For highly uniform whole genome amplification from small or precious samples
  • Easy amplification with consistent yields of up to 10 µg
  • Unbiased amplification of genomic loci
  • Reliable results due to Multiple Displacement Amplification (MDA)
  • Amplified DNA highly suitable for most downstream applications
  • No risk of DNA degradation during long-term storage

The REPLI-g Mini Kit provides optimized reagents for whole genome amplification (WGA) from small samples using innovative Multiple Displacement Amplification (MDA) technology. The typical DNA yield of a 50 μl reaction is  up to 10 μg, with an average product length greater than 10 kb (ranging between 2 kb and 100 kb). Unique REPLI-g technology delivers highly uniform WGA from a variety of small or precious sample types, including purified genomic DNA, or directly from fresh or dried blood, buccal swabs, fresh or frozen tissue, and cells. This simple and reliable method is capable of accurate and unbiased amplification of genomes and generates DNA that can be applied without further purification or quantification for downstream applications that do not require labeling. In contrast to PCR-based WGA technologies, high fidelity rates are increased up to 1000-fold, avoiding costly false positive or negative results. For more information about WGA technology, visit our WGA resource page, and for other dedicated REPLI-g products, refer to Table 1.

Quantity Product Cat. no. Price
 
 
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Quantity Product Cat. no. Price Sum
 
REPLI g Human Control Kit 25
Human control DNA for 25 x 50 µl whole genome amplification reactions
150090
$35.30
$0.00
 
REPLI-g Mini Kit (25)
DNA Polymerase, Buffers, and Reagents for 25 x 50 µl whole genome amplification reactions (typical yield 10 µg per reaction)
150023
$204.00
$0.00
 
REPLI-g Mini Kit (100)
DNA Polymerase, Buffers, and Reagents for 100 x 50 µl whole genome amplification reactions (typical yield 10 µg per reaction)
150025
$627.00
$0.00
The REPLI-g Mini Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Consistent DNA yields using any sample type.|Effect of heat and alkaline denaturation on loci representation.|Consistent long-term stability.|Comparable NGS (next-generation sequencing) results obtained using purified gDNA or REPLI-g amplified DNA.|REPLI-g Mini and Midi procedure.|Accurate genotyping.|Schematic representation of REPLI-g amplification.|Unbiased amplification with Phi29 polymerase.|Highly representative amplification.|Consistent and accurate whole genome amplification.|Uniform DNA yield from various amounts of template.|Reliable SNP genotyping.|
Various starting materials, including genomic DNA, and heparin- and EDTA-preserved whole blood, were amplified using REPLI-g Midi and Mini Kits. Typical yields of 40 µg (Midi Kit) and 8–10 µg (Mini Kit) were obtained.|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.|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").|Whole genome sequencing of the Bacillus subtilis genome was performed. For analysis, 2 μg of genomic DNA or DNA amplified from 105 cells using the REPLI-g Midi Kit was sheared into 300 bp fragments. For library preparation, 1 μg of each was used. Sequencing was performed on the Illumina MiSeq instrument. [A] Comparable sequence coverage was observed for both gDNA and REPLI-g amplified DNA. [B] Alignment comparison of the genomic loci sequence demonstrates comparably high percentage of alignment for REPLI-g amplified DNA in comparison to the gDNA, which is an indication of minimized levels of junk DNA after WGA (whole genome amplification). Comparison of nonamplified and REPLI-g amplified DNA revealed error rates (mismatch, high-quality error, indels, or chimeras) in a similar percentage range.
(Alignment comparison performed using SMALT [Welcome Trust Sanger Institute]).|Amplification of genomic DNA using the REPLI-g Mini 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.|Twenty DNA samples amplified using REPLI-g technology, without subsequent DNA purification, were subjected to genotyping analysis using 3 STR loci (CSF1PO, TPOX, and THOI). Results were compared with those obtained for unamplified genomic DNA. The DNA was separated by polyacrylamide gel electrophoresis, and visualized by silver staining. A lane with one band represents a homozygote, while a lane with two bands represents a heterozygote for the specific STR locus.|Ph 29 polymerase moves along the DNA template strand displacing the complementary strand. The displaced strand becomes a template for replication, enabling high yields of high-molecular-weight DNA to be generated.|[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 Phi29 polymerase, which displaces secondary structures enabling accurate and highly uniform amplification of the entire genome.|The relative representation of eight loci was determined using real-time quantitative PCR for DNA amplified using [A] REPLI-g technology, [B] DOP-PCR, and [C] PEP. Locus representation was determined by comparison to 1 µg of unamplified control DNA. (Dean, F.B. et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99, 5261. © 2002 National Academy of Sciences, USA.)|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.|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.|DNA amplified using REPLI-g technology, without subsequent purification, was subjected to SNP genotyping at 2 randomly selected loci (WIAF-1004 and WIAF-622) using a TaqMan analysis. Tight clusters of alleles allow reliable determination of genotyping of homo- and heterozygote genotypes.|
Performance
High yields from a variety of samples, suitable for numerous applications

With the REPLI-g Mini 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 10 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, 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 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 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”).

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").

Principle

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 typical yield of the REPLI-g Mini Kit is up to 10 µg, and can be easily scaled down according to your needs with the REPLI-g Midi 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”). 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").

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 Mini 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").

Procedure
Simple, one tube procedure

The REPLI-g Mini 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 minute incubation (see figure "REPLI-g Mini and Midi procedure"). After neutralization, master mix (including REPLI-g Mini DNA Polymerase) is added and the isothermal amplification reaction proceeds overnight at 30°C. REPLI-g amplified DNA can be stored long-term at –20°C with no negative effects (see figure "Consistent long-term stability").

Select the REPLI-g Kit most suited to your specific requirements from our complete range of dedicated REPLI-g products (see table).

Specifications for the wide range of REPLI-g Kits
REPLI-g Single Cell REPLI-g Mini REPLI-g UltraFast Mini REPLI-g Midi REPLI-g Screening REPLI-g FFPE REPLI-g Mitochondrial DNA
Starting material Single cells, gDNA Purifed gDNA, blood, cells Purifed gDNA, blood, cells FFPE tissue, purified gDNA from FFPE tissue Purified gDNA
(Protocols for other starting materials available from www qiagen.com)
Input amount Single cells, 2–1000 cells, tissue, purified gDNA (1–10 ng) >10 ng gDNA, 0.5 µl blood or cells (>600 cells/µl) >10 ng gDNA, 0.5 µl blood or cells (>600 cells/µl) Section (1 cm diamter, 10–40 µm thick); >100 ng gDNA >1 ng purified gDNA
Yield (µg/reaction) 40 10 7–10 40 8 Standard yield: ≤10; High yield: ≤40 3–5
Reaction time 8–16 h 10–16 h 1.5 h 8–16 h 12–16 h Standard yield: 4 h; High yield: 10 h 8 h
Hands-on time 15 min 15 min 15 min 15 min 15 min 40 min 15 min
Format Tube Tube Tube Tube Plate Tube Tube
Applications

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 
Feature
Specifications
Amplification Whole genomic DNA
Applications Genotyping, hybridization, RFLP
Denaturation step Alkaline
Maximum input volume >10 ng DNA, 0.1– 0.5 µl whole blood, >600 cells/µl
Minimal pipetting volume needed 0.5 µl
Quality assessment No
Reaction time 8–16 hours (overnight)
Reaction volume 50 µl
Samples per run; throughput Mid
Starting amount of DNA >10 ng purified genomic DNA
Starting material Genomic DNA, blood, cells, tissue
Technology Multiple Displacement Amplification (MDA)
Yield 10–40 µg

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Kit Handbooks
1
For whole genome amplification from purified genomic DNA, blood, and cells
Show details