Genotyping encompasses a range of applications used to analyze genetic differences between individuals or cells. Researchers often face the problem of limited or insufficient DNA quantity for downstream analysis. Using REPLI-g for whole genome amplification solves this problem by providing high yields of unbiased DNA from small or precious samples.
REPLI-g amplified DNA enables highly reliable genotyping results
PCR-based methods (e.g., DOP-PCR and PEP) for whole genome amplification can produce nonspecific amplification artifacts and give incomplete coverage of loci. In several cases, DNA less than 1 kb long may be generated that cannot be used in many downstream applications. In general, the resulting DNA is generated with a much higher mutation rate due to the use of the low-fidelity enzyme Taq
DNA polymerase, which can lead to error-prone amplification that results in, for example, single base-pair mutations, STR contractions, and expansions.
In contrast to these disadvantages, REPLI-g provides highly uniform amplification across the entire genome, with minimal locus bias and minimized mutation rates during amplification (see figures Highly representative amplification
and Consistent and accurate whole genome amplification
). The average product length of REPLI-g amplified DNA is typically more than 10 kb, with a range between 2 kb and 100 kb, enabling downstream applications such as complex restriction enzyme analysis and long-range PCR to be carried out. REPLI-g amplified DNA is highly suited for genotyping applications, such as SNP genotyping with TaqMan primer/probe sets (see figure Reliable SNP genotyping
), sequencing, and STR/microsatellite analysis (see figure Accurate genotyping
DNA from formalin-fixed, paraffin-embedded samples that have been amplified using the REPLI-g FFPE Kit is also highly suited for use in real-time PCR, and for genotyping analyses including SNP genotyping and microsatellite analysis (see figure Reliable microsatellite analysis
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Copy number analysis
Gene copy number can vary within individual genomes. Studying gene copy number changes in specific cell types (e.g., tumor cells) has become increasingly important in cancer and cytogenetic research.
Copy number changes are classified as DNA “gains” or “losses” and show characteristic patterns that include mutations at chromosomal and subchromosomal levels. These chromosomal changes are frequently analyzed using comparative genome hybridization (CGH) techniques.
Breakpoint analysis enables the identification of chromosomal regions where “gains” or “losses” of chromosomal fragments have occurred. It also allows the identification of the genes that have been amplified or deleted. The high-quality DNA obtained using multiple displacement amplification (MDA) is more representative of the true genome than other whole genome amplification (WGA) techniques. Research by Hosono et al (2003) (1) and Paez et al (2004) (2) clearly shows that the copy number of different loci is very well preserved after amplification using MDA. In addition, any changes in copy number that are introduced through the process of amplification can be eliminated through normalization of the sample DNA against amplified control DNA.
1. Hosano, S. et al. (2003) Unbiased whole genome amplification directly from clinical samples. Genome Res. 13
2. Paez, J.G. et al. (2002) A full-coverage, high-resolution human chromosome 22 genomic microarray for clinical and research applications. Hum. Mol. Genet. 11