TECHNOLOGY USED TO PERFORM DNA TESTING

• Jurisdiction: Australia

[82.600] Introduction

Scientists estimate that only a small fraction of our DNA (around 0.3% or about 10 million nucleotides) differs between people and makes us unique individuals. These variable regions of DNA provide the capability of using DNA information for human identity purposes. Methods have been developed to locate and characterise this genetic variation at specific sites in the human genome.

The most common method for examining a specific region of human DNA is an amplification technique known as the polymerase chain reaction or PCR. PCR utilises two primers, which are short DNA sequences that contain a specific order of nucleotides known to match the DNA sequence in an area of interest such as an STR marker. DNA (known as template) is added to a solution which is heated to separate the two strands of DNA. The solution is cooled to allow small pieces of complementary DNA (primers) to bind the template in targeted locations. An enzyme called a polymerase then uses DNA building blocks (nucleotides) to add to the primers creating a piece of DNA that is complementary to the template. The solution is then heated again and the primers will now bind to the original DNA strands and the polymerase generated strands and the process repeats resulting in a logarithmic increase in the copies of DNA as one copy becomes two which becomes four and then eight and 16 and so forth. By attaching a fluorescent dye on the end of one of the PCR primers, labelled PCR product is created enabling very sensitive detection in DNA analysis instruments.

Figure 4

PCR Amplification with Specific Primers labelled primer STR Repeal 5? 3? Flanking region 1 2 3 4 (Maternal allele) 5? 3? 1 2 3 4 5 6 (Paternal allele) forward primer hybridization region reverse primer hybridization region (size in base pairs) 75 80 100 120 140 160 180 200 220 240 260 Fluorescent signal 1000 500 4 DMA Separation and Detection 139bp

In Figure 4 above, the primers define the locus to be amplified or copied, by binding to the flanking regions on either side. During copying the fluorescently labelled primer is incorporated into the copied DNA. The paternal allele has six repeating units and is therefore longer than the maternal allele with only four units. These two alleles can be separated using capillary electrophoresis on the basis of their different lengths as shown at the bottom. To detect the DNA, a laser beam excites the fluorescent tag on the DNA, enabling a charge coupled device (ccd) camera...