Variants of PCR represent a diverse array of techniques that have evolved from the basic polymerase chain reaction (PCR) method, each tailored to specific applications in molecular biology, such as genetic analysis, DNA sequencing, and disease diagnosis, by modifying factors like primer design, temperature conditions, and enzyme usage.
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 (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 repetitive areas of the genome that exhibit length variation. Analysis of the genotypes in the samples 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 a double-stranded DNA target. It is used in some sequencing methods and hybridization probing to generate one DNA strand as product. Thermocycling is carried out exactly as in conventional PCR, but with a limiting amount or leaving out one of the primers. When the limiting primer becomes depleted, replication increases arithmetically rather than exponentially through extension of the excess primer. A modification of this process, named Linear-After-The-Exponential-PCR (or LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer in order to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction. See also 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 (qPCR) 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), sometimes simply called Real-Time PCR (RT-PCR), refers to a collection of methods that use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time as the amplification progresses.
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