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Amplification of genome-representative DNA from limited sources with GenomePlex WGA technology for use in genetic alterations studies

ed 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
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