The PCR Process: Amplifying DNA for Analysis

Advances in DNA testing technology enable scientists to transform a particle of DNA into a probability of paternity. Science has made paternity testing quick, convenient and affordable. So, exactly how do we get all those numbers from a cheek swab? In 1983, Kary Mullis invented a method to replicate, or amplify, small sections of DNA. He termed this process the polymerase chain reaction (PCR).

DNA Worldwide utilizes PCR technology for its paternity testing. PCR replicates small fragments of human DNA referred to as loci, or markers. DNA Worldwide paternity tests examine the 13 FBI CODIS markers, an amelogenin marker for sex determination and two additional markers – commonly referred to as a 16-marker test.

The lab technician puts an extract of DNA into a special solution containing primers, small synthetic DNA fragments that can identify and copy the DNA markers, and proteins, used to build copies of the DNA. PCR begins by separating the double-stranded DNA – simply by turning up the heat. As the solution cools, the primers bind to complementary regions of DNA. One primer contains a fluorescent dye so it will be detectable on a DNA analyzer.

A DNA polymerase enzyme elongates each bound primer. The enzyme “reads” the fragment and synthesizes a complimentary strand of DNA – a reverse copy of the base pairs. It follows the rule that adenine (A) bases are complementary with thiamine (T) bases and guanine (G) bases are complementary to cytosine (C) bases. Hence, a T on one strand of DNA is bonded to an A on the opposite strand. Likewise, a G on one strand of DNA is bonded to a C on the opposite strand.

By the end of the first cycle a single target of DNA has been duplicated into two identical strands of DNA. These steps, heating and cooling the DNA solution and copying the DNA strands, are repeated 28 times to allow a minute quantity of DNA markers to amplify into enough copies to be viewed with a DNA analyzer.

After PCR is complete the sample has successfully amplified all 16 loci of interest. Individual variation at each marker will correlate to different DNA fragment sizes. Sophisticated DNA analyzers detect the fluorescent dye bound to each fragment and, with the assistance of analysis software, the sizes of the fragments is measured. The fragments are then represented by the numbers (alleles) that appear on a paternity analysis report. The allele information from each of the 16 loci (markers) is used to determine a biological relationship between individuals. A child’s results will be a combination of half the father’s alleles and half the mother’s alleles.