Rolling Circle Amplification (Yale University)
|Paul Lizardi||Yale University||Messenger RNA Profiling by Single Molecule Counting||R33|
Obtaining mutational profiles from clinical tissue would help stage and categorize the progression of various human cancers. However, the need for diagnostic and molecular manipulations of miniscule or fixed clinical samples has been a major limiting factor. Consequently, much effort has been applied to identifying molecular alterations in human cancers using small amounts of samples. Technologies now exist for amplifying entire genomes faithfully and rapidly from single cells and assessing thousands of genes simultaneously, which will greatly facilitate efforts to use small amounts of existing human samples in a potentially high-throughput manner.
Dr. Paul Lizardi began his work on rolling circle amplification (RCA) prior to his IMAT award, R33CA081671, and that work was published inNature Geneticsin 1998.To date, that seminal publication has been cited 267 times. Among the specific aims of Dr. Lizardi’s R33 IMAT grant was the plan to use the RCA technology to identify single nucleotide polymorphisms (SNPs) involved in early cancer development. RCA is a technique that takes advantage of the capacity of certain polymerases to use circularized ssDNA as a template. RCA can be used to robustly amplify a signal for in situ detection of fixed samples or identify SNPs. While working on the RCA technology, Dr. Lizardi developed a method to rapidly amplify an entire genome (isothermal whole-genome amplification [iWGA]). His work on iWGA led to one patent application. iWGA kits are now available commercially from Amersham Biosciences and Molecular Staging Inc.
Dr. Lizardi combined iWGA with simultaneous ligation of thousands of genes using comparative genomic hybridization array analysis to detect low-abundance mutations in cancer development.This advancement has provided a potential solution for detecting the single mutant allele within a pool of “normal” tissue or cells and helps overcome smaller clinical sample availability. Furthermore, these technologies are amenable to robotic automation.
PubMed lists 134 articles that mention the use of rolling circle amplification in their abstracts, 9 of which are related to cancer.
1 Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC. Mutation detection and single-molecule counting using isothermal rolling-circle amplification.Nat GenetJul;19(3):225-32, 1998.
2 Lage JM, Leamon JH, Pejovic T, Hamann S, Lacey M, Dillon D, Segraves R, Vossbrinck B, Gonzalez A, Pinkel D, Albertson DG, Costa J, Lizardi PM. Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH.Genome ResFeb;13(2):294-307, 2003