The Importance Of Precision In Accurate Genotyping

STR genotyping is performed by comparison of the size of a sample's alleles to size of alleles in allelic ladders for the same loci being tested in the sample. A high degree of precision is needed between multiple runs in order to make an accurate comparison of data from two runs, where one run is the allelic ladder standard and the other run is the questioned sample. The precision for a measurement system is determined by analyzing replicate samples or allelic ladders under normal operating conditions.

Precision for the separation and detection platform must be less than +/-0.5 bp to accurately distinguish between microvariant (partial repeat) alleles and complete repeat alleles that differ by a single nucleotide (Gill et al. 1996). In general the greater the molecular weight of the PCR products, the larger the measurement error. Thus, alleles from larger STR loci such as FGA and D18S51 will generally have a larger size measurement variation than smaller STR loci such as D3S1358 and TH01.

For ABI 310 users, there is a reliance on a high degree of precision for run-to-run comparisons since a number of samples are run in a sequential fashion through the capillary between each injection of the allelic ladder (Lazaruk et al. 1998). Even though the samples are analyzed in parallel on a slab gel, a high degree of precision between samples run in different lanes is also necessary since an allelic ladder is present only a few times on each gel. This same principle applies for multi-capillary array systems.

The precision on an ABI 310 instrument is typically better than 0.1 bp (Wallin et al. 1998, Applied Biosystems 1998). However, a temperature variation of as little as 2 or 3°C over the course of a number of runs can cause allele peaks to migrate slightly differently from the internal sizing standard and therefore size differently over time. To alleviate this problem, the allelic ladder may be run more frequently (e.g., every 10 injections instead of every 20 injections) and the samples can be typed to the allelic ladder sample that was injected nearest them.

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Stammering Its Cause and Its Cure

Stammering Its Cause and Its Cure

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