Arriving at a DNA profile from analysis of a body fluid sample or stain follows a simple linear process. The body fluid is located using a screening test and possibly a confirmatory one. The DNA is extracted. The STR region is amplified. The amplified DNA is separated from the mixture, and the alleles identified. The DNA profile consists of the identified alleles. The first step has been dealt with in the blood and semen basics. The second is simple wet chemistry, and the remainder of this part of the module focuses on the last three steps.
The amplification and product separation steps can be viewed as black box procedures in which sample is introduced to the equipment and the desired result comes out the other end, but because of its importance we shall now take a brief look at the PCR reaction. If you have not already reviewed the video on the PCR process, do so no or as soon as you have completed this activity. You should realise that the PCR reaction mimics the replication process used by cells to copy their DNA. The key points are that the STR amplified is determined by the primers used in the process. The replication is a three-step process where the template DNA is heat denatured into separate strands.
The sample is cooled, and the primers anneal to their complementary sequences at the five prime ends. And then the temperature is raised and the Taq polymerase extends the primers. The PCR cycle is repeated up to 30 times, and each cycle doubles the number of copies in the reaction mix. The reliability of the amplification falls off if it is repeated too many times, which is the reason for an upper limit of 30. The desired end from this PCR application is a production of STRs in sufficient quantity to identify the alleles present. Quantitative PCR used to measure the amount of DNA in the stain extract is a little different.
It measures the rate of production of product which is proportional to the concentration of DNA in the introduced sample. Now let’s see these steps being implemented in the examination of the blood stain from the crime scene car in our case. The first video shows the seat cover being removed from the car in the crime scene garage and the preliminaries for its examination in the laboratory. Note that the cover is securely sealed and has a label that establishes the chain of custody. The next is the screening step where the stain is swapped with philtre paper on the Kastle-Meyer test applied.
Important steps not seen in the clip are the recording the label information before opening the package, the recording of the physical appearance of the cover, and the swabbing of a non-bloodstained area to act as a negative control sample to show no contamination. The control swab after application of the reagent is seen in the video. The rapid development of a strong pink red colour is what could be expected from a blood stain. Samples are removed from the stained area and a nearby unstained control area, and any DNA present extracted into a small vial. The extracts are taken to the DNA lab for the amplification stage.
First of all, the amount of DNA in the extract is measured since the amplification is fairly sensitive to the quantity of DNA present. The method shown uses a technique called quantitative PCR, which is very briefly described in the section on amplification. The extract is diluted as required and introduced to an instrument called a thermal cycler along with the required reagents for the STR amplification step. The amplified product is introduced to an instrument called the genetic analyzer for separation of the STRs by a process called capillary electrophoresis. The genetic analyzer also identifies the STRs present.
The amplification and product separation steps can be viewed as black box procedures in which sample is introduced to the equipment and the desired result comes out the other end, but we shall take a brief look at how these critical steps work. First of all, let’s see how PCR works to create the required copies of the target STRs. A few definitions are required. Primers are short nucleotide sequences complimentary to the nucleotides that lie either side of the target STR strand. Directionality– three prime and five prime are used to describe the end to end orientation of a DNA strand. The designation refers to the position of the hydroxyl groups, the OH group, and the sugar and the DNA backbone.
Directionality is important because the polynucleotide strand is synthesised in the five prime to three prime direction. The thermal cycler is a device that can rapidly heat and cool a sample to a precise temperature. Taq polymerase is the DNA polymerase used in the PCR reaction. It is so named because it is found in the bacterium Thermus aquaticus, which lives in hot springs water and resists chemical degradation at the high temperatures used in the PCR reaction. The product of the PCR procedure is called an amplicon. The description given has focused on the identity and amplification of a single STR region.
DNA profiles are composed of the results of analyses of 10 to 20 STRs, and in the early days of the technique, this require separate amplifications for each one. Then the technique was tweaked to allow four or so STRs to be amplified at once and the resulting amplicons identified. Today, simultaneous amplification of multiplexes of 20 or more STRs are routine. This is achieved by a mixture of careful selection of the size of the amplicons so that they do not overlap together with selective use of dyes to tag groups of STRs. The multiplexes also include a region associated with the amelogenin gene that permits identification of the gender of the sample donor.
When carried out as described, DNA profiling based on STR analysis is a rapid, informative, and reliable technique. It is also extremely sensitive and will work with quantities of DNA present in biological fluid stains that are too small to see. It will also work with DNA in coughing or sneezing, in dandruff, and in sweat. That’s why crime scene examiners wear hats, suits, gloves, and footwear protection. In the videos showing analysis of the seat cover, the analyst should have been wearing a hair covering. Did you spot this? Not wearing all of the protective clothing can have implications to the interpretation of the DNA analytical results as contamination can easily occur.
At the end of the amplification stage, we have a soup of amplicons and reagents, out of which we have to identify the alleles for each STR. This is achieved by separation using capillary electrophoresis. Key points of this method are that dyes are used to label the amplicons, separation of amplicons is by size, and there is a built in sizing reference standard. STRs with alleles that are in a similar size range are differentiated by the colour of the dye. The capillary electrophoresis equipment usually comes with software that identifies and names the amplicons produced in the amplification stage. The software assigns the alleles present for each of the STRs in the multiplex kit from the combination of their size and dye.