Polymerase Chain Reaction

This method is used to amplify DNA sequences without using any living system. PCR amplifies DNA in an in vitro environment. In 1984, an American biochemist, Karry Mullis, invented this method (Fig. 8.1). After the invention of PCR, it became an essential technique for performing molecular biology experiments. The primary application of PCR is to synthesize a million copies of selected DNA sequences. Since its inception, it has brought tremendous changes in gene cloning procedures. 

1.0Principle of PCR

• The principle of PCR is divided into three main steps: 
• Denaturation (separation)
• Renaturation (annealing)
• Synthesis (amplification) 
• The double-standard target DNA is denatured, later annealed in the presence of an external primer, and finally amplified using deoxyribonucleotides in the presence of polymerase enzymes. 

2.0Procedure of PCR

• A reaction mixture containing the DNA from which a segment is to be amplified, an excess of the two primer molecules, all four kinds of dNTPs, MgCl₂, and Taq polymerase, is mixed with the DNA. 
• The DNA segment is amplified involving the following three steps:

Step 1 Denaturation

• The reaction mixture is heated to a high temperature (94-96ºC) so that the DNA molecule is denatured, i.e., the two strands of the DNA duplex are separated. 
• Each strand of the target DNA then acts as a template for DNA synthesis.

Step 2 Annealing

• The mixture is then cooled by lowering the temperature upto 55-65ºC. 
• At this temperature, the two primers anneal to each of the single-stranded template DNA. 
• Annealing occurs due to complementary sequences located at the 3' ends of the template DNA.

Step 3 Extension

• In this step, the temperature is adjusted so that the Taq polymerase becomes active. Synthesis of the new DNA strand begins between the primers, dNTPs, and Mg2+. 
• The optimum temperature for this polymerization is kept at 72ºC. 
• The next PCR amplification cycle begins as soon as all the stages of the previous cycle end. 
• During PCR operation, the extension product of one cycle serves as a template for subsequent cycles, and the amount of DNA doubles each time. 
• Thus, a single template molecule of DNA generates 2n molecules at the end of n cycles.

3.0Limitations  of PCR

• Contamination of the PCR reaction mixture, even by a single gene, can result in an impure product of the PCR reaction. 
• Female workers are required to diagnose male-specific genes; otherwise, male workers' skin cells can also cause contamination. 
• PCR cannot be performed directly on food samples, and processing of samples is required to extract and study the DNA. DNA recovery in complex food samples (milk, oysters) may be only a few per cent. Food substances copurified with bacterial DNA may reduce amplification efficiency. 
• If cell growth is allowed to increase DNA, target DNA recovery probability increases, interference by food substances reduces, but time for analysis increases. 
• Dead cells may contain amplifiable DNA; hence, positive PCR does not mean viable cells in the sample. Thus, pasteurized milk may give positive PCR, and the technique of using reverse transcriptase is lost. PCR can directly detect mRNA and can be significant in distinguishing between live and dead cells. 
• PCR is most helpful in amplifying DNA segments less than 2 kb in length. 
• Base substitution errors by Taq polymerase are 1 per 9000 bp, and frameshift errors are 1 per 40000. These errors are significant if PCR extends over 30 cycles when an error occurs in every 300 bp product. Taq's high error rate is due to its lack of 3' 5' exonuclease activity (proofreading). 
• Shuffle clones (recombinant products) may be formed if the product formed is only partially extended in one cycle to reanneal to a different template in a later cycle.

4.0Types of PCR 

• We will use different types of PCR variants to help amplify the target DNA and the primers used to carry out this technique. 
• In some situations, we would amplify mRNA molecules to produce the complementary DNA (cDNA), which is reverse transcription PCR. 
• Apart from amplifying the DNA sequences by using the modern approaches in PCR, one can even quantify the amount of DNA amplified inside the thermal cycler. 
• This technique is known as quantitative PCR. Let us discuss these variants of PCR techniques one by one. 

Inverse PCR

• This type of PCR is employed when the researcher needs to learn about the DNA sequence being amplified. In such situations, the researcher would use primers that are not specific to the target DNA. 
• The first step of this PCR method is to amplify the border sequences of target DNA by inserting it into a vector known as a plasmid. By doing so, we will invert the target DNA into a circular form; hence, it is known as inverse PCR. 
• After inserting the target DNA into the circular vector, we will perform amplification, which amplifies both the target DNA and the vector.

Nested PCR 

• This PCR variant's significant advantage is reducing the amplification of undesirable DNA sequences. 
• Two types of primers are used to perform this method, where the primary primers will bind to the nonspecific sequences or, you can say, general co-sequences present in the target DNA. 
• However, the second type of primers being used are target-specific; hence, they can bind with the DNA template produced after amplification with the first primers. 
• This PCR method will enhance the accuracy, specificity, and sensitivity of the technique and reduce nonspecific binding, which indirectly avoids undesired amplification. 

Reverse Transcription PCR (RT-PCR)

• This type of PCR is widely used in gene expression studies. 
• To perform PCR, the messenger RNA (mRNA) must be converted into two complementary DNA (cDNA) using the enzyme reverse transcriptase. 
• Since we use reverse transcriptase enzyme, this technique is named reverse transcription PCR. The cDNA is the template or target DNA for the normal PCR reaction. 

Real time PCR (q PCR)

• This PCR method is applied when the researcher is looking to quantify the amount of DNA sequence being amplified. 
• That is the level of genes that are being expressed. Moreover, real-time PCR allows you to monitor the amplification of target DNA while amplification is taking place. 
• Another advantage of using a thermal cycler is that it can rapidly heat and cool samples. 
• This will minimize the undesired changes in the physical and chemical properties of the target and amplifier DNA sequences. 
• In addition to all the PCR requirements, fluorescence dyes like SYBR green are used to detect and monitor the progress of qPCR. 

Advantage of using Real-Time PCR 

• Traditional PCR is measured at the End-point (plateau), while Real-Time PCR collects data in the exponential growth phase.  
• The reporter fluorescent signal increases directly proportionally to the number of amplicons generated. 
• The cleaved probe provides a permanent record amplification of an Amplicon. Increase dynamic range of detection. 
• No-post PCR processing. Detection is capable of down to a 2-fold change. 

5.0Application of PCR 

• Diagnosis of disease by detection of bacteria/viruses.  
• Diagnosis of genetic diseases.  
• DNA fingerprinting for forensic medicine.  
• For research purposes.  
• For molecular mapping. 
• For preparation of molecular markers.  
• To monitor genetic engineering and gene therapy experiments.  
• Study of polymorphism. Detection of pathogens in food, water, and other samples.  
• Detection of sex of livestock embryos.