The Polymerase Chain Reaction Dna Amplification

Out of a natural laziness, I always start with the easiest possible protocol and work from there. Better yet, I suggest that someone start from there, and I come back in a month to see how things worked out.

(Kary Mullis, inventor of PCR)

Forensic science and DNA typing laboratories have greatly benefited from the discovery of a technique known as the polymerase chain reaction or PCR. First described in 1985 by Kary Mullis and members of the Human Genetics group at the Cetus Corporation (now Roche Molecular Systems), PCR has revolutionized molecular biology with the ability to make millions of copies of a specific sequence of DNA in a matter of only a few hours. The impact of PCR has been such that its inventor, Kary Mullis, received the Nobel Prize in Chemistry in 1993 - less than 10 years after it was first described.

Without the ability to make copies of DNA samples, many forensic samples would be impossible to analyze. DNA from crime scenes is often limited in both quantity and quality and obtaining a cleaner, more concentrated sample is normally out of the question (most perpetrators of crimes are not surprisingly unwilling to donate more evidence material). The PCR DNA amplification technology is well suited to analysis of forensic DNA samples because it is sensitive, rapid, and not limited by the quality of the DNA as the restriction fragment length polymorphism (RFLP) methods are.


PCR is an enzymatic process in which a specific region of DNA is replicated over and over again to yield many copies of a particular sequence (Saiki et al. 1988, Reynolds et al. 1991). This molecular 'xeroxing' process involves heating and cooling samples in a precise thermal cycling pattern over ~30 cycles (Figure 4.1). During each cycle, a copy of the target DNA sequence is generated for every molecule containing the target sequence (Figure 4.2). The boundaries of the amplified product are defined by oligonucleotide primers that are complementary to the 3'-ends of the sequence of interest.

Figure 4.1

Thermal cycling temperature profile for PGR. Thermal cycling typically involves three different temperatures that are repeated over and over again 25-35 times. At 94°G, the DNA strands separate, or 'denature'. At 60°G, primers bind or 'anneal'' to the DNA template and target the region to be amplified. At 72°G, the DNA polymerase extends the primers by copying the target region using the deoxynucleotide triphos-phate building blocks. The entire PGR process is about 3 hours in duration with each cycle taking ~5 minutes on conventional thermal cyclers: 1 minute each at 94°G, 60°G, and 72°G and about 2 minutes ramping between the three temperatures.

Figure 4.2 DNA amplification process with the polymerase chain reaction. In each cycle, the two DNA template strands are first separated (denatured) by heat. The sample is then cooled to an appropriate temperature to bind (anneal) the oligonucleotide primers. Finally the temperature of the sample is raised to the optimal temperature for the DNA polymerase and it extends the primers to produce a copy of each DNA template strand. For each cycle, the number of DNA molecules (with the sequence between the two PGR primers) doubles.



94 °C

94 °C 94 °C

\ 72 °C

72 °C

\ 72 °C \

60 °C

60 °C Single Cycle

60 °C


Typically 25-35 cycles performed during PCR

The denaturation time in the first cycle is lengthened to -10 minutes when using AmpliTaq Gold to perform a 'hot-start' PCR

P Starting DNA

3 aemplate


Forward primer V strands

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