The polymerase chain reaction (PCR) is a technology using thermo-stable DNA polymerase to amplify a few copies of DNA across several orders of magnitude, generating millions of copies. The method relies on thermal cycling, consisting of cycles of repeated temperature changes and enzymatic replication of DNA. One pair of primers (containing short sequences complementary to the target region) along with a DNA polymerase are key components to enable selective and repeated amplification. Two primers are synthesized to correspond to the beginning and ending of the DNA stretch to be copied. Polymerase is moved along the segment of DNA, reading its code and assembling a copy.

Polymerase Chain Reaction

1. Multiplex PCR

 

Multiplex PCR is a method using multiple primer sets targeting different DNA sequences within a single PCR tube. Because multiple genes are amplified at once, primer dimer and interference should be avoided, and amplicons should be visualized by gel electrophoresis.

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2. Real-time PCR

 

Real-time PCR monitors DNA during amplification in a real-time manner. A precise quantitative gene expression analysis is possible with this method, as well as a qualitative analysis, such as pathogenic infection. In general there are two detection chemistries in quantitative PCR: non-specific fluorescent dyes that intercalate with any double-stranded DNA (e.g., SYBR green), and sequence-specific DNA probes consisting of fluorophore and quencher. (e.g., Taqman probe)

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3. ASO PCR

 

Allele Specific Oligonucleotide (ASO) PCR is a technique to detect mutations of single base in DNA. Primers are designed based on nucleotide sequences of different alleles. Especially, primers should be carefully designed for perfectly matching to oligonucleotide in 3’-end of a specific allele. ASO is used to detect the presence of a mutation in one's DNA sequence, and can be used to establish whether an individual has a certain genetic disorder.

4. Methylation Specific PCR (MSP)

 

DNA methylation in human typically occurs at cytosine-phosphate-guanine(CpG) sites and can be involved in regulation of gene expression. There are certain regions, known as CpG islands that are associated with the promoters. CpG islands are not methylated under normal gene expression. Both hypermethylation and hypomethylation have become a cause of cancer by expression of oncogenes or inhibition of tumor suppressor genes. Because methylation-specific PCR (MSP) can rapidly assess the methylation status within CpG sites, we can find out the cancer-induced region.

Multiplex PCR is useful if there are multiple gene targets in a sample. This technique is cost-effective since it quantifies pathogens and determines genotypes in a single test. Thus, multiplex PCR is widely used for sexually transmitted diseases (STD), HPV detection andgenotype determination and detection of Tuberculosis and multi-drug-resistant tuberculosis. This method is also used for forensics and paternity test using short tandem repeats (STR) marker.

SYBR Green is cheaper than Taqman probe but it binds to all dsDNA PCR products, including nonspecific PCR products. Melting curve analysis should be conducted to assess dissociation-characteristics of double-stranded DNA after PCR. TaqMan probes bind to a DNA region amplified by a set of primers. During extension, the 5' to 3' exonuclease activity of Taq polymerase degrades the probe that has annealed to the template. Degradation of probe releases the fluorophore and breaks the close proximity to the quencher, therefore relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in quantitative PCR is directly comparative to the fluorophore released and the amount of DNA template present in the PCR.

ASO PCR method can be applied in Single Nucleotide polymorphism (SNP) analysis. SNP is a single DNA nucleotide sequence variation at specific sites of genome which commonly occurs within a population. Humans are genetically 99.9% identical, but 0.1% difference in the sequence determine human race and other characteristics. For example, Koreans are more vulnerable to gastric and liver cancers than Westerners.

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A bisulfite treatment on Methylated DNA can change non-methylate cytosine into uracil. Depending on this, methylated-specific and unmethylated-specific primers are used for PCR. Methylated-specific primers amplify methylated DNA while unmethylated-specific primers amplify non-methylated DNA.