Polymerase Chain Reaction

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Chapter: Biochemistry : Biotechnology and Human Disease

PCR is a test tube method for amplifying a selected DNA sequence that does not rely on the biologic cloning method.


POLYMERASE CHAIN REACTION

PCR is a test tube method for amplifying a selected DNA sequence that does not rely on the biologic cloning method. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source, including viral, bacterial, plant, or animal. The steps in PCR are summarized in Figures 33.19 and 33.20.


Figure 33.19 Steps in one cycle of the polymerase chain reaction.


Figure 33.20 Multiple cycles of polymerase chain reaction.

 

A. Steps of a polymerase chain reaction

PCR uses DNA polymerase to repetitively amplify targeted portions of genomic or cDNA. Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase (2n, where n = cycle number) in DNA with repeated cycles of amplification. The amplified DNA products can then be separated by gel electrophoresis, detected by Southern blotting and hybridization, and sequenced.

 

1. Primer construction: It is not necessary to know the nucleotide sequence of the target DNA in the PCR method. However, it is necessary to know the nucleotide sequence of short segments on each side of the target DNA. These stretches, called flanking sequences, bracket the DNA sequence of interest. The nucleotide sequences of the flanking regions are used to construct two, single-stranded oligonucleotides, usually 20–35 nucleotides long, which are complementary to the respective flanking sequences. The 3I -hydroxyl end of each oligonucleotide points toward the target sequence (see Figure 33.19). These synthetic oligonucleotides function as primers in PCR reactions.

 

2. Denature the DNA: The DNA to be amplified is heated to separate the double-stranded target DNA into single strands.

 

3. Annealing of primers to single-stranded DNA: The separated strands are cooled and allowed to anneal to the two primers (one for each strand).

 

4. Chain extension: DNA polymerase and deoxyribonucleoside triphosphates (in excess) are added to the mixture to initiate the synthesis of two new strands complementary to the original DNA strands. DNA polymerase adds nucleotides to the 3I -hydroxyl end of the primer, and strand growth extends across the target DNA, making complementary copies of the target. [Note: PCR products can be several thousand bp long.] At the completion of one cycle of replication, the reaction mixture is heated again to separate the strands (of which there are now four). Each strand binds a complementary primer, and the cycle of chain extension is repeated. By using a heat-stable DNA polymerase (for example, Taq polymerase from the bacterium, Thermus aquaticus that normally lives at high temperatures), the polymerase is not denatured and, therefore, does not have to be added at each successive cycle. Typically 20–30 cycles are run during this process, amplifying the DNA by a million-fold (220) to a billion-fold (230). [Note: Each extension product includes a sequence at its 5I -end that is complementary to the primer (see Figure 33.19). Thus, each newly synthesized strand can act as a template for the successive cycles (see Figure 33.20). This leads to an exponential increase in the amount of target DNA with each cycle, hence, the name “polymerase chain reaction.”] Probes can be made during PCR by adding labeled nucleotides to the last few cycles.

 

B. Advantages of polymerase chain reaction

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.

 

C. Applications

PCR has become a very common tool for a large number of applications.

 

1. Comparison of a normal gene with a mutant form of the gene: PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laboriously cloning the altered DNA.

 

2. 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 real time PCR (qRT-PCR) allows quantification of starting amounts of the target nucleic acid as PCR progresses (in real time) rather than at the end and is useful in determining viral load (the amount of virus).]

 

3. 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 short tandem repeat polymorphisms. These are very similar to the VNTRs described previously ( but are smaller in size. [Note: Determination of paternity uses the same techniques.]

 

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 33.21 illustrates how the results of such a PCR test can distinguish between homozygous normal, heterozygous (carriers), and homozygous mutant (affected) individuals.

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 or more exons in a gene with many exons such as the gene for CFTR, which has 27 exons.


Figure 33.21 Genetic testing for cystic fibrosis using the polymerase chain reaction (PCR). CFTR = cystic fibrosis transmembrane conductance regulator; bp = base pairs.

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