Once your DNA sample is submitted to the laboratory for testing, an analyst first verifies the names on the sample and the case number match those on file. They also check for any signs of sample tampering. Once it is concluded that the samples have not been tampered with, they begin testing.
The buccal swabs are first prepared for DNA extraction. The cotton tip of the swabs are placed into cell lysis buffer, which causes the cells to break apart and release the DNA into the solution. This solution then undergoes Polymerase Chain Reaction (PCR). During PCR, the extracted DNA is placed into a buffer solution that contains DNA polymerase, extra nucleotides, and primers specific to the DNA sequence. Primers are small sequences of DNA designed to attach target STR loci found in the DNA using the specific pairing of A, T, G, and C nucleotide bases. Thirteen STR loci are tested during DNA tests. The primers are tagged with a fluorescent dye, which is used to detect the amplified DNA sequences. The nucleotides are the building blocks for the replicated DNA molecules and the DNA polymerase is an enzyme used to add the nucleotides to the primer.
The first step of PCR is to heat the DNA enough to break the hydrogen
bonds between the base pairs. This is done in order to separate the two DNA strands that make up the double-stranded DNA molecule, which is called denaturation. The separation of the two DNA strands is similar to unzipping a zipper. The separated strands serve as templates for replicating the target DNA molecules. The temperature is lowered to 60°C in order for the single-stranded primers to attach to the target DNA sequences, which is known as annealing. Complementary bases of A and T and G and C would attach to the bases found on the original single strands. The temperature is raised again in order to destroy any weaker non-specific pairings that may have occurred during the annealing step. The temperature is not high enough to destroy the hydrogen bonds between the primers and the DNA target sequence like it was in step one. DNA polymerase then begins the elongation process, in which two copies of the DNA have been created. This entire process occurs for 30 cycles. At each cycle the amount of DNA fragments is doubled, creating billions of copies of each target sequence. The target sequences are the 13 STR loci being studied.
After PCR is complete, the amplified DNA is separated according to size in a capillary tube filled with polymer beads. Smaller DNA fragments pass through the polymer beads faster than larger DNA fragments. A laser then causes the fluorescent tags on the DNA fragments to glow as they pass through the capillary tube. The signals are detected and used to create a DNA profile for each individual tested, which are compared to one another. The maternal alleles of the child’s DNA are identified and the rest of the child’s alleles are compared to the alleged father’s DNA profile. Non-matches at three or more STR loci results in no biological relationship and the alleged father is excluded. Exclusions are tested twice for confirmation.
To schedule a DNA test today or learn more information please call 781-460-6020 or visit www.ARCpointLabs.com/Woburn.