THE ANALYSIS OF SHORT TANDEM REPEATS | Kickoff

THE ANALYSIS OF SHORT TANDEM REPEATS

12 Apr

THE ANALYSIS OF SHORT TANDEM REPEATS

Figure 6.7 During electrophoresis the computer software records the fluorescence levels at regular time points and these are recorded as data points. The DNA fragments that make up the internal- lane size standards are plotted against the data points. An example of the sizing curves that are produced from (a) the GeneScanTM-500 standard (Applied Biosystems) and (b) the ILS600 (Promega) are shown using the local Southern method to generate the size calling curve The loci used in forensic casework have been well characterized and multiple alleles have been sequenced to determine the allelic structure and verify that the size of the peaks is a good indicator of the alleles they represent. However, because the migration of PCR products and internal-lane size standard varies slightly with factors such as temperature and the electrophoretic conditions, and because some STR alleles differ by only one base pair, the use of allelic ladders that contain all the common alleles (Figure 6.9) at each locus has been adopted by the forensic community to ensure accurate profiling [22, 42]. Unlike the internal-lane size standards the allelic ladders cannot be analysed in the same injection as the samples but are run periodically during the analysis of a batch of samples. When assigning the alleles, the unknown peaks are compared to the allelic ladder and should fall within a one base-pair window that is +/− 0.5 bp of the allelic ladder size – if the unknown alleles differ by more than this then they are classified as off-ladder (OL)

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