DNA amplification method tolerant to sample degradation.
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.
Wang
Gang
G
Department of Radiation Oncology, and Arthur and Rochelle Belfer Cancer Genomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Maher
Elizabeth
E
Brennan
Cameron
C
Chin
Lynda
L
Leo
Christopher
C
Kaur
Manjit
M
Zhu
Penny
P
Rook
Martha
M
Wolfe
Jia Liu
JL
Makrigiorgos
G Mike
GM
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
D017931:DNA Primers; D004270:DNA, Circular; D004273:DNA, Neoplasm