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What does the diagnosis of hereditary diseases, the detection and diagnosis of infectious diseases, personalized DNA sequencing, DNA cloning, genetic functional analysis, genetic fingerprinting and DNA-based phylogeny have in common?
The all employ a widely used molecular technique called polymerase chain reaction or PCR.
The idea was conceived by Kary Mullis in the early 1980s and was first described, albeit briefly, in an article investigating the mutation that causes sickle cell anemia . The details of the method and its uses were discussed in greater detail over the next two years [2-3]. PCR revolutionized molecular genetics by allowing rapid duplication and analysis of DNA.
The PCR method
PCR is used to amplify a specific region of DNA in order to produce a large number of nearly identical copies. The method uses a heatstable DNA replication enzyme called a DNA polymerase, the four deoxynucleotide building blocks of DNA and two small single-stranded DNA segments called primers, which flank the “target” region of DNA to be amplified and are complementary to each strand (meaning the matching strand to which its bases pair).
There are 3 basic steps in PCR that are carried out at different temperatures to create conditions optimal for:
- DNA denaturation (meaning to separate the double-stranded DNA into single strands).
- Primer binding or hybridization to each of the single strands of DNA at either the beginning or the end of the target sequence, depending upon the single-strand of DNA. Hybridization combines complementary, single-stranded DNA into a single molecule. This process is called annealing.
- DNA polymerase elongation. The enzyme attaches to the primer-single-stranded DNA duplex and synthesizes the complementary strand of DNA, using the existing single-strand as a template.
Newly synthesized DNA strands can serve as additional template for complementary strand synthesis. PCR rapidly amplifies DNA; because both strands are copied, there is an exponential increase in the number of copies. Assuming there is only a single copy of the target gene before cycling starts:
Cycle 1 4 copies (22)
Cycle 2 8 copies (23)
Cycle 3 16 copies (24)
Cycle 35 68.7 billion copies (236)
After 35 cycles of PCR, there will be over 68 billion copies! In reality, PCR starts with many copies of the target gene, so the end result is typically higher. Each cycle only takes a few minutes. Factoring in the time to change temperatures, the entire process can be done in several hours.
More recently, a new method of PCR quantification called real-time PCR or quantitative real-time PCR (qRT-PCR) was developed . qRT-PCR enables the detection and quantification of a specific DNA sequence using a fluorescent reporter (either a dye or a modified DNA oligonucleotide probe), which increases in direct proportion to the amount of DNA amplified in a reaction.
It’s easier to understand how PCR works with pictures. Visit DNA Interactive to view an animation of PCR. DNA Interactive is an award-winning educational web site created in 2003 to celebrate the 50th anniversary of the discovery of the DNA double helix structure.
The advent of PCR and recombinant DNA technologies have enabled numerous applications in basic and clinical research. PCR is often regarded as one of the most important scientific advances in molecular biology. Indeed, Kary Mullis holds the only Nobel Prize ever awarded to a scientist in the biotechnology industry. He received the Nobel Prize for Chemistry in 1993.
Saiki et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350-4.
Mullis et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73.
Mullis and Faloona. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335-50.
Higuchi et al. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y). 1993 Sep;11(9):1026-30.