Blackett Family DNA Activity 2
Methods of Analysis of STRs
We will assume that you have a basic understanding of the Polymerase Chain Reaction (PCR), and gel electrophoresis, especially as applied to DNA sequence analysis. We will focus here on the special features of PCR and gel electrophoresis as they are applied to STR characterization. If you are unfamiliar with these techniques, you should still be able to complete this activity.
Methods in Analysis of the 13 CODIS STR loci1. DNA extraction
DNA can be extracted from almost any human tissue. Buccal cells from the inside of the cheek are most commonly used for paternity tests. Sources of DNA found at a crime scene might include blood, semen, tissue from a deceased victim, cells in a hair follicle, and even saliva. DNA extracted from items of evidence is compared to DNA extracted from reference samples from known individuals.2. PCR Amplification
DNA primers have been optimized to allow amplification of multiple STR loci in a single reaction mixture. By carefully adjusting the distance of the primers from the tetrameric repeat sequence, products from different loci will not overlap during gel electrophoresis.
In the partial results shown above, the three STRs D3S1358, vWA, and FGA are being analyzed simultaneously. The lengths of the amplified DNAs are shown by the scale from 100 bp to 280 bp at the top of the figure. The middle panels with multiple peaks are reference standards with the known alleles for each STR locus. Notice that the alleles for the three different loci do not overlap. The lower panel shows the alleles for Bob Blackett's mother Norma for the D3S1358, vWA, and FGA loci. Norma's alleles have been compared by computer to the refrence standards, and labeled. To interpret this result, Norma's genotype is 15, 15 at the locus D3S1358, 14, 16 at vWA, and 24, 25 at FGA.3. Detection of DNAs after PCR Amplification
The PCR primers in the commercial kits used for STR analysis have fluorescent molecules covalently linked to the primer. To extend the number of different loci that can be analyzed in a single PCR reaction, multiple sets of primers with different "color" fluorescent labels are used. Following the PCR reaction, internal DNA length standards are added to the reaction mixture and the DNAs are separated by length in a capillary gel electrophoresis machine. As DNA peaks elute from the gel they are detected with laser activation. The sequencing machines used for allele separation and detection are the same type currently being used in the Human Genome Sequencing project, with digital output that can be analyzed by special computer software.
In the AmpFLSTR Profiler Plus PCR Amplification Kit from Applied Biosystems used by Bob Blackett, 9 STRs are analyzed by using three sets of primers. Each set has a different colored fluorescent label. In the figure above, three sets of STRs are represented by blue, three by green, and three by yellow (shown as black) fluourescent peaks. The red peaks are the DNA size standards. Special computer software is used to display the different colors as separate panels of data and determine the exact length of the DNAs. A tenth marker called AMEL is used to distinguish male DNA as X, Y or female DNA as X, X.
A second kit, called Cofiler Plus, is used in a second PCR reaction to ammplify 4 additional STR loci, plus repeat some of the loci from the Profiler Kit. The result from 2 PCR reactions is the analysis of the entire CODIS set of 13 STRs, with overlap of some loci, and a test for the sex chromosomes. The results are obtained as discrete, digital alleles determined from the exact size of the amplified products compared to known standards.
University of Arizona
October 27, 2000
All contents copyright © 1996-2000. All rights reserved.