A procedure for highly specific, sensitive, and unbiased whole-genome amplification.
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 v2 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.
Pan
Xinghua
X
Department of Genetics, Yale University School of Medicine, New Haven, CT 06520-8005, USA. xinghua.pan@gmail.com
Urban
Alexander Eckehart
AE
Palejev
Dean
D
Schulz
Vincent
V
Grubert
Fabian
F
Hu
Yiping
Y
Snyder
Michael
M
Weissman
Sherman M
SM
D004247:DNA / Q000032:analysis*; D016678:Genome; D023281:Genomics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
D004247:DNA