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).
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).