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GenomiPhi amplification of FFPE tissue DNA for array CGH

ing a high molecular weight (~20 kb) product. We have also verified the specificity of FFPE DNA amplification from GenomiPhi amplifications by performing conventional PCR on select human genes that yield products ranging from 150 to 1100 bp (data not shown).

Amplified DNA yield from FFPE tissue
FFPE-extracted DNA amplification using GenomiPhi DNA Amplification Kit consistently gave higher yields of product (~37 g per reaction), whereas amplification yield from the DOP-PCR method was lower and more variable (Table 1 and data not shown). The higher yields from amplified product show that this method is tolerant to degraded DNA samples such as those extracted from FFPE slides. The amplification consistency is caused by the strand-displacement activity of Phi29 DNA polymerase on starting template, which continues until all nucleotides in the reaction are exhausted.

Cy3 and Cy5 probe preparation
The sizes and yield of Cy3- and Cy5-labeled probes (analyzed on 2% agarose gel) were identical when both GenomiPhi and DOP-PCR amplified templates were used for labeling (Fig 3). The high molecular weight DNA made by GenomiPhi DNA Amplification Kit was found to have no impact on Cy3 and Cy5 dye incorporation; sizes and yields of probes were similar with or without Dpn II restriction enzyme treatment of amplified DNA (Fig 3). For array CGH analysis of FFPE-amplified DNA, two Cy3 and Cy5 labeling reactions were combined (10 µg total) for hybridization to give higher signal levels, which help in downstream data processing.

Hybridization of Cy3- and Cy5-labeled probes to BAC CGH arrays
The Cy3 and Cy5 labeled probes from both amplification techniques were hybridized separately on SpectralChip 2600, which is a high-density array CGH chip consti
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TAG: GenomiPhi amplification FFPE tissue DNA for array CGH

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