Whole genome amplification for array comparative genomic hybridization using DNA extracted from formalin-fixed,...

Area of Science:

• Genomics
• Molecular Biology
• Oncology

Background:

• Array comparative genomic hybridization (CGH) detects genome-wide chromosomal imbalances.
• Limited DNA from small tumor areas on paraffin sections restricts array CGH.
• Whole genome amplification (WGA) can provide sufficient DNA but may introduce bias.

Purpose of the Study:

• To optimize a whole genome amplification (WGA) protocol for array CGH.
• To minimize amplification bias when using DNA from formalin-fixed, paraffin-embedded tumor sections.
• To enable reliable detection of genetic alterations using WGA-amplified DNA.

Main Methods:

• Optimized WGA protocol using formalin-fixed, paraffin-embedded tumor sections with known genetic alterations (TP53 mutations, LOH 1p, 10q, 19q, EGFR amplification).
• Incorporated a ligation step before WGA and a short reaction time (<1.5 hours) with Phi29 DNA polymerase.
• Performed array CGH (Agilent 105K) on DNA before and after WGA; analyzed results using Pearson correlation.

Main Results:

• Optimized WGA protocol generated >4 µg of WGA-DNA from >150 ng template with minimal amplification bias (< threefold).
• Detected known genetic alterations after WGA, confirming protocol efficacy.
• Significant positive correlation (P < 0.0001) observed between array CGH results before and after WGA.

Conclusions:

• Optimized WGA protocol enables reliable array CGH analysis of DNA from paraffin-embedded tumor sections.
• A ligation step and short Phi29 reaction time are crucial for minimizing WGA bias.
• Carefully optimized WGA protocols are essential for accurate genetic analyses from challenging samples.