Castle Philip E, Garcia-Closas Montserrat, Franklin Tracie, Chanock Stephen, Puri Vinita, Welch Robert, Rothman Nathaniel, Vaught Jim
Division of Cancer Epidemiology and Genetics,National Cancer Institute, Bethesda, MD 20892, USA.
Am J Hum Genet. 2003 Sep;73(3):646-51. doi: 10.1086/378077. Epub 2003 Aug 13.
Buccal cells were collected from 29 participants, by use of mouthwash rinses, and were split into equal aliquots, with one aliquot irradiated by electron-beam (E-beam) irradiation equivalent to the sterilizing dosage used by the U.S. Postal Service and the other left untreated. Aliquots were extracted and tested for DNA yields (e.g., TaqMan assay for quantifying human genomic DNA), genomic integrity, and amplification-based analysis of genetic variants (e.g., single-nucleotide polymorphisms [SNPs] and single tandem repeats [STRs]). Irradiated aliquots had lower median DNA yields (3.7 microg/aliquot) than untreated aliquots (7.6 microg/aliquot) (P<.0005) and were more likely to have smaller maximum DNA fragment size, on the basis of genomic integrity gels, than untreated aliquots (P<.0005). Irradiated aliquots showed poorer PCR amplification of a 989-bp beta-globin target (97% for weak amplification and 3% for no amplification) than untreated aliquots (7% for weak amplification and 0% for no amplification) (P<.0005), but 536-bp and 268-bp beta-globin targets were amplified from all aliquots. There was no detectable irradiation effect on SNP assays, but there was a significant trend for decreased detection of longer STRs (P=.01) in irradiated versus untreated aliquots. We conclude that E-beam irradiation reduced the yield and quality of buccal-cell specimens, and, although irradiated buccal-cell specimens may retain sufficient DNA integrity for some amplified analyses of many common genomic targets, assays that target longer DNA fragments (>989 bp) or require whole-genome amplification may be compromised.
从29名参与者中通过漱口水收集颊细胞,并将其分成等量的几份,其中一份用相当于美国邮政服务使用的灭菌剂量的电子束(E-beam)照射,另一份不进行处理。提取这些样品并检测DNA产量(例如,用于定量人类基因组DNA的TaqMan分析)、基因组完整性以及基于扩增的遗传变异分析(例如,单核苷酸多态性[SNPs]和单串联重复序列[STRs])。与未处理的样品(7.6μg/份)相比,照射后的样品DNA产量中位数较低(3.7μg/份)(P<0.0005),并且根据基因组完整性凝胶分析,照射后的样品比未处理的样品更有可能具有更小的最大DNA片段大小(P<0.0005)。与未处理的样品(弱扩增占7%,无扩增占0%)相比,照射后的样品对989bpβ-珠蛋白靶标的PCR扩增效果较差(弱扩增占97%,无扩增占3%)(P<0.0005),但所有样品都能扩增出536bp和268bp的β-珠蛋白靶标。在SNP分析中未检测到照射效应,但在照射后的样品与未处理的样品中,较长STR的检测有显著下降趋势(P=0.01)。我们得出结论,电子束照射降低了颊细胞标本的产量和质量,并且,尽管照射后的颊细胞标本可能保留足够的DNA完整性用于许多常见基因组靶标的一些扩增分析,但针对较长DNA片段(>989bp)或需要全基因组扩增的分析可能会受到影响。