Str Genotyping Issues

The technology for DNA profiling and the methods for estimating frequencies and related statistics have progressed to the point where the reliability and validity of properly collected and analyzed DNA should not be in doubt.

In Chapters 12 and 13, we discussed how short tandem repeat (STR) amplification products labeled with fluorescent dyes are separated and detected. In Chapter 14, we examined several commonly used instrument approaches for collecting the STR data. However, the data collection process leaves the analyst with only a series of peaks in an electropherogram or bands on a gel. The peak information (DNA size and quantity) must be converted into a common language that will allow data to be compared between laboratories. This common language is the sample genotype. This chapter will review the process of taking multi-color fluorescent peak information and converting it into STR genotypes.

A locus genotype is the allele, in the case of a homozygote, or alleles, in the case of a heterozygote, present in a sample for a particular locus and is normally reported as the number of repeats present in the allele. A sample genotype or STR profile is produced by the combination of all of the locus genotypes into a single series of numbers. This profile is what is entered into a case report or a DNA database for comparison purposes to other samples. Chapter 21 will cover how statistical calculations, such as random match probabilities, are performed using a STR profile.

STR alleles from the same sample that are amplified with different primer sets or analyzed by different detection platforms will differ in size. However, by using locus-specific allelic ladders, such as those described in Table 5.4, allele peak sizes may be accurately converted into genotypes (Smith 1995). These genotypes then provide the universal language for comparing STR profiles.

THE GENOTYPING PROCESS

Sample data collected from the ABI 310 or other instruments described in Chapter 14 are usually represented in the form of peaks that correspond to the various STR alleles amplified from the DNA sample. These peaks are present at various locations in a sample's electropherogram and usually plotted as fluorescent signal intensity verses time passing the detector (in the case of the ABI 310 or 3100) or position on the gel (in the case of the FMBIO II gel imager). The steps for converting those fluorescent peaks into an allele call are shown in Figure 15.1. Computer programs, such as those listed on the right side of Figure 15.1, play an important role in this process (see Butler et al. 2004). Expert systems, such as True Allele and GeneMapperiD, will be discussed in Chapter 17.

The multiplex STR kits in use today take advantage of multiple fluorescent dyes that can be spectrally resolved (see Chapter 13). The various dye colors are separated and the peaks representing DNA fragments are identified and associated with the appropriate color. The DNA fragments are then sized by comparison to an internal sizing standard (Figure 15.2). Finally, the polymerase chain reaction (PCR) product sizes for the questioned sample are correlated to an allelic ladder that has been sized in a similar fashion with internal standards. The allelic ladder contains alleles of known repeat content and is used much like a measuring ruler to correlate the PCR product sizes to the number of

Figure 15.1 Genotyping process for STR allele determination. Software packages for DNA fragment analysis and STR genotyping perform much of the actual analysis, but extensive review of the data by trained analysts/examiners is often required.

Matrix file

Internal sizing standard (e.g., GS500-ROX)

Allelic ladder sample

Matrix file

Internal sizing standard (e.g., GS500-ROX)

Allelic ladder sample

repeat units present for a particular STR locus. From this comparison of the unknown sample with the known allelic ladder, the genotype of the unknown sample is determined.

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