Lipsky R H, Mazzanti C M, Rudolph J G, Xu K, Vyas G, Bozak D, Radel M Q, Goldman D
Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20852, USA.
Clin Chem. 2001 Apr;47(4):635-44.
Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this principle.
We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion).
DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15-167 bp in length and differing by only a single nucleotide substitution.
The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.
几种检测单核苷酸多态性（SNP）的方法（如变性梯度凝胶电泳和变性高效液相色谱）间接基于异源双链DNA差异解链的原理。我们提出了一种直接基于该原理检测SNP的方法。
我们在一个高效系统（PE 7700序列检测仪）中使用双链DNA特异性荧光染料SYBR Green I（SYBR），该系统以96孔板形式控制和监测DNA解链。我们测量了DNA双链变性时伴随的荧光强度降低，使用四个天然多态性（三个SNP和一个单碱基缺失/插入）评估片段长度、染料浓度、DNA浓度和序列背景的影响。
DNA解链分析（DM）成功用于变异检测，我们还通过这种方法发现了两个以前未知的SNP。SYBR荧光很容易监测DNA扩增子的浓度，并且DNA扩增子浓度具有高度可重复性，变异系数为2.6%。我们很容易检测到长度为15 - 167 bp、仅相差一个单核苷酸替换的同源双链和异源双链片段之间解链温度的差异。
DMA的效率和灵敏度使其非常适合大规模检测序列变异。
