Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples.
Analysis of genomic DNA derived from cells and fresh or fixed tissues often requires whole genome amplification prior to microarray screening. Technical hurdles to this process are the introduction of amplification bias and/or the inhibitory effects of formalin fixation on DNA amplification. Here we demonstrate a balanced-PCR procedure that allows unbiased amplification of genomic DNA from fresh or modestly degraded paraffin-embedded DNA samples. Following digestion and ligation of a target and a control genome with distinct linkers, the two are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. We demonstrate genome-wide retention of allelic differences following balanced-PCR amplification of DNA from breast cancer and normal human cells and genomic profiling by array-CGH (cDNA arrays, 100 kb resolution) and by real-time PCR (single gene resolution). Comparison of balanced-PCR with multiple displacement amplification (MDA) demonstrates equivalent performance between the two when intact genomic DNA is used. When DNA from paraffin-embedded samples is used, balanced PCR overcomes problems associated with modest DNA degradation and produces unbiased amplification whereas MDA does not. Balanced-PCR allows amplification and recovery of modestly degraded genomic DNA for subsequent retrospective analysis of human tumors with known outcomes.
Wang
Gang
G
Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
Brennan
Cameron
C
Rook
Martha
M
Wolfe
Jia Liu
JL
Leo
Christopher
C
Chin
Lynda
L
Pan
Hongjie
H
Liu
Wei-Hua
WH
Price
Brendan
B
Makrigiorgos
G Mike
GM
D016477:Artifacts; D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D002872:Chromosome Deletion; D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005260:Female; D005557:Formaldehyde; D018628:Gene Dosage; D020869:Gene Expression Profiling / Q000379:methods; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D016707:Tissue Fixation
D017931:DNA Primers; D005557:Formaldehyde; D004247:DNA