International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)


POSTER 197 - COMPREHENSIVE MAMMALIAN GENOME AMPLIFICATION USING MULTIPLE DISPLACEMENT AMPLIFICATION

Dr. Stephen Kingsmore
Molecular Staging Inc.
300 George Street, Suite 701
New Haven
CT 06443
USA

Co-Authors: Dean FB, Hosono S, Fang L, Wu X, Faruqi AF, Bray-Ward P, Sun Z, Zong Q, Du Y, Du J, Egholm M, & Lasken RS
Institutions: Molecular Staging

Fundamental to most genetic analysis is availability of genomic DNA of adequate quality & quantity.  Since DNA yield from mammalian samples is frequently limiting, much effort has been invested in methods for propagating & archiving genomic DNA.  Methods include the creation of EBV-transformed cell lines or whole genome amplification (WGA) by degenerate oligonucleotide-primed PCR.  However, WGA methods suffer from high cost or insufficient coverage & inadequate average DNA size.  We describe a novel method for WGA using an isothermal, strand-displacing amplification process termed multiple displacement amplification (MDA).  Like the rolling circle amplification method recently described for circular DNA propagation(Gen Res 11:1095, 2001), MDA uses phi29 DNA polymerase and random primers.  Within 4 hours, 30pg (~9 genomic copies) of genomic DNA was amplified to ~0.04mg.  The average fragment length was >10kb.  The amplified DNA exhibited normal representation for 10 single nucleotide polymorphisms (SNPs).  Representation bias among 8 chromosomal loci was <3-fold in contrast to 4-6 orders of magnitude for PCR-based WGA methods.  MDA-amplified DNA is useful for several common methods of genetic analysis, including SNP genotyping, chromosome painting, Southern blotting & RFLP analysis, subcloning, DNA sequencing.  MDA-based WGA is a simple & reliable method that could have significant implications for genetic studies & long-term sample storage.


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