Introduction

Types of PCR

Multiplex PCR employs different primer pairs in the same reaction for simultaneous amplification of multiple targets. This type of PCR often requires extensive optimization of annealing conditions for maximum amplification efficiency of the different primer–template systems and is often compromised by nonspecific PCR artifacts. A stringent hot-start procedure and specially optimized buffer systems are absolutely crucial for successful multiplex PCR.

Compared with standard PCR systems using only 2 primers, an additional challenge of multiplex PCR is the varying hybridization kinetics of different primer pairs. Primers that bind with high efficiency could utilize more of the PCR reaction components, thereby reducing the yield of other PCR products. This often results in unamplified DNA sequences and absence of expected PCR products. Commercial PCR kits are available that are specifically designed to overcome the challenges of multiplex PCR and it is recommended that, where possible, such a kit is used.

PCR products of up to 4 kb can be routinely amplified using standard PCR protocols using Taq DNA polymerase. However, amplification of PCR products longer than 4 kb often fails without lengthy optimization. Reasons for failure include nonspecific primer annealing, secondary structures in the DNA template, and suboptimal cycling conditions — all factors which have a greater effect on the amplification of longer PCR products than on shorter ones. Preventing DNA damage, such as DNA depurination, is of particular importance for amplification of long PCR products, as a single DNA lesion within the template is sufficient to stall the PCR enzyme. DNA damage during PCR cycling can be minimized with specific buffering substances that stabilize the pH of the reaction. Commercial PCR kits are available that are specifically designed to overcome the challenges of long-range PCR, for example, by using an optimized mixture of Taq DNA polymerase and proofreading enzymes, and it is recommended that, where possible, such a kit is used.
Single-cell PCR provides a valuable tool for genetic characterization using a limited amount of starting material. By flow cytometry or micromanipulation, individual cells of interest can be isolated based on cell-surface markers or physical appearance. Amplification of low-abundance template molecules — which can be as low as one or two gene copies — requires a PCR system that is highly efficient, specific, and sensitive. Again, commercial PCR kits are available that are specifically designed for single-cell PCR.
Faster PCR amplification enables increased PCR throughput and allows researchers to spend more time on downstream analysis. The demand for reducing time-to-result is met by the recent development of faster PCR techniques. Fast PCR can be achieved using new thermal cyclers with faster ramping times or through innovative PCR chemistries that allow reduced cycling times due to significantly shortened DNA denaturation, primer annealing, and DNA extension times. Fast-cycling PCR reagents must be highly optimized to ensure amplification specificity and sensitivity.

MSP enables the methylation status of target DNA to be determined after sodium bisulfite treatment. The method requires two sets of primers to be designed: one set that anneals to unchanged cytosines (i.e., methylated in the genomic DNA) and one set that anneals to uracil resulting from bisulfite treatment of cytosines not methlyated in the genomic DNA. Amplification products derived from the primer set for unchanged sequences indicates the cytosines were methylated and thus protected from alteration (6).

Stringent and highly specific PCR conditions must be used to avoid nonspecific primer binding and the amplification of PCR artifacts. This is particularly important as the conversion of unmethylated cytosines to uracils reduces the complexity of the DNA and increases the likelihood of nonspecific primer–template binding.

See Hot-start DNA polymerase for more information.
See High-fidelity DNA polymerase for more information.
RAPD is a PCR-based tool enabling the study of organisms at the molecular level. It uses small, nonspecific primers to amplify seemingly random regions of genomic DNA. Successful primer pairs produce different banding profiles of PCR products between individuals, strains, species, etc., when analyzed using an agarose gel.

In RAPD, the primers are only ~10 bases long. As a result, annealing temperatures required are <40°C.

RACE is a variant of RT-PCR and is a procedure for amplification of nucleic acid sequences from a messenger RNA template between a defined internal site and unknown sequences at either the 3' or the 5' -end of the mRNA. RACE only requires the knowledge of a short sequence within the mRNA of interest. It is often used for cloning the remainder of incomplete cDNAs. There are two techniques:

5' RACE — amplifies 5' cDNA ends

3' RACE — amplifies 3' cDNA ends

The first step is common to both types of RACE and involves the conversion of RNA to single-stranded cDNA using a reverse transcriptase. The second steps are unique to each type of RACE; although each generates information that may yield the full-length cDNA sequence.

Because RACE uses an “anchor site” within the mRNA as a point of reference, it is sometimes known as “anchored PCR”.

In situ PCR is a PCR reaction that occurs inside the cell on a slide, thus combining the sensitivity of PCR or RT-PCR amplification with in situ hybridization. In situ PCR allows cellular markers to be identified and further enables the localization to cell-specific sequences within cell populations, such as tissues and blood samples. Therefore, it is a powerful tool in applications such as the study of disease progression.

Fresh or fixed cells or tissue samples can be used in the procedure, although preparation of the sample is critical to the result, with fixation having a direct influence on PCR signal. The procedure is suitable for use with radiolabeled, fluorescently labeled or biotin-labeled nucleic acid probes.

The PCR process is essentially the same as a standard PCR, but with some modified reaction conditions (e.g., Mg2+ concentration).

Differential display PCR is based on RT-PCR and is used to compare and identify differences in mRNA (and therefore gene) expression patterns between two cell lines or populations.

In this technique, first-strand cDNA synthesis is primed with an anchored primer complementary to ~13 nucleotides of the poly(A) tail of mRNA and the adjacent 2 nucleotides of the transcribed sequence. After reverse transcription and amplification, amplified products are visualized using gel electrophoresis. The banding patterns observed can be compared to identify differentially expressed cDNAs in the 2 populations.

Invented in the 1990s, the technique fast became a key tool in gene expression analysis. However, it has been more recently superseded by RNA-seq, microarrays, and qRT-PCR.

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