Beckett Sara M, Laughton Stephen J, Pozza Luciano Dalla, McCowage Geoffrey B, Marshall Glenn, Cohn Richard J, Milne Elizabeth, Ashton Lesley J
Children's Cancer Institute Australia for Medical Research, Randwick, New South Wales, Australia.
Am J Epidemiol. 2008 May 15;167(10):1260-7. doi: 10.1093/aje/kwn012. Epub 2008 Mar 7.
Self-collection of buccal cells provides a noninvasive method for obtaining biologic samples for genetic analyses in pediatric studies. Nevertheless, low yields, microbial contamination, and degradation of buccal samples present challenges for epidemiologic studies incorporating genetic investigations. The aims of this study were to compare the quality and yield of DNA extracted from buccal specimens with BuccalAmp swabs (Epicenter BioTechnologies, Madison, Wisconsin) or FTA cards (Whatman, Inc., Clifton, New Jersey) and to investigate the use of whole-genome amplification (WGA) for increasing DNA yields for single nucleotide polymorphism analyses. Buccal specimens were collected from 55 children with acute lymphoblastic leukemia and 52 control children without acute lymphoblastic leukemia in New South Wales, Australia, in 2003-2004. Real-time polymerase chain reaction was used to evaluate polymorphisms in the genes encoding the cytochrome p450 enzyme CYP3A4 (CYP3A4 A392G, also known as CYP3A4*1B) and the steroid xenobiotic receptor (SXR C25385T). Results showed that DNA could be isolated from buccal specimens collected by use of both methods and that yields could be substantially improved with WGA without introducing genotyping error. However, DNA quality was poorer in samples collected by BuccalAmp swabs, and the presence of polymerase chain reaction inhibitors in these samples reduced the sensitivity of quantitative real-time PCR analysis. These findings show that different methods for collecting buccal samples impact on the downstream success of genetic investigations and influence DNA quality after WGA.
自行采集颊黏膜细胞为儿科研究中的基因分析提供了一种获取生物样本的非侵入性方法。然而，颊黏膜样本的低产量、微生物污染和降解给纳入基因研究的流行病学研究带来了挑战。本研究的目的是比较使用颊黏膜扩增拭子（Epicenter生物技术公司，威斯康星州麦迪逊）或FTA卡（Whatman公司，新泽西州克利夫顿）从颊黏膜标本中提取的DNA的质量和产量，并研究使用全基因组扩增（WGA）来提高单核苷酸多态性分析的DNA产量。2003年至2004年，在澳大利亚新南威尔士州，从55名急性淋巴细胞白血病患儿和52名无急性淋巴细胞白血病的对照儿童中采集颊黏膜标本。使用实时聚合酶链反应评估编码细胞色素P450酶CYP3A4（CYP3A4 A392G，也称为CYP3A4*1B）和类固醇外源性受体（SXR C25385T）的基因中的多态性。结果表明，两种方法采集的颊黏膜标本均可分离出DNA，且WGA可显著提高产量，且不会引入基因分型错误。然而，颊黏膜扩增拭子采集的样本中DNA质量较差，这些样本中聚合酶链反应抑制剂的存在降低了定量实时PCR分析的灵敏度。这些发现表明，不同的颊黏膜样本采集方法会影响基因研究的下游成功率，并影响WGA后的DNA质量。
