Navigation Links
GenomiPhi: protocol for BAC amplification and fabrication of slides for array CGH

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).


GenomiPhi amplification
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.


Conclusions
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.


References
1. Mamone, J. T. A method for representatively amplifying genomic DNA. Genomic/Proteomic Technology April/May 2003. [Online.] http://www.amershambiosciences.com/applic/upp00738.nsf/vLookup Doc/253872879-B500/$file/GPT_Mamone_Reprint.pdf

2. Kallioniemi, A. et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258, 818–821 (1992).

3. Solinas-Toldo, S. et al. Matrix-based comparative genomic hybridization: biochips to screen for genomic imbalances. Genes Chromosomes Cancer 20, 399–407 (1997).

4. Fiegler, H. et al. DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones. Genes Chromosomes Cancer 36, 361–374 (2003).

5. Snijders, A. M. et al. Assembly of microarrays for genome-wide measurement of DNA copy number. Nat. Genet. 29, 263–264 (2001).

6. Smirnov, D. A. et al. Method for manufacturing whole-genome microarrays by rolling circle amplification. Genes Chromosomes Cancer 40, 72–77 (2004).

7. Application note: Use of GenomiPhi DNA Amplification Kit for array CGH, GE Healthcare, 63-0056-26, Edition AA (2005).

For more information, visit www.amershambiosciences.com/genomiphi






back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. CELLocate protocol blocks for 175 m microgrid
2. CELLocate protocol blocks for 55 m microgrid
3. BioRobot MDx standardize your automated protocols in clinical laboratories
4. The TripleMaster PCR System for three different amplifications
5. Successful PCR amplification and subcloning of a GC-rich DNA fragment
6. Advantages of Roche Applied Science amplification products
7. GenomiPhi amplification of FFPE tissue DNA for array CGH
8. New Microarray Labeling Kit for Preparing cDNA Probes
9. Stratagenes Human cDNA Microarrays: Perform Gene Expression Profiling and Discover New Genes
10. An Optimal DNA Microarray Substrate for the Identification of Fetal and Somatic Liver Stem Cells of the Rat
11. Amplification of Mouse cDNAs for Microarrays Using the Eppendorf MasterTaq Kit
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/24/2016)... June 24, 2016 Epic Sciences unveiled ... cancers susceptible to PARP inhibitors by targeting homologous ... (CTCs). The new test has already been incorporated ... multiple cancer types. Over 230 clinical ... response pathways, including PARP, ATM, ATR, DNA-PK and ...
(Date:6/23/2016)... ... June 23, 2016 , ... UAS LifeSciences, one of the ... brand, UP4™ Probiotics, into Target stores nationwide. The company, which has been manufacturing ... to its list of well-respected retailers. This list includes such fine stores as ...
(Date:6/23/2016)... June 23, 2016 Houston Methodist Willowbrook ... Cy-Fair Sports Association to serve as their official ... Houston Methodist Willowbrook will provide sponsorship support, athletic ... with association coaches, volunteers, athletes and families. ... Cy-Fair Sports Association and to bring Houston Methodist ...
(Date:6/23/2016)... 23, 2016   EpiBiome , a precision microbiome ... in debt financing from Silicon Valley Bank (SVB). The ... to advance its drug development efforts, as well as ... "SVB has been an incredible strategic partner to ... traditional bank would provide," said Dr. Aeron Tynes ...
Breaking Biology Technology:
(Date:6/21/2016)... , June 21, 2016 NuData ... the new role of principal product architect and ... the director of customer development. Both will report ... technical officer. The moves reflect NuData,s strategic growth ... response to high customer demand and customer focus ...
(Date:6/9/2016)... Paris Police Prefecture and ... ensure the safety of people and operations in several locations ... Teleste, an international technology group specialised in broadband ... its video security solution will be utilised by ... across the country. The system roll-out is scheduled for the ...
(Date:6/2/2016)... 2, 2016 Perimeter Surveillance & ... Systems, Physical Infrastructure, Support & Other Service  ... offers comprehensive analysis of the global Border ... generate revenues of $17.98 billion in 2016. ... a leader in software and hardware technologies for advanced ...
Breaking Biology News(10 mins):