GenomiPhi-amplified salmon sperm. Credit: GE HEALTHCARE

Rather than amplifying just the DNA between two primers, many researchers want to amplify entire genomes, to make archive copies of the total DNA from a unique biopsy sample, for example. “If you have a piece of paper with valuable information on it, obviously you don't want to lose the paper, but instead photocopy it many times, store some copies appropriately and use others,” says Andy Betera vice-president of product management of GE Healthcare, based in Little Chalfont, UK. Whole-genome amplification (WGA) systems aim to act as photocopiers for DNA. “From the point of view of the molecular epidemiologist or molecular geneticist, the overwhelming potential advantage of the newer methods of WGA is the possibility of producing additional genomic DNA, or amplified WGA DNA, for genotyping, sequencing or other types of genetic analysis like loss of heterozygosity (LOH),” says Andrew Bergen, staff scientist at the National Cancer Institute in Bethesda, Maryland.

“For the past 12 years there have been PCR-based methods of WGA with about a 10 or 15% locus bias, meaning that 10 or 15% of the genetic loci in the genome would not be amplified,” explains Bergen. Newer methods can avoid this loss in the right conditions. But problems really arise when you have only a very small sample from a slide or biopsy to play with. Bergen says colleagues who have almost exhausted the material from a particular sample ask him: “Can this sample be rescued?” And no WGA method is really effective with less than 10 ng of genomic DNA, he says.

On the commercial front, GE Healthcare's GenomiPhi DNA amplification kit uses the highly processive single-strand displacing DNA polymerase from phage phi29. Short primers are bound randomly throughout the genome and the enzyme copies the DNA, starting from each primer and displacing the primer ahead of it. The result is a soup of genomic fragments of varying lengths, averaging around 10,000 bases. The phi29 method is able to efficiently amplify a whole genome with no loss of sequence if high molecular weight DNA is used as the starting material.

The GenomePlex WGA kit from Sigma-Aldrich uses technology licensed from Rubicon Genomics of Ann Arbor, Michigan. In this method, the genomic DNA is initially broken up into fragments of around 400 bases long, which are then attached to an identical sequence. The collection is amplified by PCR using a primer that recognizes this sequence. Because short fragments are being copied, this method can cope with starting DNA of high or low molecular weight.

P.M.