Variants of PCR - Basic Modifications

Basic Modifications

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

• Multiplex-PCR uses several pairs of primers annealing to different target sequences. This permits the simultaneous analysis of multiple targets in a single sample. For example, in testing for genetic mutations, six or more amplifications might be combined. In the standard protocol for DNA Fingerprinting, the 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 of primers, avoiding the resolution limitations of multiplex PCR. Multiplex PCR has also been used for analysis of microsatellites and SNPs.

• Variable Number of Tandem Repeats (VNTR) PCR targets areas of the genome that exhibit length variation. The analysis of the genotypes of the sample usually involves 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 preferentially amplifies one strand of the target DNA. It is used in some sequencing methods and hybridization probing, to generate one DNA strand as product. Thermocycling is carried out as in PCR, but with a limiting amount or leaving out one of the primers. When the limiting primer becomes depleted, replication increases arithmetically through extension of the excess primer. A modification of this process, named L'inear-After-The-Exponential-PCR (or LATE-PCR), uses a limiting primer with a higher Melting temperature (T_m) 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 did not generate products that were larger than about 400 bp. Taq polymerase can however amplify targets of up to several thousand bp long. Since then, modified protocols with Taq enzyme have allowed targets of over 50 kb to be amplified.

• Nested PCR is used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first PCR, one pair of primers is used to generate DNA products, which may contain products amplified from non-target areas. The products from the first PCR are then used as template in a second PCR, using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set, thus increasing specificity. 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. By measuring amplification only within the phase of true exponential increase, the amount of measured product more accurately reflects the initial amount of target. Special thermal cyclers are used that monitor the amount of product during the amplification. 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.

• Hot-start PCR is a technique performed manually by heating the reaction components to the DNA melting temperature (e.g. 95°C) before adding the polymerase. In this way, non-specific amplification at lower temperatures is prevented. Alternatively, specialized reagents 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.

• In Touchdown PCR, the annealing temperature is gradually decreased in later cycles. The annealing temperature in the early cycles is usually 3-5°C above the standard T_m of the primers used, while in the later cycles it is a similar amount below the T_m. The initial higher annealing temperature leads to greater specificity for primer binding, while the lower temperatures permit more efficient amplification at the end of the reaction.

• Assembly PCR (also known as Polymerase Cycling Assembly or PCA) is the synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments, to assemble two or more pieces of DNA into one piece. It involves an initial PCR with primers that have an overlap and a second PCR using the products as the template that generates the final full-length product. This technique may substitute for ligation-based assembly.

• In Colony PCR, bacterial colonies are screened directly by PCR, for example, the screen for correct DNA vector constructs. Colonies are sampled with a sterile pipette tip and a small quantity of cells transferred into a PCR mix. To release the DNA from the cells, the 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.

• The Digital polymerase chain reaction simultaneously amplifies thousands of samples, each in a separate droplet within an emulsion.