Uniform and unbiased amplification across the entire genome
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).
Multiple Displacement Amplification (MDA) technology.Primers (arrows) anneal to the template DNA and are extended at 30°C by Phi 29 polymerase, which moves along the DNA template strand, displacing the complementary strand while becoming a template itself for replication. In contrast to PCR amplification, MDA does not require different temperatures and ends in very long fragments with low mutation rates.
Complete genome coverage. Comprehensive analysis of 267 loci across the entire genome was performed using RT2 qPCR Primer Assays (QIAGEN) and real-time PCR following DNA amplification with the REPLI-g Single Cell Kit from 3 different single cell experiments. Low and consistent CT values, with no dropout from any marker, indicate that DNA was successfully amplified from all areas of the genome.
Consistent and accurate whole genome amplification. Real-time PCR was performed on 47 human loci (2 loci on each autosomal pair, 2 loci on the X chromosome[s], and 1 locus on the Y chromosome) from 44 different samples amplified using REPLI-g technology. Each sample was amplified approximately 10,000-fold with a maximum bias of representation between the loci being only 6-fold.
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).
Highly representative amplification. The relative representation of 8 loci was determined using real-time quantitative PCR for DNA amplified using [A] REPLI-g technology [B]OP-PCR and [C] PEP. Locus representation was determined by comparison to 1 µg of unamplified control DNA.
Comparable NGS results. Whole genome sequencing of the Bacillus subtilis genome was performed on the Illumina MiSeq instrument. For analysis, 2 μg of genomic DNA or DNA amplified from a single cell (three different single cell experiments) and 103 cells, using a single-cell WGA kit, was sheared into 300 bp fragments and 1 μg of each was used for library preparation. Comparable sequence coverage was observed for gDNA and single-cell DNA. Comparison of nonamplified and single-cell amplified DNA revealed error rates in a similar, very low, percentage range.
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.
Unbiased amplification with Phi 29 polymerase. [A] Upon encountering secondary DNA structures, Taq polymerase may pause synthesis, slip, or dissociate from the template. This can result in inaccurate DNA amplification, incomplete loci coverage, and short fragment sizes. [B] REPLI-g Kits utilize Phi 29 polymerase, which displaces secondary structures enabling accurate and highly uniform amplification of the entire genome.
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).
Unbiased amplification from a single cell. The REPLI-g Single Cell Kit or kits from Suppliers G, N, and S were used to individually amplify 6 human cells. Real-time PCR was used to analyze 3 markers to identify loss or variability in the amount of genomic loci. [A] Unlike kits from other suppliers, the REPLI-g Single Cell Kit delivered unbiased amplification of DNA in each of the 6 cells, indicated by equivalent CT values for each marker. [B] DNA amplified using the kits from Suppliers G and N also showed high dropout rates for some of the genomic markers, indicating incomplete genome coverage and biased amplification making the kits unsuitable for single cell research.