Mechanism of PCR

Variants of PCR

This page assumes familiarity with the terms and components used in the Polymerase Chain Reaction (PCR) process.

The versatility of PCR has led to a large number of variants:

Basic modifications

Often only a small modification needs to be made to the 'standard' PCR protocol to achieve a desired goal:

  • One of the first adjustments made to PCR was the amplification of more than one target in a single tube. Multiplex-PCR can involve up to a dozen pairs of primers acting independently. This modification might be used simply to avoid having to prepare many individual reactions, or could allow the simultaneous analysis of multiple targets in a sample that has only a single copy of a genome. In testing for genetic disease mutations, six or more amplifications might be combined. In the standard protocol for DNA Fingerprinting, the 13 targets assayed are often amplified in groups of 3 or 4. Multiplex Ligation-dependent Probe Amplification (or MLPA) permits multiple targets to be amplified using only a single pair or primers, avoiding the resolution limitations of multiplex PCR.

  • Variable Number of Tandem Repeats (VNTR) PCR involves few modifications to the basic PCR process, but instead targets areas of the genome that exhibit length variation. The analysis of the genotypes of the sample usually involves simple sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as Short Tandem Repeats (or STRs) is the basis for DNA Fingerprinting databases such as CODIS.
  • Asymmetric PCR is used to preferentially amplify one strand of the target DNA. It finds use in some types of sequencing and hybridization probing, where having only one of the two complementary strands of the product is advantageous. PCR is carried out as usual, but with a limiting amount of one of the primers. When it becomes depleted, continued replication leads to an arithmetic increase in extension of the other primer. A recent modification on this process, known as Linear-After-The-Exponential-PCR (or LATE-PCR), uses a limiting primer with a higher melting temperature Melting temperature (or Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction. (Also see Overlap-extension PCR).
  • Some modifications are needed to perform long PCR. The original Klenow-based PCR process had trouble making a product larger than about 400 bp. However, early characterization of Taq polymerase showed that it could amplify targets up to several thousand bp long. Since then, modified protocols have allowed targets of over 50,000 bp to be amplified.
  • Nested PCR, another early modification, can be used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first, one pair of primers is used to generate DNA products, which may also contain products amplified from non-target areas. The products from the first PCR are then used to start a second, using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set. The specificity of all of the primers is combined, usually leading to a single product. Nested PCR is often more successful in specifically amplifying long DNA products than conventional PCR, but it requires more detailed knowledge of the sequence of the target.
  • Quantitative PCR (or Q-PCR) is used to measure the specific amount of target DNA (or RNA) in a sample. The normal PCR process is performed in a way that is largely qualitative - the amount of final product is only slightly proportional to the initial amount of target. By carefully running the amplification only within the phase of true exponential increase (avoiding the later 'plateau' phase), the amount of product is more proportional to the initial amount of target. Thermal cyclers have been developed which can monitor the amount of product during the amplification, allowing quantitation of samples containing a wide range of target copies. A method currently used is Quantitative Real-Time PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product as the amplification progresses. It is often confusingly referred to as RT-PCR, the same acronym used for PCR combined with Reverse Transcriptase (see below), which itself might be used in conjunction with Q-PCR. More appropriate acronyms are QRT-PCR or RTQ-PCR.
  • Hot-start PCR is a technique that modifies the way that a PCR mixture is initially heated. During this step the polymerase is active, but the target has not yet been denatured and the primers may be able to bind to non-specific locations (or even to each other). The technique can be performed manually by heating the reaction components to the melting temperature (e.g. 95°C) before adding the polymerase. Alternatively, specialized systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody, or by the presence of covalently bound inhibitors that only dissociate 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 only activated at elevated temperatures.
  • Another simple modification can also decrease non-specific amplification. In Touchdown PCR, the temperature used to anneal the primers is gradually decreased in later cycles. The annealing temperature in the early cycles is usually 3-5°C above the standard Tm of the primers used, while in the later cycles it is a similar amount below the Tm. The initial higher annealing temperature leads to greater specificity for primer binding, while the lower temperatures permit more efficient amplification to the end of the reaction.
  • Other common modifications to PCR allow it to amplify low copy targets. The original report on Taq polymerase showed how the use of up to 60 cycles could amplify targets diluted to just one copy per reaction tube. A later report showed how multiple genetic loci could be amplified and analyzed from a single sperm. Modified protocols have allowed the identification of just one copy of the HIV genome within the DNA of up to 70,000 host cells.
  • Assembly PCR (also known as Polymerase Cycling Assembly or PCA) is the artificial synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotide building blocks alternate between sense and antisense directions, and the overlaps determine the order of oligonucleotides, thereby selectively producing the final long DNA product.
  • In Colony PCR, bacterial colonies are rapidly screened by PCR for correct DNA vector constructs. Colonies are sampled with a sterile toothpick and dabbed into a master mix. To free the DNA for amplification, PCR is either started with an extended time at 95°C (when standard polymerase is used), or with a shortened denaturation step at 100°C and special chimeric DNA polymerase. Colonies from the master mix that shows the desired product are then tested individually.
  • The Digital polymerase chain reaction simultaneously amplifies thousands of samples, each in a separate droplet within an emulsion.

Pretreatments and extensions

The basic PCR process can sometimes precede or follow another technique:

  • RT-PCR (or Reverse Transcription PCR) is a common method used to amplify, isolate, or identify a known sequence from a cell's or tissue's RNA. PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two reactions are compatible enough that they can be run in the same mixture tube, with the initial heating step of PCR being used to inactivate the transcriptase. Also, the Tth polymerase described below exhibits RT activity, and can carry out the entire combined reaction. RT-PCR is widely used in expression profiling, which determines the expression of a gene or identifies the sequence of an RNA transcript (including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, 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 an RT-PCR method named RACE-PCR, short for Rapid Amplification of cDNA Ends. (Note that the acronym RT-PCR has more recently been applied to Real-Time PCR, a version of Quantitative PCR described above.)
  • Since PCR is based on components of DNA replication, it is not surprising that it can easily be combined with DNA sequencing. In its simplest form, the products of an 'asymmetric PCR' (above) are diluted into a new reaction containing sequencing components, which are then extended by Taq polymerase.
  • Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' that are first ligated to fragments of the target DNA. PCR primers are then chosen from the linker sequences, and used to amplify the unknown target fragments. It has been used for DNA sequencing, genome walking, and DNA footprinting. A related technique, Amplified fragment length polymorphism, looks at fragments of a genome that differ in length.
  • Methylation-specific PCR (or MSP) was developed to study patterns of methylation at CpG islands in genomic DNA. Target DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two amplifications are then carried out on the modified DNA, using primer sets that distinguish between the modified and unmodified templates. One primer set recognizes DNA with cytosines to amplify the previously methylated DNA, and the other set recognizes DNA with uracil or thymine to amplify unmethylated target. MSP using Q-PCR can also be performed to obtain quantitative information about methylation.

Buffer and temperature modifications

Adjustments to the 'small' components in PCR can sometimes be useful:

  • The divalent magnesium ion (Mg++) is crucial to the activity of the polymerase used in PCR. Since many of the other components used in an amplification will also bind Mg++, it's exact concentration available to the enzyme is difficult to control. In general, lower concentrations will increase replication fidelity, while higher concentrations will introduce more mutations (either of which may be desired).
  • The use in PCR of modified dNTPs can help to control 'carryover' contamination. The PCR process can be carried out using dUTP, an analog of the normal dTTP. Later amplifications are then treated with an enzyme that destroys DNA containing the analog, but leaving the normal target DNA unmodified. Thus, targets that represent contamination from earlier amplifications are selectively destroyed.
  • A wide variety of other chemicals can be added to PCR, for a variety of effects. Mild denaturants (such as DMSO) can increase amplification specificity by destabilizing non-specific primer binding. Certain chemicals (such as glycerol) can act as stabilizers for the activity of the polymerase during amplification. Detergents (such as Triton X-100) can prevent having the polymerase stick to itself, or to the walls of the reaction tube.
  • The temperature changes carried out by the thermal cycler will also affect amplification. A particular set of primers are usually tested using different annealing temperatures to determine their optimum. The time given to the polymerase to fully copy the templates may need to be adjusted, depending on their lengths. Longer extension times can also lead to higher yields after the reaction has entered the 'plateau' phase. When amplifying low-copy targets, the total number of cycles performed must be increased.
  • The polymerases that perform replication during PCR sometime incorporate incorrect bases. This is of no consequence to most assays that test the bulk of the amplified product - the errors are scattered within the product at random, and aren't seen by the assay. However, it is best to perform high-fidelity PCR when the products are individually cloned (for sequencing or expression). A different DNA polymerase (such as Pfu, with a proofreading activity missing in Taq) might be used, and the Mg++ and dNTP concentrations might be adjusted to maximize the number of products that exactly match the original target DNA. Some researchers choose to do the opposite, purposefully running PCR under low-fidelity conditions to produce a spectrum of mutations in the amplified product.

(For additional details, see the auxiliary article PCR optimization.)

Primer modifications

Adjustments to the synthetic oligonucleotides used as primers in PCR are a rich source of modification:

  • Normally PCR primers are chosen from an invariant part of the genome, and might be used to amplify a polymorphic area between them. In Allele-specific PCR the opposite is done. At least one of the primers is chosen from a polymorphic area, with the mutations located at (or near) its 3'-end. Under stringent conditions, a mismatched primer will not initiate replication, whereas a matched primer will. The appearance of an amplification product therefore indicates the genotype. (For more information, see SNP genotyping.)
  • InterSequence-Specific PCR (or ISSR-PCR) is method for DNA fingerprinting that uses primers selected from segments repeated throughout a genome to produce a unique fingerprint of amplified product lengths. The use of primers from a commonly repeated segment is called Alu-PCR, and can help amplify sequences adjacent (or between) these repeats.
  • Primers can also be designed to be 'degenerate' - able to initiate replication from a large number of target locations. Whole genome amplification (or WGA) is a group of procedures that allow amplification to occur at many locations in an unknown genome, and which may only be available in small quantities. Other techniques use degenerate primers that are synthesized using multiple nucleotides at particular positions (the polymerase 'chooses' the correctly matched primers). Also, the primers can be synthesized with the nucleoside analog inosine, which hybridizes to three of the four normal bases. A similar technique can force PCR to perform Site-directed mutagenesis. (also see Overlap extension polymerase chain reaction)
  • Normally the primers used in PCR are designed to be fully complementary to the target. However, the polymerase is tolerant to mis-matches away from the 3' end. Tailed-primers include non-complementary sequences at their 5' ends. A common procedure is the use of linker-primers, which ultimately place restriction sites at the ends of the PCR products, facilitating their later insertion into cloning vectors.
  • An extension of the 'colony-PCR' method (above), is the use of vector primers. Target DNA fragments (or cDNA) are first inserted into a cloning vector, and a single set of primers are designed for the areas of the vector flanking the insertion site. Amplification occurs for whatever DNA has been inserted.
  • PCR can easily be modified to produce a labeled product for subsequent use as a hybridization probe. One or both primers might be used in PCR with a radioactive or fluorescent label already attached, or labels might be added after amplification. These labeling methods can be combined with 'asymmetric-PCR' (above) to produce effective hybridization probes.

Polymerase modifications

There are many choices for the all-important DNA polymerase used in PCR:

  • The Klenow fragment, derived from the original DNA Polymerase I from E. coli, was the first enzyme used to demonstrate PCR. It is inactivated in the denaturation step of PCR, and had to be replenished during each cycle.
  • The bacteriophage T4 DNA polymerase was also tested shortly after the first reports of PCR. It has a higher fidelity of replication than the Klenow fragment. Since it is also destroyed by heat, it has seen little use since the development of thermostable polymerases.
  • The DNA polymerase from Thermus aquaticus (or Taq), was the first thermostable polymerase used in PCR, and is still the one most commonly used. The enzyme can be isolated from its 'native' bacterial source, or from a cloned gene expressed in E. coli.
  • The Stoffel fragment is produced from a truncated gene for Taq polymerase, expressed in E. coli. It is missing the 'forward' nuclease activity, and may be able to amplify longer targets than the native enzyme.
  • The Faststart polymerase is a variant of Taq polymerase that only becomes active after the first denaturation step of PCR, thereby avoiding problems during the first cycle. (see Hot-start PCR above)
  • A thermostable polymerase has also been isolated from the archeozoic organism Pyrococcus furiosus. Unlike Taq polymerase, Pfu DNA polymerase includes a 'proofreading' activity, leading to about a 5-fold decrease in the error rate of replication. Since these errors accumulate during every cycle of PCR, Pfu is the preferred polymerase when products are to be individually cloned for sequencing or expression.
  • An extremely thermostable DNA polymerase has been isolated from Thermococcus litoralis, and is marketed as Vent polymerase.
  • Another thermostable polymerase has been isolated from Thermus thermophilus, and is known as Tth polymerase. In the presence of Mn++ ions, it exhibits a reverse transcriptase activity, allowing PCR amplification to be initiated by RNA targets.
  • But not Bst polymerase, isolated from the thermophilic bacterium Bacillus stearothermophilus. This was an early candidate to be tested for PCR. It was later found to be unsuitable for continued amplification - it is irreversibly inactivated during the denaturation step. This highlights the point that a good polymerase for PCR should both be active at a higher temperature (for specificity), and should also be able to survive the near-boiling temperatures of the PCR process.

Mechanism modifications

Sometimes even the basic mechanism of PCR can be modified:

  • Unlike normal PCR, Inverse PCR allows amplification and sequencing of DNA that surrounds a known sequence. It involves initially subjecting the target DNA to a series of restriction enzyme digestions, and then circularizing the resulting fragments by self ligation. Primers are designed to be extended outward from the known segment, resulting in amplification of the rest of the circle. This is especially useful in identifying sequences to either side of various genomic inserts.
  • Similarly, Thermal Asymmetric InterLaced PCR (or TAIL-PCR) is used to isolate unknown sequences flanking a known area of the genome. 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.

Isothermal amplification methods

Some amplification protocols have been developed that only remotely resemble PCR:

  • Helicase-dependent amplification is a technique that is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension steps. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.
  • PAN-AC also uses isothermal conditions for amplification, and may be used to analyze living cells.

Additional reading

PCR Events

  • Visit the next international real-time PCR Event http://www.qPCR2009.net

References

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