PCR-based WGA can lead to error-prone amplification that results in, for example, single base-pair mutations, STR contractions, and expansions, and also leads to biased and underrepresented loci. In contrast, REPLI-g technology, which uses MDA technology and Phi 29 polymerase, delivers highly uniform amplification across the entire genome with minimal locus bias during amplification — a critical factor for reliable downstream analyses.
Uniform amplification across the entire genome
The long DNA fragment lengths generated using the highly processive Phi 29 polymerase (see figure
Multiple Displacement Amplification (MDA) technology) ensure that REPLI-g amplified DNA covers the whole genome, enabling consistent and unbiased locus representation and minimized mutation rates during amplification (see figures
Complete genome coverage and
Consistent and accurate whole genome amplification).
Due to unequal amplification of different loci caused by unresolved secondary structures, PCR-based WGA methods such as DOP-PCR or PEP exhibit frequent locus dropout. REPLI-g technology shows highly representative DNA amplification and minimal risk of locus dropout (see figure
Highly representative amplification). DNA amplified using REPLI-g provides comparable sequence coverage with unamplified gDNA, making it highly suited for demanding technologies, including next-generation sequencing (see figure
Comparable NGS results).
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Unbiased locus representation
Phi 29 polymerase is a DNA polymerase with 3'→5' prime exonuclease activity (proofreading activity) that delivers up to 1000-fold higher fidelity compared to
Taq DNA polymerase. Supported by the optimized REPLI-g buffer system, Phi 29 polymerase easily solves secondary structures such as hairpin loops, thereby preventing slipping, stoppage, and dissociation of the polymerase during amplification. This enables the generation of DNA fragments up to 100 kb without sequence bias (see figure
Unbiased amplification), even from just single cells. Four WGA kits, 2 utilizing MDA technology, including the REPLI-g Single Cell Kit, and 2 PCR-based methods, were tested for sequence representation and locus dropout using the single-cell amplification protocols specific for each kit. Unlike with the REPLI-g Single Cell Kit, single cells analyzed using kits from other suppliers often failed in complete and unbiased sequence representation (see figure
Unbiased amplification from a single cell).