RT Journal
A1 Wang, Gang
A1 Maher, Elizabeth
A1 Brennan, Cameron
A1 Chin, Lynda
A1 Leo, Christopher
A1 Kaur, Manjit
A1 Zhu, Penny
A1 Rook, Martha
A1 Wolfe, Jia Liu
A1 Makrigiorgos, G. Mike
T1 DNA amplification method tolerant to sample degradation
JF Genome Research 
JO Genome Research 
YR 2004 
FD November 01 
VO 14 
IS 11 
SP 2357 
OP 2366 
DO 10.1101/gr.2813404 
UL http://genome.cshlp.org/content/14/11/2357.abstract 
AB Despite recent advances in linear whole genome amplification of intact DNA/RNA, amplification of degraded nucleic acids in an unbiased fashion remains a serious challenge for genetic diagnosis. We describe a new whole genome amplification procedure, RCA–RCA (Restriction and Circularization-Aided Rolling Circle Amplification), which retains the allelic differences among degraded amplified genomes while achieving almost complete genome coverage. RCA–RCA utilizes restriction digestion and whole genome circularization to generate genomic sequences amenable to rolling circle amplification. When intact genomic DNA is used, RCA–RCA retains gene-amplification differences (twofold or higher) between complex genomes on a genome-wide scale providing highly improved concordance with unamplified material as compared with other amplification methodologies including multiple displacement amplification. Using RCA–RCA, formalin-fixed samples of modest or substantial DNA degradation were successfully amplified and screened via array-CGH or Taqman PCR that displayed retention of the principal gene amplification features of the original material. Microsatellite analysis revealed that RCA–RCA amplified genomic DNA is representative of the original material at the nucleotide level. Amplification of cDNA is successfully performed via RCA–RCA and results to unbiased gene expression analysis (R2 = 0.99). The simplicity and universal applicability of RCA–RCA make it a powerful new tool for genome analysis with unique advantages over previous amplification technologies.