STR based DNA profiling
Short Tandem Repeats (STRs) have replaced the use of RFLP and become the gold standard for DNA profiling in forensic science. The advantages of STR analysis include greater sensitivity, requiring less DNA to produce reliable results, as well as being faster and more efficient due to its PCR-based method. STR markers are highly polymorphic, making them more useful for individual identification than RFLP. Additionally, STR markers are more stable and less prone to degradation than RFLP markers, making it the preferred method for DNA profiling in forensic science.
What are STRs?
Short Tandem Repeats (STRs), also known as microsatellites, are short repetitive sequences of DNA that are present throughout the human genome. These sequences consist of a repeating unit of 2-7 base pairs, such as AGAGAG or TATATAT, and can vary in length between individuals due to genetic variation.
For example: at a particular locus, one individual may have a sequence of AGAGAG repeated 10 times, while another individual may have the same sequence repeated 14 times. The number of repeats at each STR locus is highly polymorphic, meaning it can vary significantly between individuals in the population and provide a provide a high level of discrimination power in identifying individuals, making STRs ideal for use in forensic DNA profiling.
How many STR markers are used in DNA profiling?
The specific STR markers used for DNA profiling can vary depending on the specific laboratory, country, and the purpose of analysis. A commonly used panel in the United States is the CODIS (Combined DNA Index System) panel, which includes 20 autosomal STR loci and one gender determination marker. Here is a list of the 20 autosomal STR loci, their location on the chromosome, and the repeating sequence of each marker:
Name Location Repeating sequence
D3S1358 Chr 3 (AGAT)n
TH01 Chr 11 (AGAA)n
D21S11 Chr 21 (TCTA)n
D18S51 Chr 18 (AGAA)n
Penta E Chr 15 (TCTA)n
D5S818 Chr 5 (AGAT)n
D13S317 Chr 13 (TATC)n
D7S820 Chr 7 (GATA)n
D16S539 Chr 16 (GATA)n
THO1 Chr 11 (TCAT)n
vWA Chr 12 (AGAT)n
D8S1179 Chr 8 (TCTA)n
TPOX Chr 2 (AATG)n
CSF1PO Chr 5 (TG)n(A)n
D2S1338 Chr2 (TCTA)n
D19S433 Chr 19 (GAAT)n
Penta D Chr X (TCTA)n
D10S1248 Chr 10 (GT)n(AG)n
D1S1656 Chr 1 (AGAT)n
D12S391 Chr 12 (TCTA)n
The gender determination marker, amelogenin (AMEL), is located on both X and Y chromosomes, but it is slightly different on each chromosome due to genetic differences between males (XY) and females (XX). In males, the amelogenin gene contains both an X-specific region (AMELX) and a Y-specific region (AMELY), whereas in females, there are two copies of the X-specific region (AMELX).
STR markers used for DNA profiling in India
The Central Forensic Science Laboratory (CFSL) in India uses a panel of 17 autosomal STR markers for DNA profiling, known as the ‘India17’ panel. Here is a list of the 17 autosomal STR loci included in the India17 panel:
Name Location Repeating sequence
D3S1358 Chr 3 (AGAT)n
D1S1656 Chr 1 (GA)n
D2S1338 Chr 2 (TCTA)n
D8S1179 Chr 8 (TCTA)n
D21S11 Chr 21 (TCTA)n
D18S51 Chr 18 (AGAA)n
D16S539 Chr 16 (GATA)n
D19S433 Chr 19 (GAAT)n
D13S317 Chr 13 (TATC)n
D7S820 Chr 7 (GATA)n
D5S818 Chr 5 (AGAT)n
TPOX Chr 2 (AATG)n
TH01 Chr 11 (AGAA)n
vWA Chr 12 (AGAT)n
FGA Chr 4 (TCTA)n
Penta E Chr 15 (TCTA)n
Penta D Chr X (TCTA)n
The India17 panel includes several markers that are also present in the CODIS panel used in the United States, such as D3S1358, D21S11, D18S51, D5S818, TPOX, TH01, and vWA. However, the India17 panel has some additional markers that are specific to the Indian population, such as D19S433 and D16S539. The use of the India17 panel has been validated by the CFSL and is widely used in forensic casework in India.
PROCEDURE for STR-based DNA profiling
DNA Extraction:
The first step is to extract the DNA from the biological sample. This can be done using a variety of methods, such as phenol-chloroform extraction, salting out, or by using commercially available DNA extraction kits. The extracted DNA is then quantified to determine its quality and quantity.
Polymerase Chain Reaction (PCR):
PCR is a critical step in STR-based DNA profiling as it amplifies the DNA regions that contain the STR markers.
The reagents used in a PCR reaction are as follows:
a) DNA Template: Provides the target DNA sequence to be amplified.
b) Primers: Serve as the starting point for DNA synthesis by DNA polymerase during each PCR cycle. During PCR, primers specific to the target STR loci labeled with fluorescent dyes are used. Each dye corresponds to a specific STR locus.
c) DNA Polymerase: Enzyme responsible for synthesizing a complementary DNA strand based on the template. Taq polymerase, derived from the bacterium Thermus aquaticus, is commonly used in PCR due to its stability at high temperatures.
d) Nucleotides (dNTPs): dATP, dTTP, dCTP, dGTP serve as the building blocks for the new DNA strands synthesized during PCR.
e) Buffer Solution: Provides optimal conditions for the activity of DNA polymerase.
f) Magnesium ions (Mg2+): Required co-factor for DNA polymerase activity. Magnesium ions stabilize the interaction between primers and template DNA.
The steps involved in a PCR reaction are as follows:
a) Denaturation: The temperature is raised to around 95°C for 15-30 seconds to denature the double stranded DNA to provide single-stranded template DNA
b) Annealing: The temperature is lowered to around 50-60°C for 15-30 seconds to allow the primers to bind specifically to the flanking regions of the target DNA segment and serve as a starting point for DNA polymerase to extend the primers.
c) Extension: The temperature is raised to around 72°C for 30-60 seconds (depending on the length of the target DNA segment) to allow the heat stable enzyme, Taq DNA polymerase, to extend the primers using dNTPs and synthesise new complementary strands of DNA, resulting in the amplification of the STR marker region labeled with fluorescent dyes that are specific to each of the STR markers being analyzed.
Genetic Analysis:
These labeled DNA fragments are then separated by size using Capillary Electrophoresis (CE), which involves the use of a narrow glass capillary tube filled with a gel matrix that serves as a separation medium. The labeled DNA fragments are injected into the capillary tube and then subjected to an electric field that causes the DNA fragments to migrate through the gel matrix. The shorter fragments move faster and reach the detection point sooner than the longer fragments. At the detection point, the DNA fragments pass through a laser beam, the fluorescent dyes emit light which is detected by a detector and a electropherogram is generated that represents the size and intensity of each DNA fragment.
Data Interpretation:
The resulting electropherogram can be used to determine the length of each STR allele for each of the markers being analyzed. Different peaks in the electropherogram represent different alleles at specific STR loci. Allele calling is done and allele numbers are assigned to the observed peaks based on their sizes. This step involves interpreting the electropherogram to identify the number of repeats at each locus.
For example: A single peak at a locus (like D8S1179) with the number 10, indicates a homozygous genotype with both alleles having 10 repeats.Double peaks at the same locus, one with number 10 and another with 12, it indicates a heterozygous genotype with alleles of sizes 10 and 12 repeats.
All the loci of the resulting DNA profile can be compared to DNA profiles from other individuals to determine if there is a match. In case of paternity testing, each peak from the DNA profile of the child should be accounted for by either parents, i.e. either the mother or the alleged father.
📌 Read more: Applications of DNA profiling