Question

1)Discuss why PCR amplification and size separation using capillary electrophoresis is used in the generation of an STR Profile. write approx 700 words

Answer 1

Short tandem repeats (STRs), which are sometimes referred to as microsatellites or simple sequence repeats (SSRs), are accordion-like stretches of DNA containing core repeat units of between two and seven nucleotides in length that are tandemly repeated from approximately a half dozen to several dozen times . Although the human genome contains thousands upon thousands of STR markers, only a small core set of loci have been selected for use in forensic DNA and human identity testing. Like using a single, common currency in a financial sense, core loci permit equivalent genetic information to be shared and compared. Commercial kits are now available to generate DNA profiles containing these core STR loci. Millions of STR profiles are generated worldwide each year by government, university, and private laboratories performing various forms of human identity testing, including DNA databasing, forensic casework, missing persons/mass disaster victim identification, or parentage testing.

With STR typing, PCR is used to recover information from small amounts of available biological material. The relatively short PCR product sizes of approximately 100–500 bp generated with STR testing are generally compatible with degraded DNA that may be present due to environmental insults on the evidentiary biological material found at a crime scene. PCR amplification of multiple STR loci simultaneously, or multiplexing, is possible with different colored fluorescent dyes and different sized PCR products. Use of multiple loci enables a high power of discrimination in a single test without consuming much DNA (e.g., 1 ng or less of starting material). It is worth noting that these core STR loci occur in between genes in which a high degree of variability is tolerated and are thus not directly responsible for physical traits such as hair color or eye color or genetic diseases.

Following PCR amplification, the overall length of the STR amplicon is measured to determine the number of repeats present in each allele found in the DNA profile. This length measurement is made via a sized-based separation involving gel or capillary electrophoresis (CE). Each STR amplicon has been fluorescently labeled during PCR, since either the forward or reverse locus-specific primer contains a fluorescent dye. Thus, by recording the dye color and migration time of each DNA fragment relative to an internal size standard, the size for each STR allele may be determined following its separation from other STR alleles. Commonly used instruments for STR allele separation and sizing include the ABI PRISM 310 and ABI PRISM 3100 genetic analyzers (Applied Biosystems)

There are a number of both biological and instrumental artifacts that often must be sorted through in order to generate a complete and accurate STR profile. Biological artifacts include stutter products, split peaks from incomplete adenylation, triallelic patterns, and variant alleles containing mutations in the repeat or flanking regions that cause an allele to be off-ladder. Instrumental artifacts arise from voltage spikes, dye blobs, and bleed-through between dye colors.

While multicolor fluorescence detection CE instrumentation, such as the ABI PRISM 3100 genetic analyzer, presently dominate the field, efforts are ongoing to develop microchip CE platforms to perform high-resolution DNA separations with eventual integration of the PCR amplification and CE separations. In addition, mass spectrometry (MS) with matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) techniques have been used for STR typing without allelic ladders.