The core repeat region of each STR locus contains tandemly repeated sequences.
The flanking regions surrounding the core repeat region are also needed for STR analysis.
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STR loci are amplified using fluorescent dye-labeled primers.
A multiplex STR system utilizes multiple fluorescent dyes to label each amplicon.
The amplicons are separated via electrophoresis.
The different fluorescent dye colors are resolved by the detector, and the signals corresponding to each DNA fragment are identified using specialized computer software.
The data collection process generates an electropherogram that shows a profile of peaks corresponding to each DNA fragment.
The positions of these peaks represent the electrophoretic mobility of the DNA fragments.
A small fragment, migrating faster than a large one, peaks earlier in the electropherogram than a longer fragment. The DNA fragments are sized by comparison to an internal size standard.
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Mutations at STR Core Repeat Regions
Chromosomal and Gene Duplications
Point Mutations
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Stuttering
Stutter: A minor allele peak, whose repeat units are shorter or longer than the parental allele peak.
Stutter Ratio: The area of the stutter peak divided by the area of the parental peak.
Nontemplate Adenylation
Heterozygote Imbalance
Allelic Dropout: It occurs when an allele, usually one of the heterozygote alleles, fails to be detected.
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Pull-Up Peaks: Occurs when a minor peak of one color on an electropherogram is pulled up from a major allelic peak in another color when the colors have overlapping spectra.
Spikes: These are sharp peaks that are present in all color panels of an electropherogram. It is caused by by air bubbles and urea crystals in the capillary of an electrophoretic platform.
Environmental exposure, such as high humidity and temperature, of biological evidence can lead to DNA degradation such as the breaking of DNA molecules into small fragments.
The more severe the degradation, the more intensive the fragmentation.
When a sample experiences some degradation, large alleles are less likely to be amplified than small alleles.
As a result, the dropout of large alleles often occurs, leading to a partial DNA profile or even a failure in obtaining a DNA profile.
The PCR primers can be redesigned to anneal more proximally to the STR core repeat region than standard STR primers, yielding small amplicons also known as miniSTRs.
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It involves the testing of very small amounts of DNA in a sample.
It is often needed for samples derived from evidence such as fingerprints and tools and weapons handled by perpetrators.
STR analysis of extremely low levels of human DNA can be achieved by increasing the number of PCR cycles to improve the yield of amplicons, thus improving the sensitivity of the analysis.
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To determine the presence of a mixture: First, determine whether the source of the DNA in the sample came from one or more individuals by examining the number of alleles at multiple loci. The characteristics listed below usually indicate a mixture:
To determine the genotypes of all alleles and to identify the number of contributors: Note that the maximum number of alleles at any given locus is two per individual. In the case of homozygous or allele overlap, the number of alleles observed can be less than two per individual.
To estimate the ratios of the contributions: Determine the relative ratios of the contributions to the mixture made by each individual by comparing the peak areas or amplitudes.
To consider all possible genotype combinations: This may be done by pair-wise comparisons to determine the allele combinations that belong to the minor contributor and those that belong to the major contributor.
To compare reference samples: The final step is to compare the genotype profiles with the genotypes of reference samples from a suspect and victim. If the DNA profile of the suspect’s reference sample matches a major or minor component of the mixture, the suspect cannot be excluded as a contributor.
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