The GenomePlex® Whole Genome Amplification method uses input DNA to generate a library of amplified DNA fragments that faithfully represents the original input genome. The three step fragmentation, isothermal amplification, and limited geometric amplification method yields products ranging in size from ~100 bp to ~2 kb with an average size of ~450 bp. The method is simple, taking approximately 30 minutes of hands-on time and a total of only 3.5 hours to complete. The initial incubation step entails a unique, chemical fragmentation of DNA. The next two steps of amplification generate denatured fragments that are subsequently primed by a proprietary, partially randomized primer set. Through a series of stepped isothermal incubations, these primers generate a library of 5’ and 3’ adaptor-appended fragments, termed the OmniPlex® library. The final step is PCR amplification using a primer set targeting the newly appended 5’ and 3’ sequences. The resulting product yield ranges from 5-10 micrograms, depending on the quality of the initial DNA input. Amplification requires only a small amount of starting material: 1 ìl blood, single cheek swab, or minimum 10 ng of genomic DNA, even if in damaged condition. Based on the research, no detectable locus or allele biases are introduced by the process of amplification.
The GenomePlex® Whole Genome Amplification Kits contains all the necessary reagents to perform fragmentation and library preparation. They come with or without the optimized enzyme WGA DNA Polymerase. In addition, GenomePlex® WGA Reamplification Kit is optimized for the subsequent re-amplification of the initial WGA product. Extensive research by Sigma-Aldrich shows that successive reamplifications provide DNA with little genetic bias when compared to the original genome
We have purchased GenomePlex® WGA Kit which has enough reagents to process 50 DNA samples. Our samples were genomic DNA extracted more than 10 years ago from whole blood as well as from EBV-transformed lymphoblasts of patients. They were stored most of the time at the -20 freezer, with some period of short-term storage at +4 degrees. Some of the samples had only residual quantities - nearly empty tubes. These samples were very important to our research studies. Initial concentrations varied from a high of 60 ng/ul to a low of undetectable by the spectrophotometer. In all the cases, when the concentration was determined, 100 ng of DNA was used for the whole genome amplification. For the samples where concentration was too low to be detected, we used the whole sample. All steps of the protocol were clearly outlined and explained in the details in the kit manual. After completion of all the required steps, samples were purified with GenElute® PCR Clean-Up Kit by Sigma-Aldrich, and DNA yield quantified by measuring absorbance at 260 nm. The result was a great success. DNA yields varied from 4 ug of amplified DNA for sample where no detectable DNA was identified to the 10 ug for samples where 100 ng were used. No DNA was amplified in the negative control sample. We also ran an aliquot of the amplified and eluted DNA samples on the 1% agarose gel. A bright smear was clearly visible in the 100-1000 bp range, indicating that the procedure worked as expected.
It is important to know that the first two steps of the protocol fragmentation and library generation are supposed to be carried out without interruption, as the ends of the library DNA are easily degradable. After the OmniPlex® library DNA is generated, it can be stored at -20 degrees. In addition, a reduced amount of starting material, such as genomic DNA amounts of less that 10 ng, can lead to altered representation after the amplification.
In summary, we were able to successfully amplify old and damaged genomic DNA samples, even when very little DNA was present. The yields were very consistent with our expectations.