For highly uniform whole genome amplification and quality assessment from small or precious samples
The REPLI-g service — based on proven REPLI-g whole genome amplification technology — allows the amplification of large amounts of DNA from limited samples with minimal sequence bias. A variety of samples can be used, including genomic DNA, whole blood, dried blood cards, buffy coat, buccal swabs, fresh or frozen tissue, and tissue culture cells. A stringent quality control assay provides information on the quality of the amplified DNA, enabling reliable predictions for the success of your downstream assays. REPLI-g Service is available in 100 µg or 500 µg scales.
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Whole genome amplification may be achieved by two different methods: PCR-based amplification or multiple displacement amplification (MDA). PCR-based methods can generate nonspecific amplification artifacts, give incomplete coverage of loci, and generate DNA less than 1 kb long that cannot be used in many downstream applications. In contrast, REPLI-g using MDA provides highly uniform amplification across the entire genome, with minimal sequence bias. See figures “Highly representative amplification” and “Consistent and accurate whole genome amplification”. The average product length is typically greater than 10 kb, with a range between 2 kb and 100 kb (see figure “Uniform yield of high molecular weight DNA”). The length of the REPLI-g amplified DNA enables complex restriction enzyme analysis and long-range PCR reactions.
REPLI-g technology provides highly uniform DNA amplification across the entire genome, with minimal sequence bias. The method is based on Multiple Displacement Amplification (MDA) technology (see figure "Schematic representation of REPLI-g amplification"), which carries out isothermal genome amplification utilizing a uniquely processive DNA polymerase capable of replicating 100 kb without dissociating from the genomic DNA template. The DNA polymerase has a 3'→5' exonuclease proofreading activity to maintain high fidelity during replication and is used in the presence of exonuclease-resistant primers to achieve high yields of DNA product. The amplified DNA can be directly used in a variety of downstream applications, including, genotyping (e.g., SNP, STR [see figure “Accurate genotyping”], microarray analysis), end-point PCR, quantitative real-time PCR, and sequencing.
Ideal results are obtained using 100 ng starting template. However, a uniform concentration of amplified DNA is usually achieved regardless of the quantity of template DNA. Sample material is lysed and the DNA is denatured by adding lysis buffer (heavily degraded genomic DNA may not serve as a good template for amplification). This gentle alkaline denaturation allows uniform amplification across the whole genome with minimal sequence bias. After neutralization, a master mix-containing buffer (including exonuclease resistant primers, DNA polymerase, and distilled water) is added. The isothermal amplification reaction proceeds overnight at 30°C. This simple and robust method is capable of accurate amplification of entire genomes.
Following DNA amplification, sequence bias is assessed using a proprietary assay of specific loci that are highly sensitive to template DNA quality. A stringent quality control assay provides information on the quality of the amplified DNA, enabling reliable predictions for the success of your downstream assay to be made (see figure “Detailed quality assessment report”).
The performance of REPLI-g amplified genomic DNA has been validated for a variety of downstream applications, including:
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