( Modern technologies d...)Amplification of genome-representative DNA from limited sources with GenomePlex WGA technology for use in genetic alterations studies

Modern technologies developed in recent years have prompted new and exciting approaches to support biomedical research. The ability to study the entire human genome using limited sources of genomic material to detect genetic disordersmade possible through Sigma-Aldrich Biotechnologys GenomePlex whole-genome amplification (WGA) systemis a notable achievement.

The development of high-throughput genomics and DNA sequencing has provided researchers the ability to obtain complete and exact DNA sequences of many genomes. Functional genomics researchers use sequencing as an analysis tool for genotyping and microarray-based technologies. These techniques, however, require sufficient amounts of DNA that in some cases may not be available. WGA technology provides the ability to generate adequate amounts of genome-derived DNA to study genetic alterations and their relationship to human diseases. It has become a useful tool for many biomedical applications, such as microsatellite analysis, single nucleotide polymorphism (SNP) detection, comparative genomic hybridization (CGH) microarrays and others.

GenomePlex WGA kit overview
The GenomePlex WGA technology provides a simple and accurate method for amplifying nanogram quantities of starting material from any source, resulting in microgram amounts of DNA with no detectable allele bias. The GenomePlex WGA technology is time-efficient, allowing the researchers to amplify DNA in less than three hours. The technology is based on random chemical fragmentation of the genome, producing a series of overlapping short templates ranging in the size from 200 to 1,500 base pairs and averaging 400 base pairs.

The resulting DNA fragments are efficiently primed to generate a library of DNA fragments with defined 3' and 5' terminithe OmniPlex library. This library is replicated using linear amplification in the initial stages, followed by a limited round of geometric amplifications.

Chromosomal imbalances: trisomy 21 and trisomy 18
We combined GenomePlex WGA technology with a CGH array to detect chromosomal abnormalities in patients with mental and developmental disabilities such as Down and Edwards syndromes. Down syndrome is caused by an extra copy of chromosome 21 (trisomy 21), and Edwards syndrome is caused by an extra copy of chromosome 18 (trisomy 18). It is extremely important to establish methods that allow identification of chromosomal abnormalities without any bias when only small amounts of DNA are available for early diagnosis.

Chromosomal microarray analysis (CMA)
Combining the GenomePlex WGA kit with a CGH microarray allowed accurate measurement of copy number changes and showed that this technique is simple to use and can be applied in both academic and clinical research. CMA uses CGH with bacterial artificial chromosome (BAC) or phage-derived artificial chromosome (PAC) clones (verified by fluorescence in situ hybridization) of known genomic location attached to a glass slide. Three or more different clones were used per region of interest. Genomic DNA was isolated from peripheral blood of Down or Edwards syndrome patients and healthy individuals using the PureGene DNA Purification kit (Gentra Systems). The samples were amplified with the GenomePlex WGA kit. Each sample was hybridized twice, using a dye reversal process. The combined results were analyzed using quantitative imaging methods and analytical software to determine a loss or gain of chromosomal copies.

Detection of chromosomal alterations with confidence
To perform the CGH assay, we prepared probe samples using 250 ng of control DNA and the same amount of patient DNA combined together. Two microarray slides were run for each trisomy case, one with unamplified genomic DNA and the other with whole genomeamplified DNA. We used only 10 ng of starting material for WGA. The threshold for the designation of over- or under-representation of genetic material in these experiments was determined to be 0.2 and 0.2 respectively.

The CMA data (Fig. 1) shows that CGH microarray analysis of the Down syndrome patient is consistent with a genomic gain, as detected by 14 out of 15 determined target clones in the array for both unamplified and amplified DNA. It reflects the presence of a third chromosome 21 (Table 1). Furthermore, the array for Edwards syndrome (Fig. 2) showed a genomic gain in 30 of 33 possible test clones for the unamplified genomic DNA sample and a gain of 27 target clones from 33 possible test clones in the whole genomeamplified DNA sample in the region tested for chromosome (Table 2). This represents a strong indication of trisomy 18.

The data presented here demonstrate that the GenomePlex WGA kit amplifies limited amounts of DNA without bias, producing material that is suitable for use with the CMA assay, allowing for the identification of genome-wide copy number and chromosomal abnormalities.

Additional information is available online (http://www.sigma.com/wga).

ACKNOWLEDGMENTS
We thank S.W. Cheung, X. Lu, S.D. Bland and A. Beaudet from the microarray facilities in the Department of Molecular and Human Genetics at Baylor College of Medicine (Houston, USA). We acknowledge E. Mueller, K. Kayser, A. Favello and R. Valdes-Camin from Sigma-Aldrich Co. for helpful discussions and support.

back to top
'"/>

Source: