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Published online 21 May 2004

Nucleic Acids Research, 2004, Vol. 32, No. 9 e76
© 2004 Oxford University Press

Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples

Gang Wang, Cameron Brennan1, Martha Rook2, Jia Liu Wolfe2, Christopher Leo3, Lynda Chin1, Hongjie Pan, Wei-Hua Liu, Brendan Price and G. Mike Makrigiorgos*

Department of Radiation Oncology, 1 Department of Medical Oncology and 3 Arthur and Rochelle Belfer Cancer Genomics Center, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA and 2 Variagenics Inc. Cambridge, MA, USA

*To whom correspondence should be addressed at Dana Farber—Brigham and Women’s Cancer Center, Brigham and Women’s Hospital, Level L2, Radiation Therapy, 75 Francis Street, Boston, MA 02115, USA. Tel: +1 617 525 7122l; Fax: +1 617 587 6037; Email: mmakrigiorgos{at}partners.org
Present address:
Jia Liu Wolfe, Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA

Received February 4, 2004; Revised April 13, 2004;; Accepted April 21, 2004

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.


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