Whole genome amplification for array comparative genomic hybridization using DNA extracted from formalin-fixed, paraffin-embedded histological sections.
Array comparative genomic hybridization (CGH) is useful to assess genome-wide chromosomal imbalance, but the requirement for relatively large amounts of DNA can be a limitation, in particular for samples extracted from small tumor areas on paraffin sections. Whole genome amplification (WGA) can be performed before array CGH to obtain sufficient DNA, but the possibility of artifacts attributable to biased amplification cannot be excluded. We optimized the WGA protocol to generate sufficient DNA with minimum amplification bias. Using formalin-fixed, paraffin-embedded histological sections of tumors carrying known TP53 mutations, LOH 1p, LOH 10q, LOH 19q, and EGFR amplification, we first optimized the protocol so that these genetic alterations were detected after WGA. We found that a ligation step before WGA is important because it allows a short reaction time with Phi29 to generate WGA-DNA with greatly decreased amplification bias. Using template >150 ng of DNA, a ligation step before WGA, and a short reaction time with Phi29 DNA polymerase (<1.5 hours), we obtained WGA-DNA (>4 mug) with minimum amplification bias (less than threefold). Using this protocol, we performed array CGH (Agilent 105K) before and after WGA. Pearson correlation analysis indicated a significant positive correlation in array CGH results between DNA before and after WGA (P < 0.0001). These results suggest that genetic analyses are possible using WGA-DNA extracted from paraffin sections, but that they should be performed with a carefully optimized and controlled protocol.
Huang
Jian
J
International Agency for Research on Cancer, Lyon, France.
Pang
Jesse
J
Watanabe
Takuya
T
Ng
Ho-Keung
HK
Ohgaki
Hiroko
H
D055028:Comparative Genomic Hybridization / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005557:Formaldehyde / Q000737:chemistry; D018773:Genes, erbB-1; D015894:Genome, Human; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016612:Paraffin Embedding; D016707:Tissue Fixation
D005557:Formaldehyde; D004247:DNA