Journal
GENOME RESEARCH
Volume 14, Issue 11, Pages 2357-2366Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1101/gr.2813404
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Funding
- NCI NIH HHS [P50 CA 93638, P50 CA093683] Funding Source: Medline
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Despite recent advances in linear whole genome amplification of intact DNA/RNA, amplification of degraded nucleic acids in all 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 (R-2 = 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.
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