PCR Variations: Multiplex-PCR and VNTR PCR

• Variations of the basic PCR technique involve several modifications to the standard PCR process for specific applications.

Multiplex-PCR

• Multiplex-PCR amplifies multiple targets in a single tube using several primer pairs.
• It reduces individual reactions needed and enables simultaneous analysis of multiple targets, useful when the sample is limited.
• In genetic disease mutation testing, six or more amplifications can be combined.
• Standard DNA Fingerprinting protocols often amplify 13 targets in groups of 3 or 4.
• Multiplex Ligation-dependent Probe Amplification (MLPA) amplifies multiple targets with a single primer pair, addressing resolution limitations of standard multiplex-PCR.

VNTR PCR

• Variable Number of Tandem Repeats (VNTR) PCR targets genome areas with length variation.
• Genotypes are analyzed by sizing amplification products using gel electrophoresis.
• Analysis of smaller VNTR segments, known as Short Tandem Repeats (or STRs), is fundamental to DNA Fingerprinting databases.

Asymmetric PCR

• Asymmetric PCR preferentially amplifies one DNA strand, which is useful in sequencing and hybridization probing.
• The process involves standard PCR with a limiting amount of one primer.
• Post primer depletion, replication results in an arithmetic increase of the complementary primer.

Long PCR

• Long PCR requires modifications because the original Klenow-based PCR had limitations creating products larger than 400 bp.
• The characterization of Taq polymerase allowed amplification of targets up to several thousand bp long.
• Modified protocols now allow amplification of targets over 50,000 bp.

Nested PCR

• Nested PCR increases DNA amplification specificity, using two primer sets in successive reactions.
• The first primer pair generates DNA products that may include products from non-target areas.
• Products from the first PCR are used in a second reaction, using one ('hemi-nesting') or two nested primers within the first set.
• The combined specificity of all primers usually results in a single product.
• Nested PCR is more successful in amplifying long DNA products than conventional PCR, but requires detailed knowledge of the target sequence.

Quantitative PCR

• Quantitative PCR (or Q-PCR) measures the specific amount of target DNA or RNA in a sample.
• Standard PCR processes are largely qualitative, with final product amount only slightly proportional to initial target amount.
• Q-PCR is run to amplify only within the phase of true exponential increase in order to ensure the amount of product is proportional to the initial amount of target.
• Thermal cyclers capable of monitoring product amount during amplification have been developed for quantitation of samples containing a wide range of target copies.
• Quantitative Real-Time PCR is a commonly used method.
• QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure amplified product amount as amplification progresses.

Hot-start PCR

• Hot-start PCR modifies the way a PCR mixture is initially heated.
• During the initial heating step the polymerase is active, but the target has not yet been denatured so primers may bind to non-specific locations.
• This technique can be performed manually by heating reaction components to the melting temperature (e.g. 95°C) before adding the polymerase.
• Specialized systems that inhibit polymerase activity at ambient temperature via antibody binding or covalently bound inhibitors that dissociate after a high-temperature activation step have also been developed
• 'Hot-start/cold-finish PCR' is achieved with new hybrid polymerases that are inactive at ambient temperature and only activated at elevated temperatures.

Touchdown PCR

• Touchdown PCR is a simple modification that reduces non-specific amplification.
• The annealing temperature is gradually decreased in later cycles.
• Early cycles use an annealing temperature 3-5°C above the standard Tm of the primers.
• Later cycles use a temperature a similar amount below the Tm.
• Higher initial annealing temperature increases primer binding specificity, while lower temperatures permit more efficient later amplification.

RT-PCR

• RT-PCR (or Reverse Transcription PCR) amplifies, isolates, or identifies a known sequence from a cell’s or tissue’s RNA.
• PCR is preceded by reverse transcriptase, an enzyme converts RNA into cDNA
• Both reactions are often perfomed in the same mixture tube, using the initial heating step of PCR to inactivate reverse transcriptase.