Methylated DNA Amplification
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What is Whole Genome Amplification?
Whole Genome Amplification (WGA) is used to amplify an entire genome from a small amount of genomic DNA, so any gene in the organism can be studied while preserving the limited sample. The GenoMatrix™ Whole Genome Amplification Kit provides up to a 500-fold amplification starting from as little as 10 ng of any type of genomic DNA, while maintaining sequence representation of the starting material. GenoMatrix Kits provide all reagents needed to quickly and easily perform genome-representative amplifications. The kit is for high-efficiency whole genome amplification and has been validated for use with samples generated by ChIP-IT® Express and the MethylCollector™ Kits, which allows for a seamless transition into microarray analysis or other applications requiring a large quantity of DNA.
Epigenetic applications of Whole Genome Amplification
Epigenetic techniques such as ChIP and DNA methylation capture assays yield DNA in quantities that can be analyzed directly by real-time qPCR, where specific gene amplification is used to determine relative enrichment of portions of the genome relative to others. Other techniques involve next-gen sequencing of isolated fragments, or hybridization to microarrays. In these situations, whole genome amplification can be necessary if the quantity of recovered DNA is insufficient to support the application. The GenoMatrix Whole Genome Amplification Kit provides up to a 500-fold amplification starting from as little as 10 ng of any type of genomic DNA, while maintaining sequence representation of the starting material. Thus, whole genome amplification maintains the relative proportions of genes present in the original pool, and allows the user to infer the relative enrichment of specific genes by individual epigenetic techniques.
Use of Whole Genome Amplification with ChIP
Chromatin immunoprecipitation, or ChIP, involves a variant of classical immunoprecipitation experiments carried out using antibodies specific for histones, individual marks on the histone code, or chromatin-associated proteins. However, instead of immunoprecipitation of the protein alone, ChIP uses chromatin extracted from cells in which it has been cross-linked with formaldehyde and sheared into small fragments. The immunoprecipitation thus enriches for DNA sequences in proximity to the target of the antibody that is used. Researchers can glean which proteins or marks are present on which loci. Sometimes, the quantity of chromatin available to begin with is small, and whole genome amplification can be used to amplify this material for further study. The researcher has to use caution in these experiments, as small experimental variations have a greater impact with a small sample size. Alternatively, whole genome amplification is useful to study rare marks or proteins associated with chromatin. When a target is rare, then even with sufficient starting material, the enrichment of that target by ChIP will result in a small amount of recovered DNA associated with chromatin. Whole genome amplification can be very useful to amplify this recovered DNA for subsequent analysis.
Use of Whole Genome Amplification to study DNA methylation
DNA methylation is typically studied in one of two ways: the first involves bisulfite conversion. Active Motif's MethylDetector™ Bisulfite Modification Kit simplifies analysis of DNA methylation. It comes complete with optimized reagents for performing DNA conversion with bisulfite, plus time-saving DNA purification columns and positive control PCR primers to validate your results. In the other situation, methylated fragments are collected by affinity to either an antibody to methyl-cytosine, or the higher-affinity method of using the methyl-DNA binding domain of MBD2. The DNA recovered from these experiments may be analyzed by hybridization to microarrays, and if the starting material is insufficient, whole genome amplification can be used to amplify the recovered DNA uniformly and without sequence bias.