Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues.
BACKGROUND
Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA) is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold.


RESULTS
We amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC) tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH)-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb). When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar.


CONCLUSIONS
Large fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.
Aviel-Ronen
Sarit
S
Department of Pathology, University Health Network, 200 Elisabeth Street, Toronto, ON M5G 2C4, Canada. saronen@uhnres.utoronto.ca <saronen@uhnres.utoronto.ca>
Qi Zhu
Chang
C
Coe
Bradley P
BP
Liu
Ni
N
Watson
Spencer K
SK
Lam
Wan L
WL
Tsao
Ming Sound
MS
D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005557:Formaldehyde; D018628:Gene Dosage; D015894:Genome, Human; D001411:Geobacillus stearothermophilus / Q000737:chemistry; D006801:Humans; D008168:Lung / Q000737:chemistry / Q000166:cytology; D008175:Lung Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D008297:Male; D009447:Neuroblastoma / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D016707:Tissue Fixation / Q000379:methods*
D004273:DNA, Neoplasm; D005557:Formaldehyde; D004259:DNA-Directed DNA Polymerase