MARCO ISLAND, Fl., Feb 7 - As Amersham Pharmacia Biotech researcher John Nelson described TempliPhi, a new DNA amplification polymerase he is currently developing, a wave of excitement and disbelief rolled over the audience of genomics researchers at the Advances in Genome Biology and Technology conference held this week in Marco Island, Fl.
" I've never had any DNA polymerase that works like this," Nelson said. " It stayed on the molecule for 700,000 bases."
During his presentation, Nelson showed a picture of a test tube so filled with amplified DNA that its contents were oozing out slowly in a goopy, gluey form. He claimed that this new polymerase, formerly known as Phi 29 DNA polymerase, will take every nucleotide in a given sample and turn it into DNA.
" Typically, the products of such reactions on quite modest levels of input DNA (1ng of M13) will become viscous and difficult to pipette after only a few hours of incubation," Nelson wrote in a poster presentation of his results.
TempliPhi works through a technique known as rolling circle amplification. Its structure is a rolling circle that attracts DNA strands. The strands attach to the outside of the circle, with their ends hanging off like arms. These ends in turn attract new strands, which attach to them, causing a large pinwheel-like structure to form, in which more strands of
DNA are attached to the larger and larger branches.
Nelson said the method eliminated the need for growing cells and purifying a template for sequencing, turning an 18-step process into a two-step one. From amplification, " you can go directly into the sequencing reaction," Nelson said.
Furthermore, the polymerase prefers to amplify bacterial plasmid DNA, which researchers use for small bits of DNA they are trying to sequence. This offers the advantage in that any chromosomal DNA from contamination will not be amplified at the same rate, Nelson said.
Molecular Staging also uses Rolling Circle Amplification Technology in its diagnostic nucleic acid bioarrays. Motorola owns a stake in the New Haven, Ct.-based company.
After Nelson’s presentation, a number of conference participants wondered if the product could replace PCR technology. AP Biotech representatives said they hoped so, but others were more skeptical, noting that there is no hard data comparing the efficacy of TempliPhi with that of PCR.
" It would be hard for anything to do the same job as PCR," said Wendy Price, a Qiagen marketing representative.
Currently, TempliPhi is being beta-tested at the Sanger Center, the Washington University Genome Sequencing Center, and the Department of Energy's Joint Genome Institute.
AP Biotech hopes to roll out TempliPhi kits in the second quarter, but has not yet released pricing information, other than to say it will be priced competitively with other template preparation kits.