Unbiased whole-genome amplification directly from clinical samples
Unbiased whole-genome amplification directly from clinical samples
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DOI：
10.1101/gr.816903
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发表时间：
2003-05-01
期刊：
影响因子：
7
通讯作者：
Lasken, RS
中科院分区：
文献类型：
--
作者：
Hosono, S;Faruqi, AF;Lasken, RS
从临床样本中制备基因组DNA是基因分型和DNA测序分析的瓶颈，常常受到可用标本数量的限制。我们使用多重位移扩增（MDA）直接从少量全血、干血、颊细胞、培养细胞和黄皮毛标本中扩增10,000倍的全基因组，产生大量用于基因检测的DNA。基因组DNA均匀扩增，具有完整的覆盖范围和所有基因的一致代表。从44个个体中分析的所有47个位点在扩增的DNA中都代表了起始基因组DNA模板中拷贝数的0.5至3.0倍。高保真DNA聚合酶确保DNA序列的准确表示。在20个个体的3种不同临床样本类型的5次SNP和10次微卫星DNA分析中，扩增的DNA与原始基因组DNA模板无法区分。直接从细胞中扩增基因组DNA具有高度可重复性，消除了DNA模板纯化的需要，并允许从小型临床样本中进行基因检测。与老式的基于pcr的方法相比，MDA的低扩增偏倚代表了在扩增全基因组能力方面的巨大技术进步。
Preparation of genomic DNA from clinical samples is a bottleneck in genotyping and DNA sequencing analysis and is frequently limited by the amount of specimen available. We use Multiple Displacement Amplification (MDA) to amplify the whole genome 10,000-fold directly from small amounts of whole blood, dried blood, buccal cells, cultured cells, and buffy coats specimens, generating large amounts of DNA for genetic testing. Genomic DNA was evenly amplified with complete coverage and consistent representation of all genes. All 47 loci analyzed from 44 individuals were represented in the amplified DNA at between 0.5- and 3.0-fold of the copy number in the starting genomic DNA template. A high-fidelity DNA polymerase ensures accurate representation of the DNA sequence. The amplified DNA was indistinguishable from the original genomic DNA template in 5 SNP and 10 microsatellite DNA assays on three different clinical sample types for 20 individuals. Amplification of genomic DNA directly from cells is highly reproducible, eliminates the need for DNA template purification, and allows genetic testing from small clinical samples. The low amplification bias of MDA represents a dramatic technical improvement in the ability to amplify a whole genome compared with older, PCR-based methods.