High yields from a variety of samples, suitable for numerous applications
Various clinical and non-clinical research samples can be used with REPLI-g Mini and Midi Kits, including genomic DNA, fresh or dried blood, fresh or frozen tissue and cells. Typical DNA yields per 50 µl reaction consistently reach 10 µg (mini kit) or 40 µg (midi kit) (see figure " Consistent DNA yields using any sample type"), while a uniform yield of amplified DNA is usually achieved regardless of the quantity of template DNA (see figure " Uniform DNA yield from various amounts of template"). Obtaining uniform DNA yields from varying template concentrations is always important, but particularly essential for high-throughput applications, which require subsequent genetic analyses to be possible without additional measurement or adjustment of DNA concentration.
The average DNA yield from the REPLI-g UltraFast Mini Kit is 7–10 µg per 20 µl reaction in 90 minutes with standard quality samples. If time is very limited, sufficient product is available for downstream genetic analysis after just 45 minutes (see figure " Consistent and high yields of amplified DNA"). Negative control samples (without template DNA) are not amplified, providing a reliable indicator of the presence/absence of sample or contaminating DNA (see figure " Consistent and high yields of amplified DNA").
Typical DNA yields from a REPLI-g Screening Kit reaction are 8 µg per 40 µl reaction. The amplified DNA can be used directly in a variety of downstream applications, including genotyping (e.g., SNP, STR, deletions and insertions), end-point PCR, quantitative real-time PCR, microarray and sequencing. Unlike other whole genome amplification procedures that require the reaction setup to be performed on ice, the REPLI-g Screening Kit uses room temperature setup with just 20 minutes of handling time, making it especially suited for processing multiple samples in parallel. Obtaining uniform DNA yields from varying template concentrations is particularly important for high-throughput applications to allow subsequent genetic analysis without the need to measure or adjust DNA concentration. 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 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").
"In the past two years, we have been using the REPLI-g Ultra Fast Mini Kit extensively for multiple projects. The multiple displacement amplification performed with the REPLI-g Kit was able to faithfully amplify all DNA products at very low error rate and high coverage. The downstream applications we have used the kit for include most often NGS, but also qPCR and microarray. Moreover, we are working on implementing this technology in microfluidic or lab-on-a-chip systems, since the method requires no sophisticated thermal cycling program." Dr. Rong Fan, Assistant Professor of Biomedical Engineering, Yale University, US.
Successfully used in next-generation sequencing
Numerous publications have demonstrated the successful utilization of REPLI-g amplified DNA for next-generation sequencing (NGS) applications that range from exome and whole genome sequencing of tumor cells, to metagenomics research, to single cell analysis (for a range of recent publications that successfully used REPLI-g in NGS, please see our WGA resource page). Since the use of whole genome amplified DNA for NGS and array applications has been debated, we detected potential factors that could influence the success of using amplified DNA for these downstream applications. We determined that the quality of input material strongly influences the success of downstream NGS experiments. If working with low quality DNA (e.g., degraded DNA) or aged tissue material, the resulting amplified DNA may not give reliable results (data not shown). However, WGA, using REPLI-g technology, on intact cells or non-degraded purified DNA shows that NGS results are comparable to those obtained with purified gDNA. Sequence coverage and alignment comparison of the genomic loci sequence indicates minimized levels of junk DNA after WGA, whereas error rates are in a similar percentage range for both amplified and genomic DNA (see figure “ Comparable NGS (next-generation sequencing) results obtained using purified gDNA or REPLI-g amplified DNA”). The QIAseq FX Single Cell RNA and QIAseq FX Single Cell DNA Library Kits utilize the REPLI-g MDA reaction for high quality NGS libraries when starting with 10 pg – 1 ng of RNA or DNA.
High fidelity whole genome amplification
REPLI-g technology provides highly uniform DNA amplification across the entire genome. Phi29 polymerase can replicate up to 70 kb without dissociating from the genomic DNA template (see figure " Schematic representation of REPLI-g amplification"). In contrast to PCR-based whole genome amplification (WGA) technologies, Phi29 polymerase has 3'→5' exonuclease proofreading activity and maintains up to 1000-fold higher fidelity compared to Taq DNA polymerase during replication. Exonuclease-resistant primers provided in the kit ensure high yields of DNA product, and the WGA buffer system is optimized for very long read length and unbiased locus representation.
REPLI-g outperforms PCR-based WGA methods
Traditional methods of genomic DNA amplification include the time-consuming process of creating EBV-transformed cell lines followed by whole genome amplification using random or degenerate oligonucleotide-primed PCR. Also, PCR-based methods (e.g., DOP-PCR and PEP), as generally used by other suppliers, 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 using REPLI-g technology" and " Consistent and accurate whole genome amplification").