A procedure for highly specific, sensitive, and unbiased whole-genome amplification
A procedure for highly specific, sensitive, and unbiased whole-genome amplification
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DOI：
10.1073/pnas.0808028105
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发表时间：
2008-10-07
影响因子：
11.1
通讯作者：
Weissman, Sherman M.
中科院分区：
文献类型：
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作者：
Pan, Xinghua;Urban, Alexander Eckehart;Weissman, Sherman M.
没有偏见或模板无关产物(TIPS)的复杂DNA池的高度特异性扩增仍然是一个挑战。我们开发了一种使用phi29 DNA聚合酶和海藻糖的方法，并优化了扩增控制，以创建从DNA的微克到亚毫微克的特定扩增片段。只要输入0.5-2.5 ng的人gDNA或几个细胞，产物就可以在基因座表示上接近天然DNA。从5和0.5 ng的DNA扩增，忠实地证明了所有已知的杂合性片段复制和缺失(3Mb到18kb)位于22号染色体上，甚至在高分辨率染色体范围比较基因组杂交中发现了小于1kb的纯合性缺失。对于550k的Infinium珠芯片SNP分型，与未扩增DNA的结果相比，>；99.7%的准确率更好。重要的是，通过本程序大大挽救了GenomiPhone v(2)中出现的染色体末端表达不足的情况，并且对于具有0.5 ng、部分降解的DNA输入的扩增片段，SNP分型的呼叫率和准确性也得到了提高。此外，根据饱和前的总产量，扩增以对数方式进行；完整细胞的扩增效率是等量提取DNA的50倍；DNA输入0.1 ng或更低的扩增片段的基因座不平衡是可变的，而输入更高的DNA扩增产物基本上是可重现的。这一程序便于用单细胞或其他痕迹的DNA进行基因组分析，并产生适合通过大规模平行测序和微阵列杂交进行分析的产物。
Highly specific amplification of complex DNA pools without bias or template-independent products (TIPS) remains a challenge. We have developed a method using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to subfemtograms of DNA. With an input of as little as 0.5-2.5 ng of human gDNA or a few cells, the product could be close to native DNA in locus representation. The amplicons from 5 and 0.5 ng of DNA faithfully demonstrated all previously known heterozygous segmental duplications and deletions (3 Mb to 18 kb) located on chromosome 22 and even a homozygous deletion smaller than 1 kb with high-resolution chromosome-wide comparative genomic hybridization. With 550k Infinium BeadChip SNP typing, the >99.7% accuracy was compared favorably with results on unamplified DNA. Importantly, underrepresentation of chromosome termini that occurred with GenomiPhi v(2) was greatly rescued with the present procedure, and the call rate and accuracy of SNP typing were also improved for the amplicons with a 0.5-ng, partially degraded DNA input. In addition, the amplification proceeded logarithmically in terms of total yield before saturation; the intact cells was amplified >50 times more efficiently than an equivalent amount of extracted DNA; and the locus imbalance for amplicons with 0.1 ng or lower input of DNA was variable, whereas for higher input it was largely reproducible. This procedure facilitates genomic analysis with single cells or other traces of DNA, and generates products suitable for analysis by massively parallel sequencing as well as microarray hybridization.