Amplification of Genome-Representative DNA from Limited Sources with GenomePlex® WGA Technology for Use in Genetic Alterations Studies

By: Victoria Rusakova, Lukasz Nosek, LSI Volume 7 Article 1

Victoria Rusakova and Lukasz Nosek
Sigma-Aldrich Biotechnology, St. Louis, Missouri, USA

Introduction

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.

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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’ termini – the OmniPlex® library. This library is replicated using linear amplification in the initial stages, followed by a limited round of geometric amplifications.

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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.

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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.

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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 genome-amplified 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 (Figure 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 (Figure 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 genome-amplified DNA sample in the region tested for chromosome (Table 2). This represents a strong indication of trisomy 18.

Figure 1. The karyotype shown indicates Trisomy 21. Microarray hybridization was performed simultaneously using patient DNA and a sample from a healthy individual as reference DNA. The effect of normalization is shown by comparing the data marked “normalized” with the “raw” data (not shown). The normalized data shows a number of clones from chromosome 21 that were displaced to the left (blue) and to the right (red) in the dye reversal, both indicating a gain of chromosomal material in the female patient DNA versus the female reference DNA. In the “combined” column, the sign of one of the two reversed hybridizations is changed and the data are averaged with gains to the right and losses to the left. The circled regions show the increase in the total genomic data and indicate the presence of an extra chromosome 21.


Figure 2. The karyotype shown indicates Trisomy 18 in an Edwards syndrome female DNA sample. The hybridizations and normalization of data were performed as indicated in Figure 1. The circled regions show the increase in the total genomic data and indicate the presence of an extra chromosome 18.


Table 1. Quantified data for trisomy 21a

aResults for signal values after hybridization for critical diagnostic clones above the designated threshold. The threshold value for determination of loci overrepresentation in these experiments was set to 0.2 (cutoff log2 ratios > 0.2). The targets whose signals did not meet those criteria were omitted and not presented in the tables.


Table 2. Quantified data for trisomy 18a

aSee Table 1 footnote.
Additional information is available online (http://www.sigma.com/wga).


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.

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Acknowledgements

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.

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