For most of the STR loci that we use, the four-base sequence GATA is repeated in tandem. So, for example, on one of your chromosomes 18 you may have a D18S535 gene that has eleven GATA repeats. Since chromosomes occur in pairs (one coming from your mother and one from your father) you will also have another D18S535 gene that might have 13 GATA repeats on your other chromosome 18. These alternate forms of the same gene are called alleles.

In order to amplify an STR region the sequence of the flanking DNA must be known. The flanking DNA sequences are used to design forward and reverse primers for the amplification process. In the amplification process, primers are small sequences of single-stranded DNA (usually 20-25 nucleotides long) that are complementary to one of the strands of the double-stranded DNA that is to be amplified. A primer is designed to define each end of the amplified fragment of DNA. One primer must be complementary to one strand of the double helix while the other primer is complementary to the other strand.

You may be asking why these short pieces of single-stranded DNA (commonly referred to as oligonucleotides) are called primers. The reason is that they serve to prime the reaction in which new DNA is synthesized. An enzyme called DNA polymerase catalyzes this reaction. The DNA polymerase cannot synthesize a new DNA strand from the existing double-stranded DNA unless it separates into single strands that have a short double-stranded primer with a free end to synthesize from.

All DNA strands have polarity. In other words, the two ends are different. We call one end of a DNA strand the 5’ (5-prime) end and the other end the 3’ (3-prime) end. These names are derived from the chemical structure of the sugar molecule deoxyribose that is part of the DNA molecule. In nature, DNA is synthesized from free 3’ ends of primers. In a laboratory amplification reaction, this synthesis takes place as long as temperature is controlled properly and the appropriate chemicals are added to the reaction. Of course, the building blocks of DNA (A, T, G, and C) must be present along with the enzyme and the cofactor magnesium that is necessary for the DNA polymerase to function.

Another important part of the amplification process is the denaturation of the double helix that is to be amplified. Denaturation of a double helix is the process of separating it into single strands. The easiest way to denature a DNA molecule is to heat it. When a double helix is heated to 95° (close to the boiling point of water) it separates into single strands. When it is cooled back down, these single strands re-anneal to form a double helix again. This typically starts to occur at a temperature between 60° C and 70° C. The process of heating and cooling DNA to denature and renature it is an important part of the PCR process. In order to heat DNA to a temperature of 95° C, it is necessary to use a DNA polymerase that is not inactivated by this high temperature. Most enzymes are destroyed by exposure to temperatures this high. Fortunately, bacteria that survive in deep-sea thermal vents or hot springs have DNA polymerases that are not heat inactivated. It is this type of enzyme that makes PCR possible.

So how is PCR carried out? If an amplification mixture (template DNA = an individual’s extracted genomic DNA + primers + DNA polymerase + A, T, C, and G building blocks + magnesium cofactor) is heated to 95° C, the template double helixes will denature. If the mixture is then cooled to about 60° C, the primers will be able to anneal to the ends of the STR region. Subsequent heating to 72° C provides an optimal temperature for the DNA polymerase to extend the primer into a longer strand of DNA. The PCR occurs in cycles using temperatures approximating these. The temperatures are optimized for the sequences of the template and primer DNA. The reactions are carried out in an instrument called a Thermal Cycler which can very accurately change the temperature of a metal block in which the amplification tubes are inserted.

Theoretically, each cycle of the PCR process will double the number of copies of the region being amplified. If a PCR reaction is carried out for 30 cycles (each cycle lasts about 5 minutes) the number of copies of the template could increase by a billion-fold in about 2.5 hours. In reality, we typically obtain less of an increase than this, but it is still an impressive amplification process. We start the PCR process with the extracted DNA from about 1500 cells.

We also take advantage of the fact that we can amplify more than one STR region in a single reaction. This is because we will separate amplified DNA fragments by fragment size as well as color of the label that is attached to each fragment. We use three different fluorescent label colors and a size range of 80 to 340 base pairs to enable us to differentiate 11 different STR’s. The labels are actually attached to the 5’ end of one primer for each system.