Polymerase Chain Reaction : Advantages and Applications

Advantages
The major advantages of PCR over biologic cloning as a mechanism for amplifying a specific DNA sequence are sensitivity and speed. DNA sequences present in only trace amounts can be amplified to become the predominant sequence. PCR is so sensitive that DNA sequences present in an individual cell can be amplified and studied. Isolating and amplifying a specific DNA sequence by PCR is faster and less technically difficult than traditional cloning methods using recombinant DNA techniques.
 Applications
PCR has become a very common tool in research, forensics, and clinical diagnostics.
1. Comparison of a normal gene to its mutant form: PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laborious biologic cloning of the DNA.
2. Forensic analysis of DNA samples: DNA fingerprinting by means of PCR has revolutionized the analysis of evidence from crime scenes.
DNA isolated from a single human hair, a tiny spot of blood, or a sample of semen is sufficient to determine whether the sample comes from a specific individual. The DNA markers analyzed for such fingerprinting are most commonly a type of polymorphism known as short tandem repeats. These are very similar to the VNTR described previously  but are smaller in size. [Note: Paternity testing uses the same techniques.]
3. Detection of low-abundance nucleic acid sequences: Viruses that have a long latency period, such as human immunodeficiency virus (HIV), are difficult to detect at the early stage of infection using conventional methods. PCR offers a rapid and sensitive method for detecting viral DNA sequences even when only a small proportion of cells harbors the virus. [Note: Quantitative PCR (qPCR), also known as real-time PCR, allows quantification of the amount (copy number) of the target nucleic acid after each cycle of amplification (that is, in real time) rather than at the end and is useful in determining viral load (the amount of virus).]
4. Prenatal diagnosis and carrier detection of cystic fibrosis: Cystic fibrosis is an autosomal-recessive genetic disease resulting from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common mutation is a three-base deletion that results in the loss of a phenylalanine residue from the CFTR protein . Because the mutant allele is three bases shorter than the normal allele, it is possible to distinguish them from each other by the size of the PCR products obtained by amplifying that portion of the DNA. Figure 34.22 illustrates how the results of such a PCR test can distinguish between homozygous normal, heterozygous (carriers), and homozygous mutant (affected) individuals.

Figure 1:  Genetic testing for cystic fibrosis (CF) using the polymerase chain reaction (PCR). [Note: CF is also diagnosed using allele-specific oligonucleotide analysis .] CFTR = cystic fibrosis transmembrane conductance regulator; bp = base pairs.

The simultaneous amplification of multiple regions of a target DNA using multiple primer pairs is known as multiplex PCR. It allows detection of the loss of ≥1 exons in a gene with many exons such as the gene for CFTR, which has 27 exons.

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