REPLI-g Midi Kit

High yields from a variety of samples, suitable for numerous applications

With the REPLI-g Midi Kit, various clinical and non-clinical research samples can be used, including genomic DNA, fresh or dried blood, fresh or frozen tissue, and cells. Typical DNA yields per 50 µl reaction consistently reach 40 µg (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, butparticularly essential for high-throughput applications, which require subsequent genetic analyses to be possible without additional measurement or adjustment of DNA concentration.

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").

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 the 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”).

High fidelity whole genome amplification

REPLI-g technology provides highly uniform DNA amplification across the entire genome. Phi29 polymerase can replicate up to70 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 polymeraseduring replication. Exonuclease-resistant primers provided in the kit ensure high yields of DNA product, and theWGA 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").

Unique REPLI-g technology uses the innovative, high-fidelity enzyme Phi 29 polymerase to amplify complex genomic DNA using MultipleDisplacement Amplification (MDA) combined with a gentle alkaline denaturation step to amplify genomic loci uniformly.The typical yield of the REPLI-g Midi Kit is up to 40 µg, and can be easily scaled down according to your needs with the REPLI-g Mini Kit, since both kits are based on the same protocol and use the same reaction volumes. The easy reaction set-up and very low handling time of approximately 15 minutes makes REPLI-g an easy and reliable method to use when complete and unbiased locus representation is needed from limited or precious samples.

Amplification principle

REPLI-g uses isothermal genome amplification, termed Multiple Displacement Amplification (MDA), which involves the binding of random hexamers to denatured DNA followed by strand displacement synthesis at a constant temperature with the enzyme Phi29 polymerase. Additional priming events occur on each displaced strand that serve as a template, enabling generation of high yields of amplified DNA (see figure “Schematic representation of REPLI-g amplification”). Phi29 polymerase, a phage derived enzyme, 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 unique, optimized REPLI-g buffer system, Phi29 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 with Phi 29 polymerase").

Alkaline denaturation of DNA

Genomic DNA must be denatured before use in enzymatic amplification procedures, which is often accomplished using harsh methods such as incubation at elevated temperatures (heat incubation) or increased pH (chemical alkaline incubation). The REPLI-g Midi Kit uses gentle alkaline incubation, allowing uniform DNA denaturation with very low DNA fragmentation or generation of abasic sites. This results in amplified DNA with very high integrity, and maximizes the length of amplified fragments so that genomic loci and sequences are uniformly represented. With the REPLI-g Midi Kit, reliable results without false positive or negative data are ensured in subsequent downstream applications, unlike with other WGA technologies that use heat-induced denaturation that can damage template DNA, leading to biased and underrepresented loci (see figure "Effect of heat and alkaline denaturation on loci representation").

The REPLI-g Midi Kit uses a simple and reliable method to achieve accurate genome amplification from small quantities of isolated target genomic DNA, or directly from whole blood, dried blood cards, buffy coat, and tissue culture cells (see figure "REPLI-g Mini and Midi procedure). The addition of lysis buffer, which both lysis the sample material and denatures the DNA, is followed by a short incubation (see figure "REPLI-g Mini and Midi procedure"). After neutralization, master mix (including REPLI-g MidiDNA Polymerase)is added and the isothermal amplification reaction proceeds overnight at30°C.REPLI-g amplified DNA can be stored long-term at –20°C with no negative effects (see figure "Consistent long-term stability").

REPLI-g amplified genomic can be used in a variety of downstream applications, including:

• SNP genotyping with TaqMan® primer/probe sets
• qPCR- and PCR-based mutation detection
• Next-generation sequencing
• STR/microsatellite analysis
• Sanger sequencing
• RFLP and Southern blot analysis
• Array technologies, such as comparative genomic hybridization