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Discovery

Multiplying DNA Over and Over and Over Again

Greg Walker

Imagine if you wished to find out if a newborn baby carried the AIDS virus in a very small number of its blood cells or you wished to link a particular suspect in a rape case with a single sperm cell. To do this, requires checking out often-minute amounts of DNA.

It's not just in biological applications that identification of DNA fragments has its uses -- you can use DNA-tracking techniques to find out which oil tanker is responsible for a damaging spill, which factory produced the explosives in a terrorist's bomb, or which jeans are real and which are counterfeit. You can do this by "spiking" a product, such as oil, explosives or clothing, with a recognisable DNA code that can be used as an identifier.

In the past, these useful applications have been limited because of the minute amounts of DNA available in many cases (such as at crime scenes or within specific cells); often the amount of DNA involved was too small to be easily identified. The development of a new technique, termed polymerase chain reaction (PCR), has provided a means of effectively multiplying the DNA available, opening up a huge number of applications.

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The PCR concept is a simple one -- take a tiny amount of DNA and multiply it millions of times to produce a quantity large enough to be useful for whatever application you need it for. The process is very specific in that it will only amplify the DNA from the target species. For example in a forensic sample of human DNA contaminated with DNA from soil micro-organisms, only the human DNA will be amplified.

One of the beauties of the PCR process is that it all occurs in one tube of reagents. PCR relies on the fact that when a molecule of DNA is sufficiently heated, the hydrogen bonds holding the double helix together are disrupted and the molecule is separated, or denatured, into two strands. If the DNA solution is allowed to cool, then the complementary bases can reform, reproducing the double helix of the original.

The structure of DNA is well known, looking something like a twisted ladder. The sides of the ladder consist of a series of ribose sugar and phosphate molecules, the rungs are formed from four base pairs (adenine, thymine, cytosine and guanine) which link together across the "ladder" in a specific fashion (adenine joins with thymine, cytosine with guanine). It is the arrangement of these pairs, and the proteins that they in turn produce, that defines what each segment, or gene, on a strand of DNA does.

The DNA can be broken into separate strands by heating it for several minutes at 94o-96oC. This causes the hydrogen bonds between the bases to break and form single strands. The single strands can now be accessed by primers, which are segments of single-strand DNA that will bind to specific areas on the target DNA. The primers are about 20 bases long. The material is allowed to cool for one to several minutes to 50o-65oC to allow the primers to stick to their complementary sequences on either side of the target DNA sequence.

Next, some DNA polymerase enzyme synthesises new DNA complementary to the old strand. This enzyme can read the opposing strands' base arrangement and places the bases in the correct position opposite their corresponding base pair. ie adenine with thymine and cytosine with guanine. This produces two new double-stranded pieces of DNA.

The DNA polymerase enzyme used is often extracted from the hot pool bacteria Thermus aquaticus, as it can withstand the high temperatures required for DNA separation. This part of the process occurs at 72oC for several minutes. Cooling the reaction causes the reformation of double strands.

In a similar manner to the constant cycles of cell division the process of PCR is repeated all over again. Theoretically PCR can go on indefinitely making more and more copies of the original DNA sequence, starting with one molecule of DNA to produce two, then four, then eight, 16, 32, 64 and so on. After about 20 repeats, you have a million or so copies of the original DNA; 30 cycles should result in one billion amplifications.

PCR has provided a revolutionary technique for medicine, biology, anthropology and history. It can be used to explore the genetic relatedness of living things across species boundaries. Imagine being able to test the DNA from a human 8,000 years old, or being able to unravel whether the Neanderthals were an unsuccessful off-shoot of the human evolutionary tree or whether they contributed directly to modern humans.

The future for PCR seems assured and there will continue to be new and exciting developments. Some of the more enthusiastic proponents suggest that within a few years most genetic testing will be PCR based.

Greg Walker, NZSM