STR (Short tandem repeat) genotyping is a forensic investigative tool used to identify alleles at specific loci between questioned and reference samples. STR genotyping is a method that employs polymerase chain reaction (PCR) instead of Restriction Fragment Length Polymorphism(RFLP).RFLP method uses restriction enzymes to cut the DNA.STR amplicons are separated and detected using Capillary Electrophoresis.
STEPS IN GENOTYPING AND INTERPRETATION
The peaks in the electropherogram are interpreted and analyzed by the analyst and are called the STR genotype. An STR profile is a combination of all alleles from all the locus into a single series of numbers. The steps in the process are explained below briefly with a help of a flow diagram. The computer software used in the process is labeled on the right-hand side.
The STR Kits take advantage of fluorescent dyes and are spectrally resolved. Each peak is associated with a specific dye color and is sized by comparing it to the internal size standard. The most popularly used size standards are GS500-ROX (red fluorescent dye) and GS500-LIZ (orange fluorescent dye).
Analytical Threshold and Stochastic Threshold
Data produced in the Capillary electrophoresis (CE) instrument contains some baseline noise. Analytical thresholds or peak detected detection thresholds must be set up in the CE instrument. The peaks below the peak detection or analytical threshold are considered unreliable (50 RFU). If all the peaks are above the stochastic threshold (>150 RFU) it means that all amplified alleles are detected in a particular profile. The software program in use for the analysis in CE instruments ABI 310 and 3130 is GenemapperID.
ADDITIONAL PEAKS IN THE DATA
Electropherograms might contain peak other than target alleles. These peaks can be due to technical or biological issues. The analyst identifies the peaks and edits them out in the genotypes in order to prevent incorrect analysis of the data.
Stutter:- When STR loci are amplified, a minor peak 4 bases (n-4) shorter than the main peak is observed. A software implemented stutter filter can remove any peaks within the size range ( 4bp±0.25 bp) and relative height (10% to 20%) of the STR allele peaks to remove stutter products.
Incomplete adenylation:- Incomplete adenylation occurs during the amplification when there is too much DNA or thermal cycling conditions that affect the optimization of the PCR reaction. Incomplete adenylation results in split peaks. Extrachromosomal fragments or triallelic patterns occur due to the primers anneals get duplicated in one of the chromosomes. A mixed sample (more than one individual) contributes to the DNA profile results in more than two peaks in a loci.
Pull up or Bleedthrough:- The inability of an instrument to properly resolve a dye color. The peak observed in one dye is recorded by the sensor as another dye color and a second peak is also generated which is a technical artifact. The artifacts can go unrecognised considering them as true allele if they are of substantial height.
Dye-blobs And Noise :- Dye blobs can result when excessive dyes can clump together in a electropherogram and masks a true allele. Noise is present in all samples and can be seen as small peaks. It is a serious problem when the peak is big.
Other artifacts:- Air bubbles, Urea crystals, voltage spikes and sample contamination can also give rise to small peaks in CE instrument while interpreting the data.
Fundamentals of Forensic DNA Typing