DETERMINANTES SOCIALES DE LA SALUD Y VIH/SIDA

Polymerase Chain Reaction somewhat mimics what happens in vivo during DNA replication. A designated set of primers added in solution establishes the DNA sequence to copy. The other components necessary for PCR are the extracted DNA template, the DNA polymerase, dNTPs, magnesium and a buffer capable of keeping a stable pH for a wide range of temperatures. Some commercial kits used in forensic laboratories also include additives that minimize the effects of inhibition present in the samples.

One of the first thermo-resistant DNA polymerase used in PCR was extracted from the bacterial species Thermus aquaticus (Taq) which inhabits in hot springs. The Taq resistance to high temperatures is important because in PCR, the DNA is firstly melted with an increase in temperature which would degrade most DNA polymerases (94). Nowadays the polymerases used for PCR are still called Taq polymerases, even though most of them are genetically modified enzymes descendent from Taq. Another important characteristic of Taq polymerases used in forensic sciences is the fact that they require an activation step, usually performed at the beginning of the PCR reaction. The need for an activation step prevents unspecific amplification by the polymerase at room temperature. Enzymes that require the initial activation step are called Hot Start polymerases (95).

Despite of the differences in polymerases developed due to biotechnological advances, all polymerases need magnesium to properly amplify DNA, therefore an optimal concentration of magnesium is necessary in PCR (96). The primers commonly used are small oligonucleotide sequences (18-32 bp) that are complementary to the flanking regions of the DNA sequence to be copied. For each sequence to copy two primers are necessary – a forward and a reverse primer – both flanking the beginning and end of the region to be amplified. Typically the primers should have a GC (guanine- cytosine) content around 50% and a melting temperature of around 60 ºC (95). The melting temperature (TM) of a DNA template or of a primer:template region is the

temperature necessary to provide to the system in order to cause the separation of half of the primer from the template DNA, respectively. The melting temperature depends on the GC content and the length of the primer. Primers offer the 3’-OH necessary for

the template by addition of dNTPs to the nascent strand. For STR analysis, the primers anneal either to microsatellite regions of the genomic DNA and some to specific regions of the Y or X chromosome.

The PCR reaction is carried on through several steps, namely denaturation, annealing and extension. Each step has a designed temperature and duration, which are precisely achieved by instruments called thermocyclers. The design of thermocyclers allows each PCR tube to be surrounded by metal that quickly transitions its temperature at a rate of several degrees Celsius per second. The tubes are composed of thin plastic that allows an improved conductance of hear to the entire volume of the PCR solution. During denaturation, the solution is heated to 95 ºC or above to ensure that the double stranded DNA is melted to single stranded DNA. This melting of DNA mimics in vitro the unwinding of DNA by the replication machinery that occurs in vivo. The next step, called annealing, happens at an optimal temperature determined by the primer sequence and length. Typically, the annealing temperature can be set on the PCR instrument as 5 ºC below the TM of the primers. At this temperature the primers anneal to the template

DNA thus creating the template:primer substrate for DNA polymerase. The instrument will then raise the temperature to 72 ºC which is the optimal temperature for Taq polymerase to extend the primers and form a new copy of the template. The cycle of temperatures is repeated several times until several copies of the same sequence have been made (95). Each double stranded PCR product will denature in the next cycle and serve as a template for the polymerase. For that reason, in a reaction that is occurring in optimal conditions, the number of PCR products increases exponentially with the number of cycles. For the first cycles, the DNA serving as template is the genomic DNA added to

the mixture. With the increase in PCR products, the template DNA soon becomes the PCR products themselves. The fact that PCR products from the initial cycles become a template is an important issue in the use of PCR in forensic laboratories since any

amplification errors made in the first few cycles are perpetuated until the end of the PCR (97).

In forensic laboratories several DNA regions are amplified in one reaction, which is called multiplex PCR. The simultaneous amplification of multiple DNA regions is possible through the use of primers which have a similar TM and are designed to not

anneal to each other. Since the primers are labeled with fluorophores, the PCR products corresponding to each set of primers can be distinguished from each other after PCR and during analysis. Besides the use of multiplex PCR, another feature of PCR in forensic laboratories is that it uses low amounts of DNA. When a low amount of template is added to the PCR, it is possible that not all primers will be able to anneal to it with the same efficiency. Therefore some products may be more predominant at the end of the reaction, causing an artifact known as allele dropout. If the template added to the PCR is degraded, meaning the genomic DNA is fragmented due to the action of extreme heat or other environmental factors, the polymerase may not have an intact template to copy from. The bigger the region to copy, the more likely amplification can be interrupted because of fragmentation at that locus. The potential lack of amplification of larger amplicons as a result of DNA fragmentation can cause an imbalance of STRs in the final PCR product if the small STRs are properly amplified (95). The presence of inhibitors that are co-

extracted with DNA, if in high enough concentration, may decrease the PCR efficiency. The only way to detect inhibition is during quantitation with the use of an internal

control. Some forensic commercial kits contain additives such as bovine serum albumin (BSA) that helps to diminish the effects of inhibition (95).

The commercial kits developed for STR amplification were designed to take these limitations into consideration and therefore they require a certain amount of DNA to be used as quantified by specified methods. Before being commercialized most of those kits were tested by multiple laboratories to better predict the type of products they will produce, thus diminishing the risk of unspecific or unexpected results. However, since each DNA sample is unique because of its unique origin of crime scene, commercial kits continue to be developed in research every day.