Prasad D.K. Dhulipala1, Rao S. Takkellapati1, Kate Lavrenov1, Scott Hamilton1, and Jessica Pole2
1 GE Healthcare, Piscataway, NJ, USA
2 Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK
We present a comparative analysis of the two most commonly used methods for amplification of genomic DNA for use with genomic arrays. Ligation-mediated PCR is one of several current strategies being used for the generation of genomic arrays. In this process, a genomic DNA clone is digested with a restriction enzyme and a universal primer adaptor is ligated to serve as a priming site for PCR amplification. A different method employs degenerate oligonucleotide-primed PCR (DOP-PCR) to randomly amplify bacterial artificial chromosome DNA (BAC DNA). This is accomplished by the use of degenerate primers that selectively amplify human DNA but not contaminating E. coli DNA.
In this protocol, we used GenomiPhi DNA Amplification Kit to representatively amplify BAC DNA and then applied the product to the fabrication of slides for array comparative genomic hybridization (CGH). We present microarray data comparing the performance of the GenomiPhi method against DOP-PCR and demonstrate that the two methods provide data of similar quality. We also show that the GenomiPhi method is less expensive and easier to use than DOP-PCR.
BAC target DNA preparation
The NucleoSpin™ 96 Flash method was used to prepare BAC DNA clones spanning the p12 region of human chromosome 8 and Drosophila genomic regions (as negative controls).
The GenomiPhi DNA Amplification Kit contains four vials with sample buffer, reaction buffer, enzyme mix, and lambda control DNA. In addition, a re action buffer containing 1.2 mM 5-aminoallyl-dCTP (aa-dCTP) was prepared. This buffer was used in GenomiPhi reactions to allow spotting on CodeLink™ Activated Slides.
All reactions were performed using the following protocol: For each amplification reaction, 9 µl of sample buffer was added to 10 ng of BAC DNA (1 µl). The mixture was heated at 95 ºC for 3 min, and then cooled on ice to 4 ºC. A further 9 µl of the standard reaction buffer (for Corning UltraGAPS™ slides) or reaction buffer with aadCTP (for CodeLink Activated slides) and 1 µl of enzyme mix were added. (Alternatively, 1 µl of 25 mM aa-dCTP was added to the standard GenomiPhi reaction to print on CodeLink slides).
The mixture was briefly centrifuged and then incubated at 30 ºC for 16-18 h. The reaction was stopped by heating at 65 ºC for 10 min to denature the Phi29 enzyme. At this point, the reactions can be kept on ice or stored at -20 ºC until needed. Note that reaction volumes can be increased or decreased depending on the amount of DNA required for the intended application. All components in the amplification reaction, including the input DNA, must be scaled up or down proportionally. A minimum reaction volume of 5 µl is required for successful GenomiPhi amplification.
BAC DNA was also amplified by DOP-PCR following Sanger’s protocol for comparative analysis.
Quantitation of amplified products
The yield of amplified product cannot be directly measured by spectrophotometry because the completed reaction contains unused hexamers, deoxynucleotides in addition to amplification product. We used the PicoGreen™ dsDNA method for accurate quantitation of the unpurified amplification product.
Microarray fabrication and processing
The following method was used for BAC DNA amplified via both the Ge nomiPhi (including modified and unmodified nucleotides) and DOP-PCR methods: The DNA concentration was adjusted to 100 ng/µl with water. An equal volume of 2×printing buffer (200 mM sodium phosphate, pH 8.5) was added to the DNA sample, mixed thoroughly and spotted onto CodeLink Activated and UltraGAPS slides using the Piezorray™ spotter to generate eight replicate spots at a relative humidity of 42%. UV cross-linking (3000 µJoules) was used to immobilize the DNA onto the UltraGAPS slides. Coupling of DNA to CodeLink slides was achieved by incubating the slides in a saturated NaCl chamber for 48 h. Refer to the CodeLink user guide (GE Healthcare) for further processing of the CodeLink slides.
The GenomiPhi DNA Amplification Kit can be used successfully to generate BAC arrays. Compared to DOPPCR, the GenomiPhi method provides a simple, cheap and easy to use protocol. Analysis of microarray data showed that both methods produced similar DNA yields, sensitivity and performance. We also showed that BAC DNA generated by the GenomiPhi method detected all 8p12 chromosomal amplifications that were also detected by the DOP-PCR method in an MDA-MB-134 breast cancer line.
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For more information, visit www.amershambiosciences.com/genomiphi
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