Bergen Andrew W, Qi Ying, Haque Kashif A, Welch Robert A, Chanock Stephen J
Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
BMC Biotechnol. 2005 Sep 16;5:24. doi: 10.1186/1472-6750-5-24.
Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1-200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR Identifiler panel) and N = 49 SNP (TaqMan) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs.
The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates.
The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR Identifiler panel from wgaDNA equivalent to that of gDNA.
全基因组扩增（WGA）有望消除与基因组DNA（gDNA）数量相关的实际分子遗传学分析限制。我们使用从淋巴母细胞系中提取的gDNA（N = 27）评估了多重置换扩增（MDA）WGA的性能，WGA反应中起始gDNA输入量范围为1 - 200 ng。使用三种DNA定量方法（OD、PicoGreen和RT-PCR）对全基因组扩增DNA（wgaDNA）进行产量和组成分析。对每个gDNA和wgaDNA样本进行两组重复检测，一组为N = 15个短串联重复序列（STR，使用AmpFlSTR Identifiler试剂盒），另一组为N = 49个单核苷酸多态性（SNP，TaqMan）基因分型检测。在SNP检测中使用1、4和20 ng的gDNA和wgaDNA样本量来评估DNA样本量对SNP基因分型检测性能的影响。总共N = 6,880次STR和N = 56,448次SNP基因分型尝试提供了足够的能力来检测gDNA和wgaDNA之间以及不同gDNA模板输入量产生的wgaDNA之间STR和SNP基因分型性能的差异。
双链wgaDNA和人特异性PCR可扩增wgaDNA的比例随着WGA反应中gDNA输入量的增加而增加。WGA反应中gDNA输入量的增加改善了wgaDNA的基因分型性能。尽管这种降低并不总是具有统计学意义，但观察到所有gDNA输入水平产生的wgaDNA的基因型完成率或基因型一致性率与gDNA相比有所降低。wgaDNA基因分型性能降低主要是由于等位基因扩增方差增加，导致杂合性丧失或未确定基因型增加。MDA WGA从无模板对照样本中产生wgaDNA；此类样本表现出相当高的假阳性基因分型率。
MDA WGA反应中gDNA的输入量是wgaDNA基因分型性能的关键决定因素。要获得与gDNA相当的wgaDNA TaqMan SNP检测基因分型性能，MDA WGA反应中至少需要输入10 ng淋巴母细胞gDNA。要使用AmpFlSTR Identifiler试剂盒从wgaDNA中获得与gDNA相当的最佳STR基因分型性能，MDA WGA反应中需要输入超过100 ng淋巴母细胞gDNA。
