title: DNA amplification method tolerant to sample degradation.
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.
authors.affiliation: Department of Radiation Oncology, and Arthur and Rochelle Belfer Cancer Genomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Maher
authors.forename: Cameron
authors.initials: C
authors.lastname: Brennan
authors.forename: Lynda
authors.initials: L
authors.lastname: Chin
authors.forename: Christopher
authors.initials: C
authors.lastname: Leo
authors.forename: Manjit
authors.initials: M
authors.lastname: Kaur
authors.forename: Penny
authors.initials: P
authors.lastname: Zhu
authors.forename: Martha
authors.initials: M
authors.lastname: Rook
authors.forename: Jia Liu
authors.initials: JL
authors.lastname: Wolfe
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D045744:Cell Line, Tumor; D019411:Clinical Laboratory Techniques; D017931:DNA Primers; D004270:DNA, Circular / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis*; D020869:Gene Expression Profiling; D016678:Genome; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats; D015336:Molecular Probe Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D013048:Specimen Handling
chemical_list: D017931:DNA Primers; D004270:DNA, Circular; D004273:DNA, Neoplasm