Determining The Presence Of A Microvariant Allele

Suspected microvariants can be fairly easily seen in heterozygous samples where one allele lines up with the fragment sizes in the allelic ladder and one does not (Figure 6.6). In the example shown here, the sample contains a peak that lines up with allele 25 from the FGA allelic ladder and a second peak that is labeled

Figure 6.6

Detection of a microvariant allele at the STR locus FGA. The sample in the bottom panel is compared to the allelic ladder shown in the top panel using Genotyper 2.5 software. Peaks are labeled with the allele category and the calculated fragment sizes using the internal sizing standard GS500-RQX.

I 1 1 1 I ' I 1 I 1 I ' l 1 I 1 I 1 I 1 I 1 I ' I 1 I

236 238 240 242 244 246 248 250 252 254 256 258 260 262

' ProPlus...erSample1 1 Blue ProfilerPlus LADDER

I 1 1 1 I ' I 1 I 1 I ' l 1 I 1 I 1 I 1 I 1 I ' I 1 I

236 238 240 242 244 246 248 250 252 254 256 258 260 262

' ProPlus...erSample1 1 Blue ProfilerPlus LADDER

23 236.43

24 25

240.44

244.46

29 260.74

23 236.43

24 25

240.44

244.46

28 256.64

29 260.74

I—I—,—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—

236 238 240 242 244 246 248 250 252 254 256 258 260 262

NG88Sample22 22 Blue FGA microvariant

I—I—,—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—

236 238 240 242 244 246 248 250 252 254 256 258 260 262

NG88Sample22 22 Blue FGA microvariant

4000 3000 2000 ■1000

4000 3000 2000 ■1000

81 = S25-L25 = 244.34 - 244.46 = -0.12 bp S2 = SOL -L28 = 257.51 - 256.64 = +0.87 bp c = | 81-82 I =| -0.12-0.87 I = 0.99 bp

28.1

as an 'off-ladder allele' and lines up between the 28 and 28.2 shaded virtual bins created by the ladder. Each peak is labeled with its calculated size in base pairs determined by reference to the internal GS500 sizing standard (see Chapter 15). The relative size difference between the questioned sample and an allelic ladder marker run under the same electrophoretic conditions is then used to determine if the allele is truly a microvariant (Gill et al. 1996).

In Figure 6.6, the size difference between the sample allele 25 and the ladder allele 25 is -0.12 bp (5j) while the 'off-ladder allele' differs from the ladder allele 28 by +0.87 bp (§2). The relative peak shift between the two alleles in this heterozygous sample is 0.99 bp (|5j—52|) and therefore the 'off-ladder' allele is 1 bp larger than allele 28 making it a true 28.1 microvariant at the FGA locus.

The presence of a STR microvariant at a particular locus usually becomes evident following a comparison to an allelic ladder made up of characterized alle-les for that locus. However, not all alleles (particularly rare microvariant alleles) can be incorporated into the standard allelic ladder used for genotyping STR markers. Therefore, interpolation of data from peaks that migrate between two characterized alleles or extrapolation of data from peaks that fall outside the expected allele range may be performed. Caution is in order though if 'off-ladder' alleles are more than a one or two repeats away from the nearest allele in the ladder since tetranucleotide repeats do not always size exactly 4.0 bp apart (see Gill et al. 1996, Applied Biosystems 1999, Butler et al. 2004).

If an allele peak falls in between the nominal alleles present in the allelic ladder, the sample may be designated by the allele number followed by a ' .x' (Crouse et al. 1999). For example, the larger FGA allele shown in Figure 6.6 would be designated as a '28.x' allele. However, it is more common to label variant alleles by their calculated repeat content (e.g., 28.1). If an allele migrates above or below the defined allelic ladder, the allele is described as '>' or '<' than the nearest allele (Crouse et al. 1999).

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  • aman
    Can ystr alleles be a microvariant?
    2 years ago

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