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GenomiPhi amplification of FFPE tissue DNA for array CGH

Cy3- and Cy5-labeled dCTP and Klenow fragment. Cy3- and Cy5 labeled probes were separated on a 2% agarose gel for size analysis and imaged using a Typhoon 8600 Variable Mode Imager. Hybridization was performed on SpectralChip 2600 BAC Arrays according to the manufacturer’s recommended conditions. The arrays were scanned on a GenePix 4000B scanner and signal intensities quantitated using GenePix Pro 3.1 software.


Results
DNA extraction from FFPE tissue
DNA was extracted from human liver FFPE tissue slides using a Chelex-100 resin extraction method. The extraction resulted in genomic DNA with an average yield of 800 ng from 20-µm specimen slides. The quality of DNA was verified using conventional PCR, demonstrating amplification of specific human gene fragments from 150 to 1100 base pairs in length (5).

Amplification of FFPE DNA
DNA extracted from FFPE tissue slides was amplified using GenomiPhi DNA Amplification Kit and DOP-PCR. Approximately 10–25 ng of FFPE-extracted DNA was used for each amplification.

When DOP-PCR amplification was carried out on FFPE- isolated DNA using three primers (DOP1, DOP2, and DOP3) as described in the Sanger protocol, only the DOP2 primer was successful in amplifying FFPE template DNA (Fig 1). The sizes of amplified DNA from DOP-PCR amplifications were small, with fragments ranging from 100 to 600 bases. The amplification was found to be dependent on the quality of FFPE DNA, resulting in lower yields than the GenomiPhi method (Table 1).


In contrast, GenomiPhi DNA Amplification Kit proved more consistent and reliable in amplifying FFPE-extracted DNA (Fig 2). The success of GenomiPhi amplifications was measured by the presence of a smear on the agarose gel indicat
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