Enhanced fidelity of sub-nanogram amplification
The importance of nucleic acid amplification from trace amounts of biological sample has increased with the expansion of genomic sequencing in various clinical and research-oriented applications. This invention characterizes techniques to reduce background signal experienced while cloning sub-nanogram nucleic acid templates. The technology is able to enhance the fidelity of sub-nanogram amplification and sequencing by reducing endogenous and exogenous nucleotide contamination by using restricted-randomized primers, treated polymerase, template-dependent polymerase selection, and the sequencing of multiple independent clones in parallel.
Intellectual Property Status: Patent(s) Pending
A number of whole genome amplification strategies, such as multiple displacement amplification, have yielded important success in the cloning of sub-nanogram template material. However, these techniques are still susceptible to undesirable background signal in amplification products. The cloning technique achieves enhanced amplification of picogram and femtogram nucleic acid material. The method has shown clean signal-to-noise amplification with as little starting material as one single human cell (~6 picograms), one E. coli cell (~ 5 femtogram) or one Prochlorococcus cell (~ 3 femtogram).