Gundry Cameron N, Dobrowolski Steven F, Martin Y Ranae, Robbins Thomas C, Nay Lyle M, Boyd Nathan, Coyne Thomas, Wall Mikeal D, Wittwer Carl T, Teng David H-F
Idaho Technology Inc., 390 Wakara Way and Department of Pathology, University of Utah School of Medicine, 50 North Medical Drive 5B426, Salt Lake City, Utah 84108, USA.
Nucleic Acids Res. 2008 Jun;36(10):3401-8. doi: 10.1093/nar/gkn204. Epub 2008 Apr 29.
Genotyping by high-resolution melting analysis of small amplicons is homogeneous and simple. However, this approach can be limited by physical and chemical components of the system that contribute to intersample melting variation. It is challenging for this method to distinguish homozygous G::C from C::G or A::T from T::A base-pair neutral variants, which comprise approximately 16% of all human single nucleotide polymorphisms (SNPs). We used internal oligonucleotide calibrators and custom analysis software to improve small amplicon (42-86 bp) genotyping on the LightScanner. Three G/C (PAH c.1155C>G, CHK2 c.1-3850G>C and candidate gene BX647987 c.261+22,290C>G) and three T/A (CPS1 c.3405-29A>T, OTC c.299-8T>A and MSH2 c.1511-9A>T) human single nucleotide variants were analyzed. Calibration improved homozygote genotyping accuracy from 91.7 to 99.7% across 1105 amplicons from 141 samples for five of the six targets. The average T(m) standard deviations of these targets decreased from 0.067 degrees C before calibration to 0.022 degrees C after calibration. We were unable to generate a small amplicon that could discriminate the BX647987 c.261+22,290C>G (rs1869458) SNP, despite reducing standard deviations from 0.086 degrees C to 0.032 degrees C. Two of the sites contained symmetric nearest neighbors adjacent to the SNPs. Unexpectedly, we were able to distinguish these homozygotes by T(m) even though current nearest neighbor models predict that the two homozygous alleles would be identical.
通过对小扩增子进行高分辨率熔解分析进行基因分型既简单又具有均一性。然而，这种方法可能会受到系统中物理和化学成分的限制，这些成分会导致样本间熔解变化。对于这种方法来说，区分纯合的G::C与C::G或A::T与T::A碱基对中性变体具有挑战性，这些变体约占所有人类单核苷酸多态性（SNP）的16%。我们使用内部寡核苷酸校准物和定制分析软件来改进LightScanner上的小扩增子（42 - 86 bp）基因分型。分析了三个G/C（PAH基因c.1155C>G、CHK2基因c.1 - 3850G>C和候选基因BX647987 c.261 + 22,290C>G）和三个T/A（CPS1基因c.3405 - 29A>T、OTC基因c.299 - 8T>A和MSH2基因c.1511 - 9A>T）人类单核苷酸变体。校准将六个目标中五个目标的141个样本的1105个扩增子的纯合子基因分型准确率从91.7%提高到了99.7%。这些目标的平均熔解温度（Tm）标准差在校准前为0.067℃，校准后降至0.022℃。尽管标准差从0.086℃降至0.032℃，但我们仍无法生成一个能够区分BX647987 c.261 + 22,290C>G（rs1869458）SNP的小扩增子。其中两个位点在SNP相邻位置含有对称的最近邻碱基。出乎意料的是，尽管当前的最近邻模型预测这两个纯合等位基因会相同，但我们仍能够通过熔解温度（Tm）来区分这些纯合子。
