Optimizing DOP‐PCR for universal amplification of small DNA samples in comparative genomic hybridization

Abstract

The standard comparative genomic hybridization (CGH) protocol relies on availability of macroscopic tumor samples, which do not contain too much interfering normal cells. Recently, CGH after universal amplification of genomic DNA with degenerate oligonucleotide primed PCR (DOP-PCR) 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 incorporation of fluorochrome-conjugated nucleotides into the PCR product. Among the four polymerase enzymes tested. ThermoSequenase gave the best yield, with PCR products ranging from 100-4,000 bp. A two-step PCR-procedure was used, consisting of a preamplification with low stringency conditions followed by amplification in more stringent conditions. The method was first validated by hybridizing DOP-PCR-amplified normal DNA against nick-translated reference DNA, which showed uniform amd even hybridization result for all chromosomes. Comparison of DOP-PCR CGH to conventional CGH in MCF-7 breast cancer cell line further indicated that genetic aberrations can be reliable detected after DOP-PCR amplification. The sensitivity of the DOP-PCR-CGH was tested by serial dilution of MCF-7 DNA. Fifty picograms of sample DNA (corresponding roughly to two MCF-7 cells) was sufficient for high quality CGH. Experiments with cells microdissected from intraductal breast cancer demonstrated that carcinoma cells from 1 to 2 ducts were sufficient for a successful DOP-PCR CGH analysis. We conclude that the improved DOP-PCR-CGH protocol provides a powerful tool to study genetic aberrations in different histological subpopulations of malignant as well as precancerous lesions. DOP-PCR also improves the success rate of conventional paraffin-block CGH, because a poor quality or a too low yield of extracted DNA can be compensated by universal DNA amplification by DOP-PCR.

Genetic heterogeneity in a prostatic carcinoma and associated prostatic intraepithelial neoplasia as demonstrated by combined use of laser-microdissection, degenerate oligonucleotide primed PCR and comparative genomic hybridization

“…Methodological improvements of approaches combining microdissection and CGH analysis [24] were prerequisites for the characterization of early chromosomal changes in cervical carcinoma [15] and intraductal breast cancer [23]. However, the identification of such early events in premalignant lesions of prostatic carcinomas requires further modification of the microdissection/CGH approach, since PIN is present as very small cell groups on 5-µm sections, which means far fewer cells available than from cervical dysplasia or carcinoma in situ of the breast.…”

“…Kuukasjärvi et al [24] diluted DNA of a cell line (MCF-7) down to 50 pg and obtained reliable CGH results after DOP-PCR amplification of the template DNA. The estimated sensitivity of this experiment using diluted DNA corresponds to approximately two MCF-7 cells.…”

“…Attempts have been made to combine microdissection and CGH analysis, and especially to allow more sensitive detection of chromosomal imbalances by separating tumour areas from stromal cells [23,38,42]. Kuukasjärvi et al [24] concluded from the results of their CGH studies of a dilution series of DNA from a breast cancer cell line that chromosomal imbalances can be reliably detected from as few as two cells. However, the feasibility of CGH analysis on small cell numbers obtained from routinely formalin-fixed specimens has yet to be validated.…”

Detection of SYT-SSX fusion transcripts in both epithelial and spindle cell areas of biphasic synovial sarcoma using laser capture microdissection

“…4 The modified DOP-PCR was performed in two separate phases. 12 First, four cycles (a preamplification step) were carried out in a 5 µl reaction mixture (using ThermoSequenase; Amersham, Cleveland, Ohio, USA) in low stringency conditions, followed by 30 cycles in a 25 µl reaction volume (using AmpliTaq polymerase, LD; Perkin Elmer, Norwalk, Connecticut, USA) under high stringency conditions. UN1 primer (5'-CCG ACT CGA GNN NNN NAT GTG G-3', with N = A, C, G, or T) was used in both reactions.…”

Comparative genomic hybridisation

“…Apart from these oncological applications, CGH analysis has also been used to study chromosomal aberrations in fetal and neonatal genomes. [22][23][24] Until now, over 300 articles on CGH have been published by research groups from several countries, including reviews [25][26][27] and technical papers [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] (fig 4). This boom is caused mainly by the reduced demands on the material used for CGH.…”