DNA is located within the nucleus of cells throughout the body and the extraction step is responsible for breaking open the nucleus and releasing the DNA molecules into solution. During this step it is also possible to separate the DNA molecules from all other cellular material and any other debris that may be present in a particular biological sample. Some of these materials can be potential “inhibitors” to steps later on in the DNA testing procedure so it is important to try and isolate only the DNA molecules. Common inhibitors commonly found in forensic cases are hemoglobin and indigo dyes from denim. The methods of extraction used at the BCA for evidence samples are based on the magnetic separation of beads, to which the DNA binds, from the rest of the cell content. Several robotic platforms are in use at the BCA that perform DNA extraction: The Maxwell FSC and the Automate are used for smaller batches of samples and the Hamilton Starlet robot can perform extraction through amplification set-up in a 96-well plate format. The DNA extraction step typically takes 2 to 3 hours.

One of the standards all DNA testing laboratories must meet is to ensure that the DNA recovered from an extraction is human rather than from another source such as bacteria. This is done through quantitation where the quality and quantity of DNA present in a sample is measured and assessed as well as the amount of male DNA in relation to the amount of total DNA in the sample. Determining the amount of DNA in a sample is essential for success in the next step since most amplification systems require a narrow range of input DNA. This step is completed in the Biology Section through the use of a purchased Quantifiler Trio Human Quantification Kit and then running all samples on an instrument known as the ABI PRISM 7500 Sequence Detection System. This process takes approximately 30-60 minutes to set-up and then approximately two hours to run on the instrument.

DNA amplification is accomplished through the use of a technique known as Polymerase Chain Reaction (PCR). PCR is a process in which millions of copies of a specific sequence of DNA can be made in a matter of only a few hours. This is important for forensic DNA samples since the DNA often found at crime scenes is limited in both quantity and quality. This molecular “xeroxing” process is completed by precise heating and cooling of the samples in a thermal cycling pattern for approximately 28 cycles. The amplification kit used by the BCA amplifies 15 different regions of DNA as well as a sex-determining marker. The Biology Section uses the Globalfiler amplification kit for most samples and the Yfiler Plus amplification kit for Y-chromosomal testing. Both kits are purchased from the manufacturer. The samples are run on an instrument known as the Veriti Pro thermal cycler. This process takes approximately 15 minutes to set-up and then approximately 1.5  hours to run on the thermal cycler instrument.

After the PCR reaction is completed it results in a large mixture of amplified DNA molecules which need to be separated in order to distinguish the various molecules from one another. This is accomplished through a process known as capillary electrophoresis. DNA molecules carry a negative charge and once an electric current is applied to the sample the molecules enter a very thin capillary filled with a gel-like polymer and migrate towards the positive anode at the other end of the capillary. The PCR products are then separated by size because the smaller DNA molecules will have an easier time migrating through the polymer than the larger DNA molecules. The data from this process is then collected on a computer attached to the CE instrument and then through the use of a software program a DNA profile is developed. The Biology Section uses an instrument called the ABI Prism® 3500 Genetic Analyzer which is capable of analyzing eight samples at a time. Set-up for this step takes approximately 10 minutes and each injection on the 3500 takes approximately 45 minutes.