Comprehensive human genome amplification using multiple displacement amplification.
Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multiple displacement amplification (MDA), which provides a highly uniform representation across the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4-6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA is an isothermal, strand-displacing amplification yielding about 20-30 microg product from as few as 1-10 copies of human genomic DNA. Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells. MDA-amplified human DNA is useful for several common methods of genetic analysis, including genotyping of single nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.
Dean
Frank B
FB
Molecular Staging, Inc., 300 George Street, Suite 701, New Haven, CT 06511, USA.
Hosono
Seiyu
S
Fang
Linhua
L
Wu
Xiaohong
X
Faruqi
A Fawad
AF
Bray-Ward
Patricia
P
Sun
Zhenyu
Z
Zong
Qiuling
Q
Du
Yuefen
Y
Du
Jing
J
Driscoll
Mark
M
Song
Wanmin
W
Kingsmore
Stephen F
SF
Egholm
Michael
M
Lasken
Roger S
RS
D015139:Blotting, Southern; D004247:DNA / Q000737:chemistry*; D005821:Genetic Techniques; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA
D004247:DNA