Smith R N
Metropolitan Police Forensic Science Laboratory, London, UK.
Biotechniques. 1995 Jan;18(1):122-8.
A strategy is presented for classifying complex short tandem repeat (STR) alleles by size. Such alleles can differ in length by only 1 bp. The HUMACTBP2 locus was used as a model. Dye-labeled, PCR-amplified alleles were analyzed on an automated DNA sequencer with laser-induced fluorescence detection and fragment-sizing software. Between-gel allele sizes calculated against an in-lane allelic ladder or viral DNA size standard were too imprecise to distinguish a 1-bp difference. However, the size difference between a sample allele and its matching ladder allele provided a reliable criterion for size classification. The mean size difference +/- 3 SDs was 0.5 bp, and so an individual result within this interval signified a match. Statistically, 99.7% of the results should lie within +/- 3 SDs with virtually no chance of encountering the 9-SD difference from the mean necessary to misclassify an allele by 1 bp. The method was valid for sample alleles sized against the allelic ladder and for both sample and ladder alleles sized against the viral DNA standard. A correction for the effect of different dye labels on mobility was included in the calculations.
提出了一种按大小对复杂短串联重复序列（STR）等位基因进行分类的策略。此类等位基因的长度差异可能仅为1个碱基对。以HUMACTBP2基因座作为模型。对经染料标记、PCR扩增的等位基因，使用带有激光诱导荧光检测和片段大小分析软件的自动化DNA测序仪进行分析。相对于泳道内等位基因阶梯或病毒DNA大小标准计算得出的凝胶间等位基因大小不够精确，无法区分1个碱基对的差异。然而，样本等位基因与其匹配的阶梯等位基因之间的大小差异为大小分类提供了可靠的标准。平均大小差异±3个标准差为0.5个碱基对，因此在此区间内的单个结果表示匹配。从统计学角度来看，99.7%的结果应落在±3个标准差范围内，几乎不可能出现与平均值相差9个标准差的情况，而这种差异是将等位基因错误分类1个碱基对所必需的。该方法对于根据等位基因阶梯确定大小的样本等位基因，以及根据病毒DNA标准确定大小的样本和阶梯等位基因均有效。计算中纳入了对不同染料标记对迁移率影响的校正。
