DNA amplification method tolerant to sample degradation |
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Authors: | Wang Gang Maher Elizabeth Brennan Cameron Chin Lynda Leo Christopher Kaur Manjit Zhu Penny Rook Martha Wolfe Jia Liu Makrigiorgos G Mike |
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Institution: | Department of Radiation Oncology, and Arthur and Rochelle Belfer Cancer Genomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. |
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Abstract: | 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 (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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