DNA amplification method tolerant to sample degradation

Gang Wang, Elizabeth Maher, Cameron Brennan, Lynda Chin, Christopher Leo, Manjit Kaur, Penny Zhu, Martha Rook, Jia Liu Wolfe, G. Mike Makrigiorgos

Research output: Contribution to journalArticle

68 Citations (Scopus)

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 (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.

Original languageEnglish (US)
Pages (from-to)2357-2366
Number of pages10
JournalGenome Research
Volume14
Issue number11
DOIs
StatePublished - Nov 2004

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Genome
DNA
Gene Amplification
Microsatellite Repeats
Nucleic Acids
Formaldehyde
Digestion
Nucleotides
Complementary DNA
RNA
Technology
Gene Expression
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Genetics

Cite this

Wang, G., Maher, E., Brennan, C., Chin, L., Leo, C., Kaur, M., ... Makrigiorgos, G. M. (2004). DNA amplification method tolerant to sample degradation. Genome Research, 14(11), 2357-2366. https://doi.org/10.1101/gr.2813404

DNA amplification method tolerant to sample degradation. / Wang, Gang; Maher, Elizabeth; Brennan, Cameron; Chin, Lynda; Leo, Christopher; Kaur, Manjit; Zhu, Penny; Rook, Martha; Wolfe, Jia Liu; Makrigiorgos, G. Mike.

In: Genome Research, Vol. 14, No. 11, 11.2004, p. 2357-2366.

Research output: Contribution to journalArticle

Wang, G, Maher, E, Brennan, C, Chin, L, Leo, C, Kaur, M, Zhu, P, Rook, M, Wolfe, JL & Makrigiorgos, GM 2004, 'DNA amplification method tolerant to sample degradation', Genome Research, vol. 14, no. 11, pp. 2357-2366. https://doi.org/10.1101/gr.2813404
Wang G, Maher E, Brennan C, Chin L, Leo C, Kaur M et al. DNA amplification method tolerant to sample degradation. Genome Research. 2004 Nov;14(11):2357-2366. https://doi.org/10.1101/gr.2813404
Wang, Gang ; Maher, Elizabeth ; Brennan, Cameron ; Chin, Lynda ; Leo, Christopher ; Kaur, Manjit ; Zhu, Penny ; Rook, Martha ; Wolfe, Jia Liu ; Makrigiorgos, G. Mike. / DNA amplification method tolerant to sample degradation. In: Genome Research. 2004 ; Vol. 14, No. 11. pp. 2357-2366.
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