OPTIMIZING DOP-PCR FOR UNIVERSAL AMPLIFICATION OF SMALL DNA SAMPLES IN COMPARATIVE GENOMIC HYBRIDIZATION

The standard comparative genomic hybridization (CGH) protocol relies o n availability of macroscopic tumor samples, which do not contain too much interfering normal cells. Recently, CGH after universal amplifica tion of genomic DNA with degenerate oligonucleotide primed PCR (DOP-PC R) has been used to detect genetic aberrations in microdissected tumor specimens, However, owing to the technical difficulties, CGH results of only few microdissected samples have so far been published. We have developed an improved protocol for DOP-PCR, which includes direct inc orporation of fluorochrome-conjugated nucleotides into the PCR product . Among the four polymerase enzymes tested, ThermoSequenase gave the b est yield, with PCR products ranging from 100-4,000 bp. A two-step PCR -procedure was used, consisting of a preamplification with low stringe ncy conditions followed by amplification in more stringent conditions. The method was first validated by hybridizing DOP-PCR-amplified norma l DNA against nick-translated reference DNA, which showed uniform amd even hybridization result for all chromosomes. Comparison of DOP-PCR C GH to conventional CGH in MCF-7 breast cancer cell line further indica ted that genetic aberrations can be reliable detected after DOP-PCR am plification. The sensitivity of the DOP-PCR-CCH was tested by serial d ilution of MCF-7 DNA. Fifty picograms of sample DNA (corresponding rou ghly to two MCF-7 cells) was sufficient for high quality CGH. Experime nts with cells microdissected from intraductal breast cancer demonstra ted that carcinoma cells from 1 to 2 ducts were sufficient for a succe ssful DOP-PCR CGH analysis. We conclude that the improved DOP-PCR-CGH protocol provides a powerful cool to study genetic aberrations in diff erent histological subpopulations of malignant as well as precancerous lesions. DOP-PCR also improves the success rate of conventional paraf fin-block CGH, because a poor quality or a too low yield of extracted DNA can be compensated by universal DNA amplification by DOP-PCR. (C) 1997 Wiley-Liss, Inc.