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Amplified DNA Product Separation for Forensic Analysts

Data Analysis

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The steps for assigning an allele call to each peak are:

  1. Data collection
  2. Peak recognition
  3. Color separation
  4. Peak sizing
  5. Allelic ladder comparison
  6. Allele assignment (i.e., genotype)

DNA fragments, represented as peaks on an electropherogram, can be sized relative to the internal size standard (ISS), which is mixed in with the amplified product.

Three plots for an Identifiler™ internal size standard
Three plots for an Identifiler™ internal size standard - courtesy Phoenix Police Department
Phoenix Police Department (see reuse policy).

For forensic short tandem repeat (STR) testing, allele sizes must have a sizing precision of a single base. Variables such as temperature, sample and buffer ionic strengths, osmotic flow, and electric field can all influence DNA mobility. The DNA fragments are sized by comparing their migration time with that of flanking internal standard peaks. The size is then calculated by interpolation, assuming a linear relationship between peaks.

AmpFℓSTR Identifiler allelic ladder on the ABI Prism® 310 Genetic Analyzer
AmpFℓSTR Identifiler allelic ladder on the ABI Prism® 310 Genetic Analyzer
National Institute of Justice (NIJ) (see reuse policy).

Sequence variation and attachment of dyes affect the size of the fragments. The size of an individual fragment is determined by comparison with the internal size standard. The allelic designation is determined by comparing its assigned size with the allelic ladder. This ISS is labeled with a different colored dye so that it can be distinguished from the DNA fragments in the sample. The ISS generally contains DNA fragments spanning the size range.

ABI uses three software programs for the genotyping process:

  • GeneScan®
  • Genotyper®
  • GeneMapper® ID

GeneScan® software spectrally resolves the dye colors for each peak and is used to size the DNA fragments in each sample. These data are then imported in the Genotyper® program, which compares the sizes of alleles in the allelic ladder to those obtained for each sample. GeneMapper® ID is a program that was released by Applied Biosystems (AB) in 2003, which combines the functions of GeneScan® and Genotyper®.

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