REPLI-g Principle and Procedures

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 Phi 29 polymerase. Additional priming events occur on each displaced strand that serve as a template, enabling generation of high yields of amplified DNA (see figure REPLI-g MDA technology). Phi 29 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, 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 with Phi 29 polymerase). Typical DNA yields consistently reach 40 µg, regardless of the starting quantity of template (see figures Uniform DNA yield and Uniform yield of high-molecular-weight DNA), meaning subsequent genetic analyses can proceed without additional measurement of DNA concentration for most applications.

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Gentle alkaline denaturation
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). REPLI-g Kits use 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 REPLI-g, 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).

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Highly stable DNA
The high-molecular-weight DNA generated through a REPLI-g Kit reaction should be regarded as high-quality genomic DNA and should be stored accordingly. For example REPLI-g amplified DNA can be stored for short periods at 4°C or –20°C for longer term storage. As with any high quality genomic DNA, to preserve the DNA quality, it should not undergo many freeze thaw cycles.

The stability of REPLI-g amplified DNA after long-term storage at -20°C was also investigated. Four different storage formats were used:

• 50 µl REPLI-g reaction without any further manipulation (“50 µl REPLI-g”)
• 50 µl REPLI-g reaction aliquoted to 5 µl volumes (“5 µl REPLI-g”)
• 50 µl REPLI-g reaction purified with QIAamp Mini Kit (“50 µl QIAamp purified REPLI-g”)
• 50 µl REPLI-g reaction diluted to a concentration of 50 ng/µl (“50 µl diluted REPLI-g”)

Amplified DNA (10 ng; as determined using a PicoGreen assay [for more information, refer to the REPLI-g Mini and Midi Handbook]) from each stored sample was analyzed at two different loci using real-time PCR (see figure Consistent long-term stability). No difference in the locus representation was detectable for the various storage conditions tested.

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Easy-to-follow protocols
REPLI-g Kits use streamlined protocols that require minimal hands-on time, and can reliably amplify DNA from as little as a single cell (see figures REPLI-g Mini and Midi procedure and REPLI-g UltraFast procedure). Kits are available in 96-well format (see figure "REPLI-g Screening procedure") and for amplification of DNA from single cells (see figure REPLI-g Single Cell procedure) and mitochondria (see figure REPLI-g Mitochondrial procedure). The REPLI-g FFPE Kit provides a fast and easy protocol that starts directly from a section of a fixed tissue sample (see figure REPLI-g FFPE procedure). It offers an integrated processing reaction that results in isolation of DNA from FFPE samples and ligation of DNA fragments in a random order. The resulting DNA is then amplified using REPLI-g Polymerase as shown in the figure Random DNA ligation).