Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84132, USA.
Clin Chem. 2011 Sep;57(9):1303-10. doi: 10.1373/clinchem.2011.166306. Epub 2011 Jul 19.
Gilbert syndrome, a chronic nonhemolytic unconjugated hyperbilirubinemia, is associated with thymine-adenine (TA) insertions in the UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1) promoter. The UGT1A1 promoter genotype also correlates with toxicity induced by the chemotherapeutic drug irinotecan. Current closed-tube assays for genotyping the UGT1A1 (TA)(n) promoter polymorphism require multiple labeled probes and/or have difficulty classifying the (TA)(5) and (TA)(8) alleles.
An unlabeled 5' extension on one primer that creates a hairpin after asymmetric PCR was used to develop a snapback primer high-resolution melting assay for the (TA)(n) polymorphism. A new method that plots the local deviation from exponential decay to improve genotype clustering was used to remove background fluorescence and to analyze the data. The snapback assay was compared with small-amplicon melting and fragment length analyses in a blinded study of DNA samples from 100 African Americans.
Genotyping results obtained by small-amplicon melting and snapback primer melting were 83% and 99% concordant, respectively, with results obtained by fragment analysis. Reanalysis of the single discordant sample in the results of the snapback genotyping assay and the fragment analysis revealed an error in the fragment analysis. High-resolution melting was required for accurate snapback genotyping of the UGT1A1 (TA)(n) polymorphism. The 100% accuracy obtained with a capillary-based instrument fell to ≤81% with plate-based instruments.
In contrast to small-amplicon genotyping, snapback primer genotyping can distinguish all UGT1A1 promoter genotypes. Rapid-cycle PCR combined with snapback primer analysis with only 2 unlabeled PCR primers (one with a 5' extension) and a saturating DNA dye can genotype loci with several alleles in <30 min.
吉尔伯特综合征是一种慢性非溶血性非结合性高胆红素血症，与 UGT1A1（UDP 葡萄糖醛酸基转移酶 1 家族，多肽 A1）启动子中的胸腺嘧啶-腺嘌呤（TA）插入有关。UGT1A1 启动子基因型也与化学治疗药物伊立替康引起的毒性相关。目前用于检测 UGT1A1（TA）（n）启动子多态性的封闭管检测方法需要多个标记探针，或者难以对（TA）（5）和（TA）（8）等位基因进行分类。
使用不对称 PCR 后创建发夹结构的一个引物的未标记 5'延伸，用于开发用于（TA）（n）多态性的 snapback 引物高分辨率熔解分析。使用一种新的方法，通过绘制局部偏离指数衰减来提高基因型聚类的方法，以去除背景荧光并分析数据。在对 100 名非裔美国人的 DNA 样本进行的盲法研究中，将 snapback 检测与小片段扩增熔解和片段长度分析进行了比较。
小片段扩增熔解和 snapback 引物熔解的基因分型结果分别与片段分析结果的 83%和 99%一致。在 snapback 基因分型检测和片段分析的结果中，对单个不一致样本进行重新分析显示片段分析存在错误。高分辨率熔解对于准确的 UGT1A1（TA）（n）多态性 snapback 基因分型是必需的。基于毛细管的仪器可获得 100%的准确性，而基于平板的仪器则降至≤81%。
与小片段基因分型相比，snapback 引物基因分型可以区分所有 UGT1A1 启动子基因型。快速循环 PCR 结合带有 5'延伸的仅 2 个未标记 PCR 引物（一个带有 5'延伸）和一个饱和 DNA 染料的 snapback 引物分析可以在<30 分钟内对具有多个等位基因的基因座进行基因分型。
