far more so than the specificity of monoclonal antibodies in binding to specific antigenic determinants. Consequently, 17-mer or longer primers are routinely used for amplification from genomic DNA of animals and plants. Too long a primer length may mean that even high annealing temperatures are not enough to prevent mismatch pairing and non-specific priming.

Degenerate Primers

For amplification of cognate sequences from different organisms, or for "evolutionary PCR", one may increase the chances of getting product by designing "degenerate" primers: these would in fact be a set of primers which have a number of options at several positions in the sequence so as to allow annealing to and amplification of a variety of related sequences . For example, Compton (1990) describes using 14-mer primer sets with 4 and 5 degeneracies as forward and reverse primers, respectively, for the amplification of glycoprotein B (gB) from related herpesviruses. The reverse primer sequence was as follows:

TCGAATTCNCCYAAYTGNCCNT

where Y = T + C, and N = A + G + C + T, and the 8-base 5'-terminal extension comprises a EcoRI site (underlined) and flanking spacer to ensure the restriction enzyme can cut the product (the New England Biolabs catalogue gives a good list of which enzymes require how long a flanking sequence in order to cut stub ends). Degeneracies obviously reduce the specificity of the primer(s), meaning mismatch opportunities are greater, and background noise increases also, increased degeneracy means concentration of the individual primers decreases thus, greater than 512-fold degeneracy should be avoided. However, I have used primers with as high as 256- and 1024-fold degeneracy for the successful amplification and subsequent direct sequencing of a wide range of Mastreviruses against a background of maize genomic DNA (Rybicki and Hughes, 1990).

Primer sequences were derived from multiple sequence alignments the mismatch positions were used as 4-base degeneracies for the primers (shown as stars 5 in F and 4 in R), as shown above. Despite their degeneracy, the primers could be used to amplify a 250 bp sequence from viruses differing in sequence by as much as 50% over the target sequence, and 60% overall. They could also be used to very sensitively detect the presence of Maize streak virus DNA against a background of maize genomic DNA, at dilutions as low as 1/10 9 infected sap / healthy sap (see below).

Some groups use deoxyinosine (dI) at degenerate positions rather than use mixed oligos : this base-pairs with any other base, effectively giving a four-fold degeneracy at any postion in the oligo where it is present. This lessens problems to do with depletion of specific single oligos in a highly degenerate mixture, but may result in too high a degeneracy where there are 4 or more dIs in an oligo.

Elongation Temperature and Time

This is normally 70 - 72oC, for 0.5 - 3 min. Taq actually has a specific activity at 37oC which is very close to that of the Klenow fragment of E coli DNA polymerase I, which accounts for the apparent paradox which results when one tries to understand how primers which anneal at an optimum temperature can then be elongated at a considerably higher temperature - the answer is that elongation occurs from the moment of annealing, even if this is transient, which results in considerably greater stability. At around 70oC the activity is optimal, and primer extension occurs at up to 100 bases/sec. About 1 min is sufficient for reliable amplification of 2kb sequences (Innis and Gelfand, 1990). Longer products require longer times: 3 min is a good bet for 3kb and longer products. Longer times may also be helpful in later cycles when product concentration exceeds enzyme concentration (>1nM), and when dNTP and / or primer depletion may become limiting.

Reaction Buffer

Recommended buffers generally contain :

  • 10-50mM Tris-HCl pH 8.3,
  • up to 50mM KCl, 1.5mM or higher MgCl2,
  • primers 0.2 - 1uM each primer,
  • 50 - 200uM each dNTP,
  • gelatin or BSA to 100ug/ml,
  • and/or non-ionic detergents such as Tween-20 or Nonidet P-40 or Triton X-100 (0.05 - 0.10% v/v)

(Innis and Gelfand, 1990). Modern formulations may differ considerably, however - they are also generally proprietary.

PCR is supposed to work well in reverse transcriptase buffer, and vice-versa, meaning 1-tube protocols (with cDNA synthesis and subsequent PCR) are possible (Krawetz et al., 19xx Fuqua et al., 1990).

Higher than 50mM KCl or NaCl inhibits Taq, but some is necessary to facilitate primer annealing.

[Mg2+] affects primer annealing Tm of template, product and primer-template associations product specificity enzyme activity and fidelity. Taq requires free Mg2+, so allowances should be made for dNTPs, primers and template, all of which chelate and sequester the cation of these, dNTPs are the most concentrated, so [Mg2+] should be 0.5 - 2.5mM greater than [dNTP]. A titration should be performed with varying [Mg2+] with all new template-primer combinations , as these can differ markedly in their requirements, even under the same conditions of concentrations and cycling times/temperatures.

Some enzymes do not need added protein, others are dependent on it. Some enzymes work markedly better in the presence of detergent, probably because it prevents the natural tendency of the enzyme to aggregate.

Primer concentrations should not go above 1uM unless there is a high degree of degeneracy 0.2uM is sufficient for homologous primers.

Nucleotide concentration need not be above 50uM each: long products may require more, however.

Cycle Number

The number of amplification cycles necessary to produce a band visible on a gel depends largely on the starting concentration of the target DNA: Innis and Gelfand (1990) recommend from 40 - 45 cycles to amplify 50 target molecules , and 25 - 30 to amplify 3x105 molecules to the same concentration. This non-proportionality is due to a so-called plateau effect, which is the attenuation in the exponential rate of product accumulation in late stages of a PCR, when product reaches 0.3 - 1.0 nM. This may be caused by degradation of reactants (dNTPs, enzyme) reactant depletion (primers, dNTPs - former a problem with short products, latter for long products) end-product inhibition (pyrophosphate formation) competition for reactants by non-specific products competition for primer binding by re-annealing of concentrated (10nM) product (Innis and Gelfand, 1990).

If desired product is not made in 30 cycles , take a small sample (1ul) of the amplified mix and re-amplify 20-30x in a new reaction mix rather than extending the run to more cycles: in some cases where template concentration is limiting, this can give good product where extension of cycling to 40x or more does not.

A variant of this is nested primer PCR : PCR amplification is performed with one set of primers, then some product is taken - with or without removal of reagents - for re-amplification with an internally-situated, "nested" set of primers. This process adds another level of specificity, meaning that all products non-specifically amplified in the first round will not be amplified in the second. This is illustrated below:

This gel photo shows the effect of nested PCR amplification on the detectability of Chicken anaemia virus (CAV) DNA in a dilution series: the PCR1 just detects 1000 template molecules PCR2 amplifies 1 template molecule ( Soiné C, Watson SK, Rybicki EP, Lucio B, Nordgren RM, Parrish CR, Schat KA (1993) Avian Dis 37: 467-476).

Labelling of PCR products with digoxygenin-11-dUTP

(DIG Roche) need be done only in 50uM each dNTP, with the dTTP substituted to 35% with DIG-11-dUTP. NOTE: that the product will have a higher MW than the native product! This results in a very well labelled probe which can be extensively re-used, for periods up to 3 years. See also here.

Helix Destabilisers / Additives

With NAs of high (G+C) content, it may be necessary to use harsher denaturation conditions. For example, one may incorporate up to 10% (w or v/v) :

  • dimethyl sulphoxide (DMSO),
  • dimethyl formamide (DMF),
  • urea
  • or formamide

in the reaction mix: these additives are presumed to lower the Tm of the target NA , although DMSO at 10% and higher is known to decrease the activity of Taq by up to 50% (Innis and Gelfand, 1990 Gelfand and White, 1990).

Additives may also be necessary in the amplification of long target sequences: DMSO often helps in amplifying products of >1kb. Formamide can apparently dramatically improve the specificity of PCR (Sarkar et al ., 1990), while glycerol improves the amplification of high (G+C) templates (Smith et al., 1990).

Polyethylene glycol (PEG) may be a useful additive when DNA template concentration is very low: it promotes macromolecular association by solvent exclusion, meaning the pol can find the DNA.

CDNA PCR

A very useful primer for cDNA synthesis and cDNA PCR comes from a sequencing strategy described by Thweatt et al. (1990): this utilised a mixture of three 21-mer primers consisting of 20 T residues with 3'-terminal A, G or C, respectively , to sequence inside the poly(A) region of cDNA clones of mRNA from eukaryotic origin. I have used it to amplify discrete bands from a variety of poly(A)+ virus RNAs, with only a single specific degenerate primer upstream: the T-primer may anneal anywhere in the poly(A) region, but only molecules which anneal at the beginning of the poly(A) tail, and whose 3'-most base is complementary to the base next to the beginning of the tail , will be extended.

eg: 5'-TTTTTTTTTTTTTTTTTTTTTTTTT(A,G,C)-3'

works for amplification of Potyvirus RNA, and eukaryotic mRNA

A simple set of rules for primer sequence design is as follows (adapted from Innis and Gelfand, 1991):

primers should be 17-28 bases in length

base composition should be 50-60% (G+C)

primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming

Tms between 55-80 o C are preferred

runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided

3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product

primer self-complementarity (ability to form 2 o structures such as hairpins) should be avoided.

Examples of inter- and intra-primer complementarity which would result in problems:

Screen shots taken from analyses done using DNAMAN (Lynnon Biosoft, Quebec, Canada).

References

Compton T (1990). Degenerate primers for DNA amplification. pp. 39-45 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.) Academic Press, New York.
Fuqua SAW, Fitzgerald SD and McGuire WL (1990). A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts. BioTechniques 9 (2):206-211.
Gelfand DH and White TJ (1990). Thermostable DNA polymerases. pp. 129-141 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.) Academic Press, New York.
Innis MA and Gelfand DH (1990). Optimization of PCRs. pp. 3-12 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.) Academic Press, New York.
Krawetz SA, Pon RT and Dixon GH (1989). Increased efficiency of the Taq polymerase catalysed polymerase chain reaction. Nucleic Acids Research 17 (2):819.
Rybicki EP and Hughes FL (1990). Detection and typing of maize streak virus and other distantly related geminiviruses of grasses by polymerase chain reaction amplification of a conserved viral sequence. Journal of General Virology 71:2519-2526.
Rychlik W, Spencer WJ and Rhoads RE (1990). Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Research 18 (21):6409-6412.
Sarkar G, Kapeiner S and Sommer SS (1990). Formaqmide can drrastically increase the specificity of PCR. Nucleic Acids Research 18 (24):7465.
Smith KT, Long CM, Bowman B and Manos MM (1990). Using cosolvents to enhance PCR amplification. Amplifications 9/90 (5):16-17.
Thweatt R, Goldstein S and Reis RJS (1990). A universal primer mixture for sequence determination at the 3' ends of cDNAs. Analytical Biochemistry 190:314-316.
Wu DY, Ugozzoli L, Pal BK, Qian J, Wallace RB (1991). The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. DNA and Cell Biology 10 (3):233-238.
Yap EPH and McGee JO'D (1991). Short PCR product yields improved by lower denaturation temperatures. Nucleic Acids Research 19 (7):1713.


Commentary

Background Information

The theoretical basis of the polymerase chain reaction (PCR see chapter introduction) was probably first described in a paper by Kleppe et al. ( 1971 ). However, this technique did not excite general interest until the mid-1980s, when Kary Mullis and co-workers at Cetus developed PCR into a technique that could be used to generate large amounts of single-copy genes from genomic DNA (Saiki et al., 1985, 1986 Mullis et al., 1986 Embury et al., 1987 ).

The initial procedure entailed adding a fresh aliquot of the Klenow fragment of E. coli DNA polymerase I during each cycle because this enzyme was inactivated during the subsequent denaturation step. The introduction of thermostable Taq DNA polymerase from Thermus aquaticus (Saiki et al., 1988 ) alleviated this tedium and facilitated automation of the thermal cycling portion of the procedure. Taq DNA polymerase also permitted the use of higher temperatures for annealing and extension, which improved the stringency of primer–template hybridization and thus the specificity of the products. This also served to increase the yield of the desired product.

All applications of PCR depend upon an optimized PCR. The in this unit optimizes PCR for several variables, including MgCl2 concentration, enhancing additives—dimethyl sulfoxide (DMSO), glycerol, or Perfect Match Polymerase Enchancer (PMPE)—and prevention of pre-PCR mispriming. These and other parameters can be extremely important, as every element of PCR can affect the outcome (see 2.2 for discussion of individual parameters).

There are several PCR optimization kits and proprietary enhancers on the market (Table 3). Optimization kits generally provide a panel of buffers in which the pH, buffer, nonionic detergents, and addition of (NH4)2SO4 are varied, MgCl2 may be added at several concentrations, and enhancers (e.g., DMSO, glycerol, formamide, betaine, and/or proprietary compounds) may be chosen. The protocol presented here is aimed at keeping the costs low and the options broad.

Technical information in appendix to catalog

Boehringer-Mannheim, Invitrogen, Stratagene, Sigma, Epicentre Technologies, Life Technologies

Several buffers, Mg 2+ , and enhancers which may include DMSO, glycerol, formamide, (NH4)2SO4, and other unspecified or proprietary agents

Amersham Pharmacia Biotech

Ready-To-Go Beads “optimized for standard PCR” and Ready-To-Go RAPD Analysis Beads (buffer, nucleotides, Taq DNA polymerase)

EasyStart PCR Mix-in-a-Tube—tubes prepackaged with wax beads containing buffer, MgCl2, nucleotides, Taq DNA polymerase

PCR SuperMix—1.1× conc.—premix containing buffer, MgCl2, nucleotides, TaqDNA polymerase

Advanced Biochemicals Red Hot DNA Polymerase—a new rival for Taq polymerase with convenience features

Molecular Bio-Products HotStart Storage and Reaction Tubes—preadhered wax bead in each tube requires manual addition of one component at high temperature

HotWax Mg 2+ beads—wax beads contain preformulated MgCl2 which is released at first elevated-temperature step

StrataSphere Magnesium Wax Beads—wax beads containing preformulated Mg 2+

Hot Start/separate polymerase

TaqBead Hot Start Polymerase—wax beads encapsulating Taq DNA polymerase which is released at first elevated-temperature step

Hot-start/reversible inactivation of polymerase by antibody binding

TaqStart Antibody, TthStart Antibody—reversibly inactivate Taq and Tth DNA polymerases until first denaturation at 95°C

PlatinumTaq—contains PlatinumTaq antibody

JumpStart Taq—contains TaqStart antibody

Hot-start/reversible chemical modification

AmpliTaq Gold—activated at high temperature

Hot-start/reversible chemical modification

HotStarTaq DNA Polymerase—activated at high temperature

Boehringer Mannheim, New England Biolabs

Tth pyrophosphatase, thermostable

GC-Melt (in Advantage-GC Kits)—proprietary

Taq-FORCE Amplification System and MIGHTY Buffer—proprietary

Eppendorf MasterTaq Kit with TaqMaster Enhancer—proprietary

PCRx Enhancer System—proprietary

E.coli Single Stranded Binding Protein (SSB)

Perfect Match Polymerase Enhancer—proprietary

TaqExtender PCR Additive—proprietary

Critical Parameters and Troubleshooting

MgCl2 Concentration

Determining the optimum MgCl2 concentration, which can vary even for different primers from the same region of a given template (Saiki, 1989 ), can have an enormous influence on PCR success. In this protocol three concentrations are tested—1.5 mM (L), 3.0 mM (M), and 4.5 mM (H)—against three enhancers. Enhancers tend to broaden the MgCl2 optimal range, contributing to the success of the PCR at one of these concentrations. A 10× buffer optimized for a given enzyme and a separate vial of MgCl2 are typically provided with the polymerase, so that the user may titrate the MgCl2 concentration for their unique primer-template set.

Reagent Purity

For applications that amplify rare templates, reagent purity is the most important parameter, and avoiding contamination at every step is critical.

To maintain purity, store multiple small volumes of each reagent in screw-cap tubes.

For many applications, simply using high-quality reagents and avoiding nuclease contamination is sufficient however, avoid one common reagent used to inactivate nucleases, diethylpyrocarbonate (DEPC). Even tiny amounts of chemical left after treatment of water by autoclaving are enough to ruin a PCR.

Primer Selection

This is the factor that is least predictable and most difficult to troubleshoot. Simply put, some primers just do not work. To maximize the probability that a given primer pair will work, pay attention to the following parameters.

General considerations. An optimal primer set should hybridize efficiently to the sequence of interest with negligible hybridization to other sequences present in the sample. If there are reasonable amounts of template available, hybridization specificity can be tested by performing oligonucleotide hybridization as described in UNIT Unavailable. The distance between the primers is rather flexible, ranging up to 10 kb. There can be, however, a considerable drop-off in synthesis efficiency with distances >3 kb (Jeffreys et al., 1988 ). Small distances between primers, however, lessen the ability to obtain much sequence information or to reamplify with nested internal oligonucleotides, should that be necessary.

Design primers to allow demonstration of the specificity of the PCR product. Be sure that there are diagnostic restriction endonuclease sites between the primers or that an oligonucleotide can detect the PCR product specifically by hybridization.

Several computer programs can assist in primer design (see Internet Resources at end of unit). These are most useful for avoiding primer sets with intra- and intermolecular complementarity, which can dramatically raise the effective Tm. Given the abundance of primers relative to template, this can preclude template priming. Computer primer design is not foolproof. If possible, start with a primer or primer set known to efficiently prime extensions. In addition, manufacturers' Web sites offer technical help with primer design.

Complementarity to template. For many applications, primers are designed to be exactly complementary to the template. For others, however, such as engineering of mutations or new restriction endonuclease sites, or for efforts to clone or detect gene homologs where sequence information is lacking, base-pair mismatches will be intentionally or unavoidably created. It is best to have mismatches (e.g., in a restriction endonuclease linker) at the 5′ end of the primer. The closer a mismatch is to the 3′ end of the primer, the more likely it is to prevent extension.

The use of degenerate oligonucleotide primers to clone genes where only protein sequence is available, or to fish out gene homologs in other species, has sometimes been successful, but it has also failed an untold (and unpublished) number of times. When the reaction works it can be extremely valuable, but it can also generate seemingly specific products that require much labor to identify and yield no useful information. The less degenerate the oligonucleotides, especially at the 3′ end, the better. Caveat emptor.

Primer length. A primer should be 20 to 30 bases in length. It is unlikely that longer primers will help increase specificity significantly.

Primer sequence. Design primers with a GC content similar to that of the template. Avoid primers with unusual sequence distributions, such as stretches of polypurines or polypyrimidines, as their secondary structure can be disastrous. It is worthwhile to check for potential secondary structure using one of the appropriate computer programs that are available.

“Primer-dimers.” Primer-dimers are a common artifact most frequently observed when small amounts of template are taken through many amplification cycles. They form when the 3′ end of one primer anneals to the 3′ end of the other primer, and polymerase then extends each primer to the end of the other. The ensuing product can compete very effectively against the PCR product of interest. Primer-dimers can best be avoided by using primers without complementarity, especially in their 3′ ends. Should they occur, optimizing the MgCl2 concentration may minimize their abundance relative to that of the product of interest.

Template

Aside from standard methods for preparing DNA ( UNIT Unavailable- Unavailable), a number of simple and rapid procedures have been developed for particular tissues (Higuchi, 1989 ). Even relatively degraded DNA preparations can serve as useful templates for generation of moderate-sized PCR products. The two main concerns regarding template are purity and amount.

A number of contaminants found in DNA preparations can decrease the efficiency of PCR. These include urea, the detergent SDS (whose inhibitory action can be reversed by nonionic detergents), sodium acetate, and, sometimes, components carried over in purifying DNA from agarose gels (Gelfand, 1989 Gyllensten, 1989 K. Hicks and D. Coen, unpub. observ.). Additional organic extractions, ethanol precipitation from 2.5 M ammonium acetate, and/or gel purification on polyacrylamide rather than agarose, can all be beneficial in minimizing such contamination if the simplest method (precipitating the sample with ethanol and repeatedly washing the pellet with 70% ethanol) is not sufficient.

Clearly the amount of template must be sufficient to be able to visualize PCR products using ethidium bromide. Usually 100 ng of genomic DNA is sufficient to detect a PCR product from a single-copy mammalian gene. Using too much template is not advisable when optimizing for MgCl2 or other parameters, as it may obscure differences in amplification efficiency. Moreover, too much template may decrease efficiency due to contaminants in the DNA preparation.

Amount of template, especially in terms of the amount of target sequence versus nonspecific sequences, can have a major effect on the yield of nonspecific products. With less target sequence, it is more likely that nonspecific products will be seen. For some applications, such as certain DNA sequencing protocols where it is important to have a single product, gel purification of the specific PCR product and reamplification are advisable.

Taq and Other Thermostable DNA Polymerases

Among the advantages conferred by the thermostability of Taq DNA polymerase is its ability to withstand the repeated heating and cooling inherent in PCR and to synthesize DNA at high temperatures that melt out mismatched primers and regions of local secondary structure. The enzyme, however, is not infinitely resistant to heat, and for greatest efficiency it should not be put through unnecessary denaturation steps. Indeed, some protocols (e.g., UNIT Unavailable and the “hot start” method described here) recommend adding it after the first denaturation step.

Increasing the amount of Taq DNA polymerase beyond 2.5 U/reaction can sometimes increase PCR efficiency, but only up to a point. Adding more enzyme can sometimes increase the yield of nonspecific PCR products at the expense of the product of interest. Moreover, Taq DNA polymerase is not inexpensive.

A very important property of Taq DNA polymerase is its error rate, which was initially estimated at 2 × 10 −4 nucleotides/cycle (Saiki et al., 1988 ). The purified enzyme supplied by manufacturers lacks a proofreading 3′⇒5′ exonuclease activity, which lowers error rates of other polymerases such as the Klenow fragment of E. coli DNA polymerase I. For many applications, this does not present any difficulties. However, for sequencing clones derived from PCR, or when starting with very few templates, this can lead to major problems. Direct sequencing of PCR products ( UNIT Unavailable), sequencing numerous PCR-generated clones, and/or the use of appropriate negative controls can help overcome these problems. Alternatively, changing reaction conditions (Eckert and Kunkel, 1990 ) or changing to a non–Taq DNA polymerase (with greater fidelity) may be useful.

Another important property of Taq DNA polymerase is its propensity for adding nontemplated nucleotides to the 3′ ends of DNA chains. This can be especially problematic in cloning PCR products. It is frequently necessary to “polish” PCR products with enzymes such as other DNA polymerases before adding linkers or proceeding to blunt-end cloning. Conversely, addition of a nontemplated A by Taq DNA polymerase can be advantageous in cloning ( UNIT Unavailable).

Certain PCR protocols may work better with one thermostable polymerase rather than another. Table 4 lists currently available thermostable DNA polymerases by generic and trade names, the original source of native and recombinant enzymes, the supplier, the end generated (3′A addition versus blunt), and associated exonuclease activities. A 3′ to 5′ exonuclease activity is proofreading. Removal of the 5′ to 3′ exonuclease activity of Taq DNA polymerase (N-terminal deletion) is reported to produce a higher yield. A 5′ to 3′ exonuclease activity may degrade the primers somewhat. Proofreading enzymes synthesize DNA with higher fidelity and can generate longer products than Taq, but tend to generate low yields. Enzyme blends (Table 5) have been optimized for increased fidelity and length along with sensitivity and yield.

Taq (native and/or recombinant)

Ambion, Amersham Pharmacia Biotech, Boehringer Mannheim, Clontech, Fisher, Life Technologies, Marsh Biomedical, Perkin Elmer, Promega, Qiagen, Sigma, Stratagene

Taq, N-terminal deletion

— a a No information at this time.

Promega, Epicentre Technologies

— a a No information at this time.

Thermococcus litoralis

New England Biolabs (Vent), Promega

Thermococcus litoralis

Amersham Pharmacia Biotech, Boehringer Mannheim, Epicentre Technologies, Perkin Elmer, Promega

Thermostable DNA polymerases and other components

Expand High Fidelity, Expand Long Template, and Expand 20kb PCR Systems

KlenTaq LA Polymerase Mix

KlenTaq-1 (5′-exonuclease deficient Taq) + unspecified proofreading polymerase

KlenTaq-1 + unspecified proofreading polymerase + TaqStart Antibody

Advantage-cDNA and Advantage-GC cDNA Polymerase Mixes and Kits

KlenTaq-1 + unspecified proofreading polymerase + TaqStart Antibody GC Kit contains GC Melt

Advantage Genomic and Advantage-GC Genomic Polymerase Mixes and Kits

Tth + unspecified proofreading polymerase + TthStart Antibody GC Kit contains GC Melt

Taq +Psp + unspecified proofreading polymerase(s) + eLONGase Buffer

Platinum Taq DNA Polymerase

Taq + Psp + Platinum Taq Antibody

Platinum High Fidelity DNA Polymerase

Taq + Psp + Taq Antibody

Tbr with unspecified enhancer

GeneAmp XL PCR and XL RNA PCR Kits

OmniBase Sequencing Enzyme Mix

Unspecified proofreading polymerase(s) with thermostable pyrophosphatase

AccuTaq LA DNA Polymerase Mix

Taq + unspecified proofreading polymerase

TaqPlus Long and TaqPlus Precision PCR Systems

Pfu+Taq TaqPlus Precision Reaction Buffer (proprietary)

Accurase Fidelity PCR Enzyme Mix Calypso High Fidelity Single Tube RT-PCR System

Thermus sp. + Thermococcus sp. Calypso also contains AMV-RT

Hot Start

What happens prior to thermal cycling is critical to the success of PCR. Taq DNA polymerase retains some activity even at room temperature. Therefore, under nonstringent annealing conditions, such as at room temperature, products can be generated from annealing of primers to target DNA at locations of low complementarity or having complementarity of just a few nucleotides at the 3′ ends. The latter would in effect create new templates “tagged” with the primer sequences. Subsequent cycles amplify these tagged sequences in abundance, both generating nonspecific products and possibly reducing amplification efficiency of specific products by competition for substrates or polymerase. Thus conditions preventing polymerization prior to the first temperature-controlled steps are desirable. In this protocol, three methods of inhibiting polymerization prior to the temperature-controlled step are compared. These include physical separation of an essential reaction component prior to the first denaturation step, cooling reagents to 0°C, and reversibly blocking enzymatic activity with an antibody.

Denaturation of the template before Taq polymerase or MgCl2 is added to the reaction provides a dramatic improvement in specificity and sensitivity in many cases (Chou et al., 1992 ). The main drawback of this method is that it requires opening the reaction tubes a second time to add the essential missing component. This creates both an inconvenience and an increase in the risk of contamination, an important consideration when testing for the presence of a given sequence in experimental or clinical samples.

Cooling all components of the reaction mixture to 0°C prior to mixing is more convenient and the least expensive method but is also the least reliable. Transferring the PCR reaction tubes from the ice slurry to a 95°C preheated thermocycler block may improve the chance of success.

Reversible inhibition of Taq DNA polymerase by TaqStart antibody (Clontech) is the most convenient method and very effective (Kellogg et al., 1994 ). Complete reactions can be set up, overlaid with oil, and stored at 4°C for up to several hours prior to thermal cycling with no loss of sensitivity or specificity compared to the other hot start methods (M.F. Kramer and D.M. Coen, unpub. observ.). Cycling is initiated immediately following 5-min denaturation of the antibody at 94°C. DMSO inhibits antibody binding and should not be used with TaqStart.

Several hot-start products are now commercially available (Table 3). Success with each may depend on strict adherence to the manufacturer's protocols, even on a specific thermocycler. Wax barrier and reversible antibody binding methods are more forgiving, while chemical modifications have more stringent activation temperature requirements.

Deoxyribonucleoside Triphosphates

In an effort to increase efficiency of PCR, it may be tempting to increase the concentration of dNTPs. Don't! When each dNTP is 200 mM, there is enough to synthesize 12.5 mg of DNA when half the dNTPs are incorporated. dNTPs chelate magnesium and thereby change the effective optimal magnesium concentration. Moreover, dNTP concentrations >200 mM each increase the error rate of the polymerase. Millimolar concentrations of dNTPs actually inhibit Taq DNA polymerase (Gelfand, 1989 ).

The protocol in this unit calls for preparing 4dNTPs in 10 mM Tris⋅Cl/1 mM EDTA (TE buffer), pH 7.4 to 7.5. This is easier and less prone to disaster than neutralization with sodium hydroxide. However, EDTA also chelates magnesium, and this should be taken into account if stocks of dNTPs are changed. Alternatively, to lower the risk of contamination, a 4dNTP mix can be made by combining equal volumes of commercially prepared stocks.

Enhancers

Enhancers are used to increase yield and specificity and to overcome difficulties encountered with high GC content or long templates. Nonionic detergents (Triton X-100, Tween 20, or Nonidet P-40) neutralize charges of ionic detergents often used in template preparation, and should be used in the basic reaction mixture, rather than as optional enhancers. Higher yields can be achieved by stabilizing/enhancing the polymerase activity with enzyme-stabilizing proteins (BSA or gelatin), enzyme-stabilizing solutes such as betaine or betaine⋅HCl (TMAC), enzyme-stabilizing solvents (glycerol), solubility-enhancing solvents (DMSO or acetamide), molecular crowding solvents (PEG), and polymerase salt preferences [(NH4)SO4 is recommended for polymerases other than Taq]. Greater specificity can be achieved by lowering the TM of dsDNA (using formamide), destabilizing mismatched-primer annealing (using PMPE or hot-start strategies), and stabilizing ssDNA (using E. coli SSB or T4 Gene 32 Protein). Amplification of high-GC-content templates can be improved by decreasing the base pair composition dependence of the TM of dsDNA (with betaine Rees et al., 1993 ). Betaine is an osmolyte widely distributed in plants and animals and is nontoxic, a feature that recommends it for convenience in handling, storage, and disposal. Betaine may be the proprietary ingredient in various commercial formulations. For long templates, a higher pH is recommended (pH 9.0). The pH of Tris buffer decreases at high temperatures, long-template PCR requires more time at high temperatures, and increased time at lower pH may cause some depurination of the template, resulting in reduced yield of specific product. Inorganic phosphate (PPi), a product of DNA synthesis, may accumulate with amplification of long products to levels that may favor reversal of polymerization. Accumulation of PPi may be prevented by addition of thermostable PPase. When large numbers of samples are being analyzed, the convenience of adding PCR products directly to a gel represents a significant time savings. Some companies combine their thermostable polymerase with a red dye and a high density component to facilitate loading of reaction products onto gels without further addition of loading buffer.

Thermal Cycling Parameters

Each step in the cycle requires a minimal amount of time to be effective, while too much time can be both wasteful and deleterious to the DNA polymerase. If the amount of time in each step can be reduced, so much the better.

Denaturation. It is critical that complete strand separation occur during the denaturation step. This is a unimolecular reaction which, in itself, is very fast. The suggested 30-sec denaturation used in the protocol ensures that the tube contents reach 94°C. If PCR is not working, it is well worth checking the temperature inside a control tube containing 100 µl water. If GC content is extremely high, higher denaturation temperatures may be necessary however, Taq DNA polymerase activity falls off quickly at higher temperatures (Gelfand, 1989 ). To amplify a long sequence (>3 kb), minimize the denaturation time to protect the target DNA from possible effects, such as depurination, of lowered pH of the Tris buffer at elevated temperatures.

Annealing. It is critical that the primers anneal stably to the template. Primers with relatively low GC content (<50%) may require temperatures lower than 55°C for full annealing. On the other hand, this may also increase the quantity of nonspecific products. For primers with high GC content, higher annealing temperatures may be necessary. It can be worthwhile, although time-consuming, to experiment with this parameter. Some manufacturers have thermal cyclers on the market which are capable of forming a temperature gradient across the heating units, thus permitting annealing temperature optimization in one run. As with denaturation, the time for this step is based mainly on the time it takes to reach the proper temperature, because the primers are in such excess that the annealing reaction occurs very quickly.

Extension. The extension temperature of 72°C is close to the optimal temperature for Taq DNA polymerase (∼75°C), yet prevents the primers from falling off. Indeed, primer extension begins during annealing, because Taq DNA polymerase is partially active at 55°C and even lower temperatures (Gelfand, 1989 ).

The duration of extension depends mainly on the length of the sequence to be amplified. A duration of 1 min per kb product length is usually sufficient.

Certain protocols, including others in this chapter, end the PCR with a long final extension time in an attempt to try to make products as complete as possible.

Ramp time. Ramp time refers to the time it takes to change from one temperature to another. Using water baths and moving samples manually from temperature to temperature probably gives the shortest ramp times, which are mainly the time required for the tube's contents to change temperature. Different thermal cyclers have different ramp times basically, the shorter the better.

The Stratagene Robocycler uses a robotic arm to move samples from one constant-temperature block to another, virtually eliminating block ramp time, but a ramp time for tube contents must be calculated (∼1 sec/°C) and added to denaturation, annealing, and extension times. Rapid cyclers that utilize positive-displacement pipet tips or capillary tubes for the PCR reactions dramatically reduce the ramp times.

Generally, the more “high-performance” thermal cyclers with short ramp times are proportionally more costly. There are many new thermal cyclers on the market priced below $5000, which perform quite well (Beck, 1998 ).

Anticipated Results

Starting with ≥100 ng mammalian DNA (≥10 4 molecules), the can be used to determine which MgCl2 concentration, enhancing additive, and initial conditions will yield a predominant PCR product from a single-copy sequence that is readily visible on an ethidium bromide–stained gel. It is possible that other minor products will also be visible.

Time Considerations

The can be completed in a single day. Assembly of the reaction mixtures should take ∼1 hr. Cycling should take less than 3 hr. Preparing, running, and staining the gel should take another few hours. Further checks on specificity of the product such as restriction endonuclease digestion or Southern blot hybridization will take another few hours or days, respectively.


Mechanism of action:

The long chain of the single-stranded probe contains the fluorochrome or a radiolabelled compound on its end.

Once it finds its complementary sequence on the template DNA it binds to it, the fluorochrome is released and emits fluorescence which is measured by the detector.

Thus the amount of the fluorescence emitted is directly proportional to the probe hybridization.

Under the exact annealing temperature, the primer binds to its complementary sequence. Once the annealing happens, the Taq DNA polymerase starts inserting the dNTPs next to the 3’ end of it until it reaches the end of the strand.

At the end of the reaction, a new DNA molecule is generated.


Contents

PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp) in length, although some techniques allow for amplification of fragments up to 40 kbp. [5] The amount of amplified product is determined by the available substrates in the reaction, which becomes limiting as the reaction progresses. [6]

A basic PCR set-up requires several components and reagents, [7] including:

  • a DNA template that contains the DNA target region to amplify
  • a DNA polymerase an enzyme that polymerizes new DNA strands heat-resistant Taq polymerase is especially common, [8] as it is more likely to remain intact during the high-temperature DNA denaturation process
  • two DNA primers that are complementary to the 3′ (three prime) ends of each of the sense and anti-sense strands of the DNA target (DNA polymerase can only bind to and elongate from a double-stranded region of DNA without primers, there is no double-stranded initiation site at which the polymerase can bind) [9] specific primers that are complementary to the DNA target region are selected beforehand, and are often custom-made in a laboratory or purchased from commercial biochemical suppliers
  • deoxynucleoside triphosphates, or dNTPs (sometimes called "deoxynucleotide triphosphates" nucleotides containing triphosphate groups), the building blocks from which the DNA polymerase synthesizes a new DNA strand
  • a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
  • bivalentcations, typically magnesium (Mg) or manganese (Mn) ions Mg 2+ is the most common, but Mn 2+ can be used for PCR-mediated DNA mutagenesis, as a higher Mn 2+ concentration increases the error rate during DNA synthesis [10] and monovalent cations, typically potassium (K) ions [better source needed]

The reaction is commonly carried out in a volume of 10–200 μL in small reaction tubes (0.2–0.5 mL volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibrium. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermal cyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure Edit

Typically, PCR consists of a series of 20–40 repeated temperature changes, called thermal cycles, with each cycle commonly consisting of two or three discrete temperature steps (see figure below). The cycling is often preceded by a single temperature step at a very high temperature (>90 °C (194 °F)), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers. [11] The individual steps common to most PCR methods are as follows:

  • Initialization: This step is only required for DNA polymerases that require heat activation by hot-start PCR. [12] It consists of heating the reaction chamber to a temperature of 94–96 °C (201–205 °F), or 98 °C (208 °F) if extremely thermostable polymerases are used, which is then held for 1–10 minutes.
  • Denaturation: This step is the first regular cycling event and consists of heating the reaction chamber to 94–98 °C (201–208 °F) for 20–30 seconds. This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules.
  • Annealing: In the next step, the reaction temperature is lowered to 50–65 °C (122–149 °F) for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3′ end of each strand.
  • Extension/elongation: The temperature at this step depends on the DNA polymerase used the optimum activity temperature for the thermostable DNA polymerase of Taq polymerase is approximately 75–80 °C (167–176 °F), [13][14] though a temperature of 72 °C (162 °F) is commonly used with this enzyme. In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that is complementary to the template in the 5′-to-3′ direction, condensing the 5′-phosphate group of the dNTPs with the 3′-hydroxy group at the end of the nascent (elongating) DNA strand. The precise time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify. As a rule of thumb, at their optimal temperature, most DNA polymerases polymerize a thousand bases per minute. Under optimal conditions (i.e., if there are no limitations due to limiting substrates or reagents), at each extension/elongation step, the number of DNA target sequences is doubled. With each successive cycle, the original template strands plus all newly generated strands become template strands for the next round of elongation, leading to exponential (geometric) amplification of the specific DNA target region.
  • Final elongation: This single step is optional, but is performed at a temperature of 70–74 °C (158–165 °F) (the temperature range required for optimal activity of most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated.
  • Final hold: The final step cools the reaction chamber to 4–15 °C (39–59 °F) for an indefinite time, and may be employed for short-term storage of the PCR products.

To check whether the PCR successfully generated the anticipated DNA target region (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis may be employed for size separation of the PCR products. The size of the PCR products is determined by comparison with a DNA ladder, a molecular weight marker which contains DNA fragments of known sizes, which runs on the gel alongside the PCR products.

Stages Edit

As with other chemical reactions, the reaction rate and efficiency of PCR are affected by limiting factors. Thus, the entire PCR process can further be divided into three stages based on reaction progress:

  • Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). After 30 cycles, a single copy of DNA can be increased up to 1,000,000,000 (one billion) copies. In a sense, then, the replication of a discrete strand of DNA is being manipulated in a tube under controlled conditions. [15] The reaction is very sensitive: only minute quantities of DNA must be present.
  • Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents, such as dNTPs and primers, causes them to become more limited.
  • Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions. [16] [17] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants. [7] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. Addition of reagents, such as formamide, in buffer systems may increase the specificity and yield of PCR. [18] Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design. [19]

Selective DNA isolation Edit

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many ways, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.

Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid, phage, or cosmid (depending on size) or the genetic material of another organism. Bacterial colonies (such as E. coli) can be rapidly screened by PCR for correct DNA vector constructs. [20] PCR may also be used for genetic fingerprinting a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.

Some PCR fingerprint methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms when certain molecular clocks are used (i.e. the 16S rRNA and recA genes of microorganisms). [21]

Amplification and quantification of DNA Edit

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar and the body of English king Richard III. [22]

Quantitative PCR or Real Time PCR (qPCR, [23] not to be confused with RT-PCR) methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of gene expression. Quantitative PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

qPCR allows the quantification and detection of a specific DNA sequence in real time since it measures concentration while the synthesis process is taking place. There are two methods for simultaneous detection and quantification. The first method consists of using fluorescent dyes that are retained nonspecifically in between the double strands. The second method involves probes that code for specific sequences and are fluorescently labeled. Detection of DNA using these methods can only be seen after the hybridization of probes with its complementary DNA takes place. An interesting technique combination is real-time PCR and reverse transcription. This sophisticated technique, called RT-qPCR, allows for the quantification of a small quantity of RNA. Through this combined technique, mRNA is converted to cDNA, which is further quantified using qPCR. This technique lowers the possibility of error at the end point of PCR, [24] increasing chances for detection of genes associated with genetic diseases such as cancer. [4] Laboratories use RT-qPCR for the purpose of sensitively measuring gene regulation. The mathematical foundations for the reliable quantification of the PCR [25] and RT-qPCR [26] facilitate the implementation of accurate fitting procedures of experimental data in research, medical, diagnostic and infectious disease applications. [27] [28] [29] [30]

Medical and diagnostic applications Edit

Prospective parents can be tested for being genetic carriers, or their children might be tested for actually being affected by a disease. [1] DNA samples for prenatal testing can be obtained by amniocentesis, chorionic villus sampling, or even by the analysis of rare fetal cells circulating in the mother's bloodstream. PCR analysis is also essential to preimplantation genetic diagnosis, where individual cells of a developing embryo are tested for mutations.

  • PCR can also be used as part of a sensitive test for tissue typing, vital to organ transplantation. As of 2008, [update] there is even a proposal to replace the traditional antibody-based tests for blood type with PCR-based tests. [31]
  • Many forms of cancer involve alterations to oncogenes. By using PCR-based tests to study these mutations, therapy regimens can sometimes be individually customized to a patient. PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest-developed in cancer research and is already being used routinely. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity that is at least 10,000 fold higher than that of other methods. [32] PCR is very useful in the medical field since it allows for the isolation and amplification of tumor suppressors. Quantitative PCR for example, can be used to quantify and analyze single cells, as well as recognize DNA, mRNA and protein confirmations and combinations. [24]

Infectious disease applications Edit

PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses. [33] PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes. [33] [34]

Characterization and detection of infectious disease organisms have been revolutionized by PCR in the following ways:

  • The human immunodeficiency virus (or HIV), is a difficult target to find and eradicate. The earliest tests for infection relied on the presence of antibodies to the virus circulating in the bloodstream. However, antibodies don't appear until many weeks after infection, maternal antibodies mask the infection of a newborn, and therapeutic agents to fight the infection don't affect the antibodies. PCR tests have been developed that can detect as little as one viral genome among the DNA of over 50,000 host cells. [35] Infections can be detected earlier, donated blood can be screened directly for the virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can be quantified.
  • Some disease organisms, such as that for tuberculosis, are difficult to sample from patients and slow to be grown in the laboratory. PCR-based tests have allowed detection of small numbers of disease organisms (both live or dead), in convenient samples. Detailed genetic analysis can also be used to detect antibiotic resistance, allowing immediate and effective therapy. The effects of therapy can also be immediately evaluated.
  • The spread of a disease organism through populations of domestic or wild animals can be monitored by PCR testing. In many cases, the appearance of new virulent sub-types can be detected and monitored. The sub-types of an organism that were responsible for earlier epidemics can also be determined by PCR analysis.
  • Viral DNA can be detected by PCR. The primers used must be specific to the targeted sequences in the DNA of a virus, and PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. [33] Such early detection may give physicians a significant lead time in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below). A variant of PCR (RT-PCR) is used for detecting viral RNA rather than DNA: in this test the enzyme reverse transcriptase is used to generate a DNA sequence which matches the viral RNA this DNA is then amplified as per the usual PCR method. RT-PCR is widely used to detect the SARS-CoV-2 viral genome. [36]
  • Diseases such as pertussis (or whooping cough) are caused by the bacteria Bordetella pertussis. This bacteria is marked by a serious acute respiratory infection that affects various animals and humans and has led to the deaths of many young children. The pertussis toxin is a protein exotoxin that binds to cell receptors by two dimers and reacts with different cell types such as T lymphocytes which play a role in cell immunity. [37] PCR is an important testing tool that can detect sequences within the gene for the pertussis toxin. Because PCR has a high sensitivity for the toxin and a rapid turnaround time, it is very efficient for diagnosing pertussis when compared to culture. [38]

Forensic applications Edit

The development of PCR-based genetic (or DNA) fingerprinting protocols has seen widespread application in forensics:

  • In its most discriminating form, genetic fingerprinting can uniquely discriminate any one person from the entire population of the world. Minute samples of DNA can be isolated from a crime scene, and compared to that from suspects, or from a DNA database of earlier evidence or convicts. Simpler versions of these tests are often used to rapidly rule out suspects during a criminal investigation. Evidence from decades-old crimes can be tested, confirming or exonerating the people originally convicted.
  • Forensic DNA typing has been an effective way of identifying or exonerating criminal suspects due to analysis of evidence discovered at a crime scene. The human genome has many repetitive regions that can be found within gene sequences or in non-coding regions of the genome. Specifically, up to 40% of human DNA is repetitive. [4] There are two distinct categories for these repetitive, non-coding regions in the genome. The first category is called variable number tandem repeats (VNTR), which are 10–100 base pairs long and the second category is called short tandem repeats (STR) and these consist of repeated 2–10 base pair sections. PCR is used to amplify several well-known VNTRs and STRs using primers that flank each of the repetitive regions. The sizes of the fragments obtained from any individual for each of the STRs will indicate which alleles are present. By analyzing several STRs for an individual, a set of alleles for each person will be found that statistically is likely to be unique. [4] Researchers have identified the complete sequence of the human genome. This sequence can be easily accessed through the NCBI website and is used in many real-life applications. For example, the FBI has compiled a set of DNA marker sites used for identification, and these are called the Combined DNA Index System (CODIS) DNA database. [4] Using this database enables statistical analysis to be used to determine the probability that a DNA sample will match. PCR is a very powerful and significant analytical tool to use for forensic DNA typing because researchers only need a very small amount of the target DNA to be used for analysis. For example, a single human hair with attached hair follicle has enough DNA to conduct the analysis. Similarly, a few sperm, skin samples from under the fingernails, or a small amount of blood can provide enough DNA for conclusive analysis. [4]
  • Less discriminating forms of DNA fingerprinting can help in DNA paternity testing, where an individual is matched with their close relatives. DNA from unidentified human remains can be tested, and compared with that from possible parents, siblings, or children. Similar testing can be used to confirm the biological parents of an adopted (or kidnapped) child. The actual biological father of a newborn can also be confirmed (or ruled out).
  • The PCR AMGX/AMGY design has been shown to not only [clarification needed] facilitate in amplifying DNA sequences from a very minuscule amount of genome. However it can also be used for real-time sex determination from forensic bone samples. This provides a powerful and effective way to determine gender in forensic cases and ancient specimens. [39]

Research applications Edit

PCR has been applied to many areas of research in molecular genetics:

  • PCR allows rapid production of short pieces of DNA, even when not more than the sequence of the two primers is known. This ability of PCR augments many methods, such as generating hybridizationprobes for Southern or northern blot hybridization. PCR supplies these techniques with large amounts of pure DNA, sometimes as a single strand, enabling analysis even from very small amounts of starting material.
  • The task of DNA sequencing can also be assisted by PCR. Known segments of DNA can easily be produced from a patient with a genetic disease mutation. Modifications to the amplification technique can extract segments from a completely unknown genome, or can generate just a single strand of an area of interest.
  • PCR has numerous applications to the more traditional process of DNA cloning. It can extract segments for insertion into a vector from a larger genome, which may be only available in small quantities. Using a single set of 'vector primers', it can also analyze or extract fragments that have already been inserted into vectors. Some alterations to the PCR protocol can generate mutations (general or site-directed) of an inserted fragment.
  • Sequence-tagged sites is a process where PCR is used as an indicator that a particular segment of a genome is present in a particular clone. The Human Genome Project found this application vital to mapping the cosmid clones they were sequencing, and to coordinating the results from different laboratories.
  • An application of PCR is the phylogenic analysis of DNA from ancient sources, such as that found in the recovered bones of Neanderthals, from frozen tissues of mammoths, or from the brain of Egyptian mummies. [15] In some cases the highly degraded DNA from these sources might be reassembled during the early stages of amplification.
  • A common application of PCR is the study of patterns of gene expression. Tissues (or even individual cells) can be analyzed at different stages to see which genes have become active, or which have been switched off. This application can also use quantitative PCR to quantitate the actual levels of expression
  • The ability of PCR to simultaneously amplify several loci from individual sperm [40] has greatly enhanced the more traditional task of genetic mapping by studying chromosomal crossovers after meiosis. Rare crossover events between very close loci have been directly observed by analyzing thousands of individual sperms. Similarly, unusual deletions, insertions, translocations, or inversions can be analyzed, all without having to wait (or pay) for the long and laborious processes of fertilization, embryogenesis, etc. : PCR can be used to create mutant genes with mutations chosen by scientists at will. These mutations can be chosen in order to understand how proteins accomplish their functions, and to change or improve protein function.

PCR has a number of advantages. It is fairly simple to understand and to use, and produces results rapidly. The technique is highly sensitive with the potential to produce millions to billions of copies of a specific product for sequencing, cloning, and analysis. qRT-PCR shares the same advantages as the PCR, with an added advantage of quantification of the synthesized product. Therefore, it has its uses to analyze alterations of gene expression levels in tumors, microbes, or other disease states. [24]

PCR is a very powerful and practical research tool. The sequencing of unknown etiologies of many diseases are being figured out by the PCR. The technique can help identify the sequence of previously unknown viruses related to those already known and thus give us a better understanding of the disease itself. If the procedure can be further simplified and sensitive non radiometric detection systems can be developed, the PCR will assume a prominent place in the clinical laboratory for years to come. [15]

One major limitation of PCR is that prior information about the target sequence is necessary in order to generate the primers that will allow its selective amplification. [24] This means that, typically, PCR users must know the precise sequence(s) upstream of the target region on each of the two single-stranded templates in order to ensure that the DNA polymerase properly binds to the primer-template hybrids and subsequently generates the entire target region during DNA synthesis.

Like all enzymes, DNA polymerases are also prone to error, which in turn causes mutations in the PCR fragments that are generated. [41]

Another limitation of PCR is that even the smallest amount of contaminating DNA can be amplified, resulting in misleading or ambiguous results. To minimize the chance of contamination, investigators should reserve separate rooms for reagent preparation, the PCR, and analysis of product. Reagents should be dispensed into single-use aliquots. Pipettors with disposable plungers and extra-long pipette tips should be routinely used. [15]

Environmental samples that contain humic acids may inhibit PCR amplification and lead to inaccurate results.

  • Allele-specific PCR: a diagnostic or cloning technique based on single-nucleotide variations (SNVs not to be confused with SNPs) (single-base differences in a patient). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNV (base pair buffer around SNV usually incorporated). PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence. [42] See SNP genotyping for more information.
  • Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product. [43]
  • Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. [44] A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction. [45]
  • Convective PCR: a pseudo-isothermal way of performing PCR. Instead of repeatedly heating and cooling the PCR mixture, the solution is subjected to a thermal gradient. The resulting thermal instability driven convective flow automatically shuffles the PCR reagents from the hot and cold regions repeatedly enabling PCR. [46] Parameters such as thermal boundary conditions and geometry of the PCR enclosure can be optimized to yield robust and rapid PCR by harnessing the emergence of chaotic flow fields. [47] Such convective flow PCR setup significantly reduces device power requirement and operation time.
  • Dial-out PCR: a highly parallel method for retrieving accurate DNA molecules for gene synthesis. A complex library of DNA molecules is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by PCR. [48]
  • Digital PCR (dPCR): used to measure the quantity of a target DNA sequence in a DNA sample. The DNA sample is highly diluted so that after running many PCRs in parallel, some of them do not receive a single molecule of the target DNA. The target DNA concentration is calculated using the proportion of negative outcomes. Hence the name 'digital PCR'.
  • Helicase-dependent amplification: similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation. [49]
  • Hot start PCR: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (e.g., 95 °C) before adding the polymerase. [50] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody[12][51] or by the presence of covalently bound inhibitors that dissociate only after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
  • In silico PCR (digital PCR, virtual PCR, electronic PCR, e-PCR) refers to computational tools used to calculate theoretical polymerase chain reaction results using a given set of primers (probes) to amplify DNA sequences from a sequenced genome or transcriptome. In silico PCR was proposed as an educational tool for molecular biology. [52]
  • Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths. [53]
  • Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence. [54]
  • Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers it has been used for DNA sequencing, genome walking, and DNA footprinting. [55]
  • Methylation-specific PCR (MSP): developed by Stephen Baylin and James G. Herman at the Johns Hopkins School of Medicine, [56] and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
  • Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene. [57]
  • Multiplex ligation-dependent probe amplification (MLPA): permits amplifying multiple targets with a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).
  • Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes. That is, their base pair length should be different enough to form distinct bands when visualized by gel electrophoresis.
  • Nanoparticle-Assisted PCR (nanoPCR): some nanoparticles (NPs) can enhance the efficiency of PCR (thus being called nanoPCR), and some can even outperform the original PCR enhancers. It was reported that quantum dots (QDs) can improve PCR specificity and efficiency. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) are efficient in enhancing the amplification of long PCR. Carbon nanopowder (CNP) can improve the efficiency of repeated PCR and long PCR, while zinc oxide, titanium dioxide and Ag NPs were found to increase the PCR yield. Previous data indicated that non-metallic NPs retained acceptable amplification fidelity. Given that many NPs are capable of enhancing PCR efficiency, it is clear that there is likely to be great potential for nanoPCR technology improvements and product development. [58][59]
  • Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
  • Overlap-extension PCR or Splicing by overlap extension (SOEing) : a genetic engineering technique that is used to splice together two or more DNA fragments that contain complementary sequences. It is used to join DNA pieces containing genes, regulatory sequences, or mutations the technique enables creation of specific and long DNA constructs. It can also introduce deletions, insertions or point mutations into a DNA sequence. [60][61]
  • PAN-AC: uses isothermal conditions for amplification, and may be used in living cells. [62][63]
  • quantitative PCR (qPCR): used to measure the quantity of a target sequence (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA, or RNA. quantitative PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative PCR has a very high degree of precision. Quantitative PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (real-time PCR) but this abbreviation should be used only for reverse transcription PCR. qPCR is the appropriate contractions for quantitative PCR (real-time PCR).
  • Reverse Complement PCR (RC-PCR): Allows the addition of functional domains or sequences of choice to be appended independently to either end of the generated amplicon in a single closed tube reaction. This method generates target specific primers within the reaction by the interaction of universal primers (which contain the desired sequences or domains to be appended) and RC probes.
  • Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends).
  • RNase H-dependent PCR (rhPCR): a modification of PCR that utilizes primers with a 3’ extension block that can be removed by a thermostable RNase HII enzyme. This system reduces primer-dimers and allows for multiplexed reactions to be performed with higher numbers of primers. [64]
  • Single Specific Primer-PCR (SSP-PCR): allows the amplification of double-stranded DNA even when the sequence information is available at one end only. This method permits amplification of genes for which only a partial sequence information is available, and allows unidirectional genome walking from known into unknown regions of the chromosome. [65]
  • Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR [66] (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR [67] (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming).
  • Suicide PCR: typically used in paleogenetics or other studies where avoiding false positives and ensuring the specificity of the amplified fragment is the highest priority. It was originally described in a study to verify the presence of the microbe Yersinia pestis in dental samples obtained from 14th Century graves of people supposedly killed by the plague during the medieval Black Death epidemic. [68] The method prescribes the use of any primer combination only once in a PCR (hence the term "suicide"), which should never have been used in any positive control PCR reaction, and the primers should always target a genomic region never amplified before in the lab using this or any other set of primers. This ensures that no contaminating DNA from previous PCR reactions is present in the lab, which could otherwise generate false positives.
  • Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures a degenerate primer is used to amplify in the other direction from the unknown sequence. [69]
  • Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3–5 °C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3–5 °C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles. [70]
  • Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer—which can lead to artefactual 'noise') [71] by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends), [72] 5'RACE LaNe [73] and 3'RACE LaNe. [74]

The heat-resistant enzymes that are a key component in polymerase chain reaction were discovered in the 1960s as a product of a microbial life form that lived in the superheated waters of Yellowstone’s Mushroom Spring. [75]

A 1971 paper in the Journal of Molecular Biology by Kjell Kleppe and co-workers in the laboratory of H. Gobind Khorana first described a method of using an enzymatic assay to replicate a short DNA template with primers in vitro. [76] However, this early manifestation of the basic PCR principle did not receive much attention at the time and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis. [77]

When Mullis developed the PCR in 1983, he was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies, where he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived the idea for PCR while cruising along the Pacific Coast Highway one night in his car. [78] He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase. In Scientific American, Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat." [79] DNA fingerprinting was first used for paternity testing in 1988. [80]

Mullis and Professor Michael Smith, who had developed other essential ways of manipulating DNA, [81] were jointly awarded the Nobel Prize in Chemistry in 1993, seven years after Mullis and his colleagues at Cetus first put his proposal to practice. [82] Mullis's 1985 paper with R. K. Saiki and H. A. Erlich, "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia"—the polymerase chain reaction invention (PCR) – was honored by a Citation for Chemical Breakthrough Award from the Division of History of Chemistry of the American Chemical Society in 2017. [83] [1]

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90 °C (194 °F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures. [1] So the early procedures for DNA replication were very inefficient and time-consuming, and required large amounts of DNA polymerase and continuous handling throughout the process.

The discovery in 1976 of Taq polymerase—a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally lives in hot (50 to 80 °C (122 to 176 °F)) environments [13] such as hot springs—paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation, [14] thus obviating the need to add new DNA polymerase after each cycle. [2] This allowed an automated thermocycler-based process for DNA amplification.

Patent disputes Edit

The PCR technique was patented by Kary Mullis and assigned to Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The Swiss pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently [ when? ] holds those that are still protected.

A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the lives of the original PCR and Taq polymerase patents, which expired on March 28, 2005. [84]


Step 5: qPCR optimisation

Primer design and testing

During gene expression, DNA is first transcribed into mRNA. In eukaryotes, non-coding regions of the mRNA sequence, known as introns, are removed and the protein-coding regions, known as the exons, are joined to produce the mature mRNA that is translated into protein. This process is called RNA splicing. The human genome contains

20,000 protein coding genes, which can be processed into more than 80,000 protein-coding mRNA, and the estimated number of proteins synthesised is in the range of 250,000 to 1 million [23]. This suggests the regulation of gene expression is a complicated process. One of the regulatory processes is alternative splicing, in which particular exons of the same gene are joined to produce multiple mRNA, called splice variants, which are then translated into different protein isoforms. We recommend including all splice variants of a target gene unless the user is only interested in one particular splice variant of the target gene. It is also preferable to design primers that bind specifically to cDNA but not genomic DNA, as amplification from genomic DNA could interfere with gene expression analysis. This can be achieved by designing a primer sequence that crosses an exon-exon junction, or by including a large intron between the forward and reverse primer. This is an essential requirement if no genomic DNA elimination step is performed during RNA extraction. In certain circumstances, such as when the target gene sequence does not contain an intron or the primer cannot be located at the exon-exon junction due to the DNA sequence, genomic DNA must be removed from the RNA sample before being converted to cDNA. For example, the gene encoding human heat shock protein family A member 6 (HSPA6) does not contain any introns, and thus genomic DNA must be removed from the RNA sample to prevent the primers from amplifying both genomic DNA and cDNA simultaneously during the qPCR reaction. The advantage of using commercially-available, spin column-based kits to extract RNA is they generally include a genomic DNA elimination step.

To test if primers are specific to cDNA, one approach is to perform qPCR reactions using cDNA, a ˗RT control, or water as template. The final PCR amplicons can then be separated using electrophoresis with a 2% agarose gel. A single sharp DNA band of expected size should be present only in the reaction with cDNA if the primers are only binding to cDNA ( Fig 4 ).

DNA fragment products produced from a PCR reaction with the same primers, but using either water (Lane 2), -RT (cDNA synthesis reaction containing RNA but no cDNA, Lane 3), or cDNA (Lane 4) as template, were separated on a 2% agarose gel. Lane 1 is the 100 bp DNA ladder. A single sharp DNA band of the expected size, which is the final PCR amplicons, is present only in the reaction with cDNA.

Primer specificity can also be checked by melting curve analysis. Melting curve analysis is an assessment of the dissociation characteristics of double-stranded DNA (the product from the qPCR reaction) during heating, and can be used in qPCR reactions with intercalating dyes, such as SYBR Green. SYBR Green only fluoresces when it is bound to double-stranded DNA, but not in the presence of single-stranded DNA. At the end of a qPCR run, the thermal cycler is programmed to increase the temperature gradually from 60ଌ to 95ଌ (0.05ଌ·s -1 ) and to measure the amount of fluorescence. The double-stranded PCR amplicon begins to denature to single-stranded DNA, resulting in decreased fluorescence. The temperature at which the base-base hydrogen bonding between two DNA strands is broken depends on their length, guanine-cytosine content, and their complementarity thus, a unique melting curve of the changing rate of fluorescence (-Rn) versus temperature will be produced for each specific double-stranded DNA fragment ( Fig 5 ). If more than one DNA fragment is produced during the qPCR reaction, using cDNA and genomic DNA as the template respectively, more than one melting curve will be detected. We highly recommend including melting curve analysis with SYBR Green-based qPCR analysis, and ensuring a single specific product is produced in all reactions amplifying the same target gene. This can be easily done by adding the melting curve temperature program at the end of a qPCR run, which is a function available in most qPCR instruments.

The qPCR reaction using cDNA synthesised from Intact (pink) and Degraded RNA (red) sample show the same melting curve, indicating that the same PCR amplicon is produced. However, a different melting curve is observed when using cDNA synthesised from RNase Treated RNA sample (blue), which shows a different PCR amplicon is produced during the qPCR reaction. Our recommendations for qPCR optimisation are listed in Box 6.

Box 6

It is recommended to design qPCR primers that are specific to the target gene and only amplify cDNA (e.g., target all intended isoforms, and span a junction between exons), and the primer specificity needs to be tested as part of the qPCR validation process [1]. We recommend including a melting curve analysis to test primer specificity, as it requires no extra step. According to the MIQE guidelines, authors are required to provide sufficient information on the qPCR target and primers, such as gene symbol, sequence accession number, and amplicon length [1] (see example in Experiment 4).

Optimising qPCR performance

In a qPCR reaction, the quantification cycle (Cq) value is defined as the number of cycles required for the fluorescent signal to exceed the background fluorescence (also referred to as threshold cycle (Ct), crossing point (Cp), or take-off point (TOP) in previous publications). The qPCR software programs can set the threshold automatically after determination of the baseline fluorescence from cycle 3 to 15 across the entire reaction plate, which is known as the baseline value. By default, the QuantStudio Real-Time PCR software program (Applied Biosystems, Foster City, CA), which we use in our laboratory, sets this threshold at ten standard deviations above the mean baseline fluorescence. However, both the baseline and threshold can be adjusted manually.

To obtain high amplification efficiency (an increase in number of PCR amplicons per cycle), both the primer and cDNA concentration need to be optimised for different target genes. The recommended amplification efficiency is between 93% and 105% (the slope of the Cq against the Log of the cDNA input in a standard curve is between -3.2 and -3.5 and the R 2 value is above 0.98 see examples in Fig 6 ) [15].

A standard curve was generated using a 10-fold dilution of cDNA as template for qPCR reactions. The resulting Cq values are plotted against the Log of the cDNA input. The efficiency, as well as the R 2 value, are within the acceptable range. The efficiencies of Cyclophilin and PGC-1α are approximately equal, as the absolute value of the slope of 㥌q against the Log of the cDNA input is < 0.1.

The choice of cDNA concentration for the final qPCR reaction will depend on the qPCR kit of choice, the primers used, as well the expression level of the target gene. In our laboratory, we first dilute cDNA ten times with water before using in any qPCR reactions. When testing a new set of primers, a standard curve should be generated using a series of diluted cDNA samples as template ( Fig 6 , using Cyclophilin and PGC-1α as an example,). A “no template” control reaction should be set up using only water (template free control, TFC). qPCR amplification efficiency can then be calculated from the slope of the graph of Cq values plotted against the Log of the cDNA input (Efficiency = (10 𢄡/slope – 1) × 100). New sets of primers should be designed and tested if the amplification efficiency is not within the recommended range.

For all experiments described in this paper, we used an initial primer concentration of 300 nM. It is recommended to choose primer and cDNA concentrations within the linear dynamic range for qPCR, which results in a Cq of between 20 to 30 [15]. However, in reactions with a high Cq value (㸰, depending on the expression level of target gene and the qPCR protocol), it is necessary to run qPCR reactions with different primer concentrations, and to use the concentration that gives the lowest Cq value (indicating the reaction was performed under the most efficient conditions). Different qPCR reaction kits may recommend a different primer concentration. Our recommendations for optimising qPCR performance are listed in Box 7.

Box 7

It is suggested to keep the machine’s default setting as the threshold, but to always check if it has been set in the region of exponential amplification across all amplification plots, and that all plots are parallel and above the background noise of the baseline [15]. Each qPCR reaction should be optimised to achieve a preferred Cq value (20 to 30) and amplification efficiency (93%�%). For some target genes, it is difficult to obtain primers with the desired amplification efficiency even after testing multiple primer sets. We recommend using primers with an amplification efficiency closest to the desired range of between 93% and 105% (see primers for ACTB and GAPDH as examples in Experiment 4).


SCRIPT RT-qPCR ProbesMaster

Description:
SCRIPT RT-qPCR ProbesMaster is designed for quantitative real-time analyses of RNA templates using Dual Labeled Fluorescent Probes. The ready-to-use mix is based on a genetically engineered reverse transcriptase with enhanced thermal stability providing increased specificity, high cDNA yield and improved efficiency for highly structured and long cDNA fragments.
The 2x conc. mix contains all reagents required for RT-qPCR (except template, primers and the dual labeled fluorescent probe) to ensure fast and easy preparation with a minimum of pipetting steps. The premium quality enzymes and the optimized reaction buffer containing ultrapure dNTPs ensure superior real time PCR results.
RT-qPCR is used to amplify double-stranded DNA from single-stranded RNA templates to allow a rapid real-time quantification of RNA targets. In the reverse transcription step the reverse transcriptase synthesizes single-stranded DNA molecules (cDNA) complementary to the RNA template. In the first cycle of the PCR step the hot-start DNA polymerase synthesizes DNA molecules complementary to the cDNA, thus generating a double-stranded DNA template. The hot-start polymerase activity is blocked at ambient temperature and switched on automatically at the onset of the initial denaturation. The thermal activation prevents the extension of non-specifically annealed primers and primer-dimer formations at low temperatures during PCR setup.
One-step RT-qPCR offers tremendous convenience when applied to analysis of targets from multiple samples of RNA and minimizes the risk of contaminations.
The mix can also be used in combination with ROX reference dye (#PCR-351) in PCR instruments that are compatible with the evaluation of the ROX signal.

Content:
SCRIPT RT-qPCR ProbesMaster
Ready-to-use mix of SCRIPT Reverse Transcriptase, Hot Start Polymerase, RNase Inhibitor, dNTPs, reaction buffer and stabilizers.

Dual Labeled Fluorescent probes:
Real-time PCR technology based on dual labeled DNA probes provides a highly sensitive and specific PCR system with multiplexing capability. It requires two standard PCR primers and the DNA probe that hybridizes to an internal part of the amplicon. The sequence of the dual labeled DNA probe should avoid secondary structure and primer-dimer formation.

Continue with reverse transcription and thermal cycling as recommended.

Reverse transcription and thermal cycling:
Place the vials in a PCR cycler and start the following program.

reverse
transcription 5)
50-55 °C10-15 min1x
initial
denaturation 6)
95°C5 min1x
denaturation95°C15 sec35-45x
annealing and
elongation
60-65 °C 7) 1 min 8) 35-45x

For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary. Note that optimal reaction times and temperatures should be adjusted for each particular RNA / primer pair.


OUTLINE AND RATIONALE OF THE METHOD

The extraction kit has been selected on the basis of its efficiency for isolating genomic and plasmid plant DNA that have to be subsequently amplified by the PCR. DNA extraction consists of breaking the cell wall, eliminating the RNA, and removing proteins by precipitation. After this, the DNA is fixed on an affinity column, washed, and eluted. Similar approaches have already been applied to the detection of DNA from other plants [ 6 ] or from viruses [ 7 ].

Note that, in this experiment, PCR is used to determine whether soybean flour contains genetically modified plant material, but the technique can also be used in various contexts such as paleontology and forensic science [ 8 – 12 ]. The PCR proceeds in three phases: 1) heat denaturing the double stranded DNA, 2) annealing the primers on the sequences by cooling, and 3) elongation of the DNA by extending the primers in opposite directions. These three steps are called a cycle, and the PCR comprises a repetition of this cycle. The amount of DNA is exponentially amplified. DNA yield therefore increases as a function of 2 n , where n is the number of cycles. This means that after one cycle the amount of DNA is doubled, and after 30 cycles, theoretically the DNA has been amplified over 10 9 -fold and can easily be detected on electrophoresis gel.

For this experiment PCR primers were designed to detect two types of genes. The first primer set amplifies endogenous plant genes, a chloroplast gene and the specific soybean lectin gene. In this way, we verify the presence of the plant DNA and, in particular, soybean DNA. The second primer set amplifies sequences normally not found in plants but introduced by genetic transformation. In this case the two amplified foreign DNAs are the constitutively active 35 S promoter from cauliflower mosaic virus and the terminator of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens. If either of these two sequences is amplified, this shows that the target DNA is transgenic [ 1 ]. In principle, all the approved genetically engineered agricultural crops have been transformed with constructs containing either or both of these elements. The identification of new promoter and terminator sequences in transgenic constructs is becoming a matter of concern for scientists.

The PCR primers have been designed to create amplified products of different sizes to facilitate their identification. Table I shows the sequences of the synthesized primers. The annealing sites have been chosen to generate only a single DNA fragment to simplify interpretation. These fragments are visualized as bands on agarose gels following PCR (see the table for the sizes of the amplified sequences).


Polymerase Chain Reaction

David P. Clark , . Michelle R. McGehee , in Molecular Biology (Third Edition) , 2019

5 Inverse PCR

Another approach that uses incomplete sequence information to amplify a target gene is inverse PCR . In this case, a sequence of part of a long DNA molecule, say a chromosome, is known. The objective is to extend the analysis along the DNA molecule into the unknown regions. To synthesize the primers for PCR, the unknown target sequence must be flanked by two regions of known sequence. The present situation is exactly the opposite of that. To circumvent this problem, the target molecule of DNA is first converted into a circle.

Performing PCR on a circularized DNA template amplifies neighboring regions of unknown sequence.

A restriction enzyme, usually one that recognizes a six-base sequence, is used to make the circle. This enzyme must not cut into the known sequence, but it will cut upstream and downstream from the known region. The resulting fragment will have unknown sequence first, the known sequence in the middle, followed by more unknown sequence. The two ends of the fragment will have compatible sticky ends that are easily ligated together to make a circle of DNA ( Fig. 6.12 ). Two primers corresponding to the known region and facing outwards around the circle are used for PCR. Synthesis of new DNA will proceed around the circle clockwise from one primer and counter clockwise from the other. Overall, inverse PCR gives multiple copies of a segment of DNA containing some DNA to the right and some DNA to the left of the original known region.

Inverse PCR allows unknown sequences to be amplified by PCR provided that they are located next to DNA in which the sequence is already known. The DNA is cut with a restriction enzyme that does not cut within the region of known sequence, as shown in Step 1. This generates a fragment of DNA containing the known sequence flanked by two regions of unknown sequence. Since the fragment has two matching sticky ends, it may be easily circularized by DNA ligase. Finally, PCR is performed on the circular fragments of DNA (Step 2). Two primers are used that face outwards from the known DNA sequence. PCR amplification gives multiple copies of one linear product that includes unknown DNA from both left and right sides.


Watch the video: Multiplex PCR and RT PCR (January 2023).