REPLI-g Principle and Procedures

Learn how REPLI-g technology delivers reliable WGA
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Unique REPLI-g technology uses the innovative, high-fidelity enzyme Phi 29 polymerase to amplify complex genomic DNA using Multiple Displacement Amplification (MDA) combined with a gentle alkaline denaturation step to amplify genomic loci uniformly. The kits use a simple and reliable method to achieve accurate genome amplification small quantities of isolated target genomic DNA, or can amplify DNA directly from single cells, whole blood, dried blood cards, buffy coat, and tissue culture cells.
REPLI-g amplification principle
Gentle alkaline denaturation
Highly stable DNA
Easy-to-follow protocols
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).


Images
Effect of Heat and Alkaline Denaturation on Loci Representation.
Effect of heat and alkaline denaturation on loci representation.
Genomic DNA samples (10 ng) were denatured using heat (95°C) or the standard REPLI-g Kit alkaline lysis protocol. After amplification using REPLI-g DNA Polymerase the CT values of 2 loci were compared between samples. The low CT values of loci amplified using the REPLI-g Kit alkaline lysis protocol indicate better locus representation, meaning there has been no loss of sequence information at these loci.
Multiple Displacement Amplification (MDA) technology delivers long read lengths with isothermal amplification.
REPLI-g MDA technology delivers long read lengths with isothermal amplification.
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.
REPLI-g FFPE Principle.
Random DNA ligation.
The REPLI-g FFPE procedure involves a novel DNA processing reaction which prepares and ligates fragmented DNA. Whole genome amplification of this randomly ligated DNA is then performed using proven REPLI-g technology. REPLI-g products combine MDA with a uniquely processive DNA polymerase. Due to the high processivity and strand displacement activity, REPLI-g DNA Polymerase minimizes unequal sequence and locus representation. This provides much more reliable results when compared to PCR-based WGA methods.
REPLI-g FFPE Procedure.
REPLI-g FFPE procedure.
Formalin fixation and paraffin embedding cause DNA fragmentation. During the REPLI-g FFPE procedure, these DNA fragments are randomly ligated and then amplified using multiple displacement amplification. As indicated, DNA fragments (containing different loci [L1–L4]) are not assembled in the original order. However, the different loci are equally amplified and can be detected in downstream genotyping applications.
REPLI-g Mini and Midi procedure
REPLI-g Mini and Midi procedure.
Amplification of genomic DNA using the REPLI-g Midi Kit involves 3 basic steps. First, the sample (10 ng purified genomic DNA, 0.5 µl whole blood or tissue culture cells) undergoes gentle alkaline denaturation, avoiding fragmentation and damage of template DNA. Next the sample is neutralized, and finally incubated with REPLI-g master mix at 30°C.
REPLI-g Screening Procedure.
REPLI-g Mitochondrial DNA procedure.
Amplification of mitochondrial DNA using the REPLI-g Mitochondrial DNA Kit involves just two basic steps. First, the total DNA sample is denatured by incubation in REPLI-g mt Reaction Buffer for 5 minutes at 75°C then cooled to stop the denaturation. Next, REPLI-g DNA Polymerase is added and the isothermal amplification reaction proceeds for 8 hours at 33°C.
REPLI-g Single Cell WGA Kit procedure
REPLI-g Single Cell procedure.
Two easy-to-follow procedures enable WGA from 1–1000 cells or genomic DNA. Whole genome amplification using the REPLI-g Single Cell Kit involves 3 simple steps, regardless of whether 1–1000 cells (Protocol 1) or 10 ng genomic DNA (Protocol 2) is used as a starting material. First, the sample undergoes gentle alkaline denaturation, avoiding fragmentation and damage of template DNA. Next the sample is neutralized, and finally, incubated with the REPLI-g Single Cell master mix at 30°C. Regardless of your starting material, the REPLI-g Single Cell Kit delivers high yields of high-quality whole genome amplified DNA.
REPLI-g Screening Procedure
REPLI-g Screening procedure.
The simple and robust method is capable of accurate amplification of genomes from small quantities of isolated target genomic DNA, or directly from whole blood, cells, and other starting materials. The REPLI-g Screening Kit combines a number of features making it ideal for use in manual or automated high-throughput mutation screening, including high volume pipetting steps for increased accuracy, compatibility with 96-well microplates, and a streamlined protocol for fast and easy setup.
Purified Genomic DNA Procedure.
REPLI-g UltraFast Mini Procedure
Amplification of genomic DNA using the REPLI-g UltraFast Mini Kit involves 3 basic steps. First, the sample (10 ng purified genomic DNA, 0.5 µl whole blood, or 300 tissue culture cells) undergoes gentle alkaline denaturation, avoiding fragmentation and damage of template DNA. Next the sample is neutralized, and finally incubated with REPLI-g UltraFast DNA Polymerase at 30°C. The amplified DNA is ready to use after 60 minutes without further purification
Consistent long-term stability
Consistent long-term stability.
Real-time PCR of REPLI-g amplified DNA samples stored in 4 different formats at –20°C for the indicated time periods. Two loci, [A] locus A and [B] locus B, were assayed for each sample. gDNA: genomic DNA not amplified with REPLI-g. Storage formats: 50 µl REPLI-g reactions: 1) without further manipulation ("50 µl REPLI-g"); 2) aliquoted to 5 µl volumes ("5 µl REPLI-g"); 3) purified with QIAamp Mini Kit ("50 µl QIAamp purified REPLI-g"); and 4) diluted to a concentration of 50 ng/µl (50 µl diluted REPLI-g").
Unbiased amplification with Phi29 polymerase
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.
Uniform DNA Yield from Various Amounts of Template.
Uniform DNA yield from various amounts of template.
Various amounts of human genomic DNA were amplified in a standard REPLI-g Midi Kit reaction and aliquots taken at the indicated timepoints. The yield of amplified DNA from a 50 µl reaction was approximately 40 µg, regardless of the amount of starting material.
Uniform Yield of High-Molecular-Weight DNA.
Uniform yield of high-molecular-weight DNA.
Agarose gel (1%) electrophoresis of 40 DNA samples amplified using REPLI-g whole genome amplification and visualized by SYBR Green Staining. The average product length is typically greater than 10 kb.