REPLI-G Kits

For highly uniform whole genome amplification from 10ng of genomic DNA

Features

  • Amplified DNA can be used in NGS, microarray and PCR applications
  • Isothermal multiple displacement amplification (MDA) with proof-reading DNA polymerase
  • Whole genome amplification (WGA) yields 7 to 40 µg amplified DNA from 10 ng of DNA
  • Ultrafast format for whole genome amplification in 1–1.5 hours
  • High-throughput format for 96 samples with just 20 minutes of handling time
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REPLI-g Mini Kit (25)

Cat. No. / ID: 150023

DNA Polymerase, Buffers, and Reagents for 25 x 50 µl whole genome amplification reactions (typical yield 10 µg per reaction)
₩307,000.00
Kit
REPLI-g Kit
REPLI g Human Control Kit
Type
Mini
Midi
Screening
Reaction time
10–16 hours
0.5–1.5 hours
Reactions
25
100
Add to cart
REPLI-G Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

Limitations on the number and type of genomic analyses that can be performed due to insufficient quantities of genomic DNA can be overcome by global amplification of all DNA within a sample (whole genome amplification). REPLI‑g Kits provide DNA polymerase, buffers and reagents for highly uniform whole genome amplification with minimal sequence bias and can be used with various starting materials, including genomic DNA, fresh or dried blood, buccal swabs, fresh or frozen tissue and cells. QIAGEN REPLI-g Kits are available in several different sizes and configurations which allows researchers to build the best workflow for their application and lab space. Typical REPLI-g yields range from approx. 7 µg to 40 µg of amplified DNA, depending on which kit and workflow is used.

The REPLI-g Mini and Midi Kits offer a single tube workflow with higher yields of amplified DNA, while the REPLI-g UltraFast Kit enables highly uniform and accurate whole genome amplification in just 60–90 minutes. For high-throughput processing in 96-well format, the streamlined, room-temperature procedure of the REPLI-g Screening Kit enables straightforward liquid handling that requires just 20 minutes. The REPLI-g Screening kit is intended for rapid screening using a range of genotyping, PCR, sequencing or microarray assays.

Performance

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").

Researcher feedback

"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").

See figures

Principle

Amplification principle

Sufficient quantity of genomic DNA for genomic analysis is often lacking, restricting the analyses that can be performed. Whole genome amplification (WGA) overcomes this limitation by amplification of the entire genome within a sample, providing sufficient quantities to perform all analyses on the same DNA sample. REPLI-g technology delivers highly uniform DNA amplification across the entire genome with minimal sequence bias.

The method is based on multiple displacement amplification (MDA) technology, which uses isothermal genome amplification with a uniquely processive Phi 29 polymerase that is capable of replicating up to 100 kb without dissociating from the genomic DNA template (see figure “ Multiple displacement amplification [MDA] technology”). Phi 29 polymerase is used in the presence of exonuclease-resistant primers to achieve high yields of DNA product and has a 3'→5' exonuclease proofreading activity with 1000-fold higher fidelity than Taq polymerase to maintain high fidelity during replication. 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). 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 Kits, 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").

See figures

Procedure

REPLI-g Mini and Midi Kits use 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").

Amplification of genomic DNA using the REPLI-g UltraFast Mini Kit involves 3 basic steps (see figure " Purified genomic DNA procedure"). 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.

The very fast, simple and accurate REPLI-g Screening method is capable of parallel amplification of several genomic samples, as described in the REPLI-g Screening Handbook. Sample material is lysed and the DNA is denatured by incubating with Buffer SB1 at 65°C. Denaturation is stopped by cooling to room temperature. Buffer SB2 and DNA polymerase are added, and the isothermal amplification proceeds for at least 10 hours or overnight at 30°C.

The entire reaction setup is performed at room temperature, making it easy to use with liquid-handling instrumentation. Reaction setup for 96 samples takes only 20 minutes, saving time to concentrate on other important tasks. The REPLI-g Screening Kit combines a number of features that make it highly suitable 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 (see flowchart " REPLI-g screening procedure").

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”).

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
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 Tube Tube Tube
See figures

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

Comparison of REPLI-g Kits

Features REPLI-g UltraFast Mini Kit REPLI-g Mini Kit REPLI-g Midi Kit REPLI-g Screening Kit
Amplification Whole genomic DNA Whole genomic DNA Whole genomic DNA Whole genomic DNA
Applications Genotyping, microarray, PCR, real-time PCR, NGS Genotyping, hybridization, RFLP, NGS Genotyping, hybridization, RFLP, NGS Genotyping, sequencing
Denaturation step Alkaline Alkaline Alkaline Heat
Handling time for 96 samples 20 minutes
Maximum input volume 10 ng DNA, 0.5 µl whole blood, ~300 cells/µl >10 ng DNA, 0.1– 0.5 µl whole blood, >600 cells/µl >10 ng DNA, 0.1–0.5 µl whole blood, >600 cells/µl 10 ng DNA, 0.5 µl whole blood, >600 cells/µl
Minimal pipetting volume needed 1 µl 0.5 µl 0.5 µl 1 µl
Quality assessment No No No No
Reaction time 60–90 minutes 10–16 hours 8–16 hours 10–16 hours
Reaction volume 20 µl 50 µl 50 µl 40 µl
Samples per run (throughput) Medium Medium Medium High (1–96 samples)
Starting amount of DNA >10 ng purified genomic DNA >10 ng purified genomic DNA >10 ng purified genomic DNA >10 ng purified genomic DNA
Starting material Genomic DNA, blood, cells Genomic DNA, blood, cells, tissue Genomic DNA, blood, cells, tissue Genomic DNA, blood, cells
Technology Multiple Displacement Amplification (MDA) Multiple Displacement Amplification (MDA) Multiple Displacement Amplification (MDA) Multiple Displacement Amplification (MDA)
Yield 7–10 µg 10 µg 40 µg ~8 µg

Supporting data and figures

Resources

Brochures & Guides (1)
Second edition — innovative tools
Supplementary Protocols (1)
Kit Handbooks (1)
For whole genome amplification from purified genomic DNA, blood, and cells
Quick-Start Protocols (1)

FAQ

What is REPLI-g whole genome amplification?
The REPLI-g Whole Genome Amplification (WGA) method is a rapid and reliable method of generating unlimited DNA from a few cells or a few nanograms of genomic DNA. This technology amplifies the genome with comprehensive loci coverage and minimal bias between any loci, yielding 12+ kb fragments in a simple, scalable reaction.
FAQ ID -654
Will the random hexamers in the REPLI-g reaction interfere with downstream analysis?

The REPLI-g amplified products can be used directly for downstream analysis such as PCR, PCR-based applications, restriction enzyme digestion, cycle sequencing, and more, after appropriate dilution to adjust to work concentrations.

However, to determine DNA concentration by absorbance, the MDA product should be run through a spin column to eliminate the random hexamers, as they will contribute to the absorbance reading and give an artificially high concentration. For this reason, we recommend determining DNA concentration by PicoGreen analysis, which preferentially binds double-stranded DNA. As a result, single-stranded random hexamers will not contribute to the apparent DNA concentration in the quantitation assay. When using this method, the concentration of the MDA product can be determined directly, without any purification.

A Protocol for the use of PicoGreen to quantitate REPLI-g WGA product can be found in the REPLI-g Mini/Midi Handbook. Please follow this link .

FAQ ID -713
Are Centromeres and Telomeres amplified using REPLI-g WGA?
These regions are not amplified because only a subset of the random primers in the REPLI-g amplification mix can prime within these extensively repeated regions. Despite the high processivity of the Phi29 DNA Polymerase, centromeres and telomeres are at a competitive disadvantage during amplification, and drop out. In experiments we have done, single copy sequences within approximately 5000 bases of a poorly amplified region can be affected during amplification. If your gene is further than 5000 bases apart from a centromere or telomere, it should be amplified just fine.
FAQ ID -702
Can I use REPLI-g for SNP Genotyping?

Yes. Chromosomes are equally amplified. We and our customers use amplified DNA for SNP genotyping on a regular basis, using Illumina, TaqMan®, Sequenom, PCR, gel-based sequencing, and other techniques. Tzvetkov et al. (2005) used Affymetrix’ GeneChip Human Mapping 10K Arrays to investigate the accuracy and allele amplification bias in DNA samples subjected to MD-WGA with REPLI-g. They observed an excellent concordance (99.95%) between single-nucleotide polymorphisms (SNPs) called both in the nonamplified and the corresponding amplified DNA. Genomic DNA for this study was extracted from blood samples of four unrelated donors using the QIAamp DNA Blood Kit. High-throughput microarray genotyping of 11 555 different SNPs was performed using GeneChip Human Mapping 10K Arrays version Xba131 (Affymetrix).

For additional references, please visit our continuously expanding Citation Data Base online.

 See trademarks.

FAQ ID -700
What are possible reasons for reduced DNA yields with REPLI-g Kits?

Low yields with REPLI-g Kits for whole genome amplification (WGA) can result from a number of factors:

  • inhibitors in the template DNA, e.g., phenol and SDS, EDTA > 1 mM
  • low-quality, fragmented input DNA, e.g., degraded DNA from old samples, FFPE samples
  • improper reaction conditions, e.g., wrong volumes pipetted, insufficient reagent mixing, denaturation and neutralization steps omitted, or carried out incorrectly
  • Thermocycler incorrectly programmed, e.g., incubation time set incorrectly, reaction temperature too high (heated lid not adjusted to 70°C)

Note! When using a thermocycler model that does not allow adjusting the temperature of the heated lid, REPLI-g incubation temperature has to be reduced to 25-28°C to ensure optimal reaction conditions and amplification efficiency!

FAQ ID -2148
Can I purchase Phi29 DNA polymerase only?
We do not sell any REPLI-g reaction components separately.
FAQ ID -708
Does REPLI-g technology for whole genome amplification work with paraffin embedded samples?

Standard REPLI-g Kits, such as the REPLI-g Mini and Midi, UltraFast Mini-, Screening-, and Mitochondrial DNA Kits are not recommended for the amplification of gDNA extracted from paraffin embedded tissues. DNA recovered from paraffin embedded samples is typically strongly fragmented due to the fixation process, resulting in fragments only a few hundred base pairs long. Phi29 DNA polymerase however works most efficiently on DNA longer than 2 kb in length (ideally, at least a few fragments >10 kb should be present in the gDNA sample). If WGA product from strongly fragmented starting samples is utilized in genotyping assays, significant allele drop out and mis-genotyping can occur.

However, our new REPLI-g FFPE Kit overcomes these limitations by pre-processing of DNA directly derived from paraffin embedded tissue samples. The pre-processing reaction ligates fragments to generate suitable templates for subsequent amplification with REPLI-g Midi DNA Polymerase.

Further information can be found in the WGA Tutorial on our WGA Resource page.

FAQ ID -673
What are exo-resistant random hexamers used in the REPLI-g reaction?
These are DNA primers of random sequence, six nucleotides long, with two thiophosphate linkages at the 3' terminus to prevent digestion of the oligos by the 3' to 5' exonuclease activity of Phi29 Polymerase.
FAQ ID -710
Will REPLI-g work at high temperatures?
The reaction works at 30oC and will not work efficiently at higher temperatures. This is because the Phi29 DNA polymerase is not a thermostable enzyme and the random hexamer primers bind less efficiently as temperature is increased.
FAQ ID -656
How can I quantify the amount of REPLI-g DNA I have amplified?
  • Since REPLI-g amplification products contain single-stranded DNA as well as residual primers, it is important to utilize a DNA quantification method that is specific for double-stranded DNA. PicoGreen is a fluorescence-based nucleic acid quantitation method that is specific for double-stranded DNA and may be used to quantify the double-stranded REPLI-g products. For best results, the sample should be diluted with 2 volumes of water and thoroughly mixed prior to addition of PicoGreen.
  • before taking an OD reading on a spectrophotometer the reaction product must be purified as it contains unused primers and dNTPs
FAQ ID -694
What are the advantages of REPLI-g over conventional DNA sample processing and amplification methods?

One of the greatest strengths of REPLI-g whole genome amplification (WGA) is extremely low amplification bias at very high levels of amplification. DOP and PEP suffer from allele drop out and high levels of loci bias at greater than 1000-fold amplification. REPLI-g WGA is based on multiple displacement amplification (MDA) and can maintain accurate loci and allele representation even at 1 million-fold amplification with quality gDNA. This advantage comes from the intrinsic high processivity and fidelity of the Phi29 DNA polymerase.

MDA is a method that enhances simple, rapid, isothermal whole-genome amplification. It is carried out directly on crude lysed cells, whole blood, buccal swabs, and other biological samples, eliminating costly, time-consuming DNA purification steps.

REPLI-g WGA significantly increases the amount of DNA from specific samples, allowing to conduct a large number of downstream genetic tests. Conventional methods of sample processing limit the number of tests that can be done, as the amount of DNA available for testing is often small.

FAQ ID -686
Why is there DNA in the no-template control reaction when using the standard REPLI-g procedure, but not when using the UltraFast procedure?

In no-template (negative) control reactions, primer-dimers can form. The highly processive Phi29 DNA Polymerase will extend these primer-dimers leading to unspecific amplification products during the long incubation time with the standard REPLI-g procedure. Amplification in no-template controls takes place much more slowly than the amplification of template DNA. Using the REPLI-g UltraFast Mini Kit, the incubation time is too short to allow the extension of primer-dimers.

In any case, non-specific amplification products will not compromise results in downstream genetic analysis and do not appear if template DNA is present.

 

FAQ ID -1327
Has anyone verified whole genome amplification accuracy with Sequencing?

Paez et al. 2004 have shown in direct sequencing experiments sampling 500 000 bp, that the estimated error rate (9.5 x 10-6) was the same in WGA generated samples as in paired unamplified samples.

FAQ ID -701
Can I use my own primers for REPLI-g WGA to amplify a specific chromosomal region?
The REPLI-g kit is designed for whole genome amplification using random hexamers. Addition of specific primers instead of random hexamers may introduce amplification bias, or the preferential amplification of specific DNA fragments at the expense of others. Currently, specific primers alone cannot be used to amplify a specific region of the genome with REPLI-g.
FAQ ID -712
Where can I find background information and literature on Whole Genome Amplification with REPLI-g Kits?

Please visit our Whole Genome Amplification Resource Page for access to comprehensive information on WGA using REPLI-g Kits and REPLI-g Services.

Our WGA tutorial provides further information about Whole Genome Amplification and discusses the various techniques that are used. Additional detailed information is provided about REPLI-g Multiple Displacement Amplification technology (MDA), and recommendations are given on how to achieve the best results.

 

FAQ ID -1690
Are samples that are contaminated with high molecular weight DNA (e.g. from a paramedic or police officer) a problem with REPLI-g amplification?
We are not concerned about mixed samples, because there is no preferential amplification with REPLI-g WGA. A small level of contamination from a secondary source cannot become a major component of the amplified material.
FAQ ID -683
What is the length of the MDA whole genome amplification product?

MDA-amplified product has an average length of 10-12 kb, enabling Southern Blots, RFLP, and other downstream analyses that require large DNA fragments.

Note that the ligation procedure employed in the REPLI-g FFPE Kit results in the formation of very high-molecular-weight DNA and enables uniform whole genome amplification from formalin-fixed, paraffin-embedded (FFPE) tissue.

Further information on yield and length of amplified DNA can be found in the WGA Tutorial on our Whole Genome Amplification Resource Page.

FAQ ID -690
Do I need to clean up the REPLI-g amplification product for downstream analysis?
No. Dilute the amplified REPLI-g WGA product in 1x TE to a concentration identical to the gDNA concentration applied in downstream genotyping experiments. There are some residual random primers and dNTPs in the amplified mix, but these will not interfere with most genotyping methods after they are diluted. However, in some cases, purifying amplified DNA before use in a highly multiplexed PCR may yield better results.
FAQ ID -691
Do you have WGA protocols for starting materials not mentioned in the REPLI-g Handbooks?

Yes, we have a set of Specialized Protocols, using the REPLI-g Midi Kit, for direct whole genome amplification from the following starting materials:

  • flash-frozen tissue sections
  • laser-microdissected cells
  • cells contained in plasma and serum
  • dried blood spots
  • buccal cells

These protocols are available as Supplementary Protocols on our website on the REPLI-g Midi product page.

The protocols for the REPLI-g Midi Kit may be adapted for use with the REPLI-g Mini Kit, using the same reaction setup. In rare cases, potential inhibitors present in the starting material may have inhibitory effects on amplification when using the REPLI-g Mini Kit. In these cases, we recommend using the REPLI-g Midi Kit. Alternatively, upstream gDNA purification can be performed, for example, using a QIAamp Kit, with subsequent whole genome amplification of the purified DNA following the standard protocol in the REPLI-g Mini/Midi Handbook.

Since QIAGEN customers are a major resource of information regarding advanced or specialized use of our products, we are looking forward to hear back from you if you would like to share your experience on a new application with us.

 

 

 

FAQ ID -1160
How can I determine the quality of my REPLI-g amplified products?
After the REPLI-g reactions are completed, 1 ug of the WGA reaction product can be analyzed by electrophoresis through a 1.0% agarose gel in TBE buffer (90 mM Tris-borate, pH 8.0, 2 mM EDTA), stained with GelStar, ethidium bromide, or SYBR Green, and imaged with a UV-box or a Phosphor-Imager. The majority of product should be above 10 Kb in length, and generate a trail of smear by electrophoresis down to about 2 Kb.
FAQ ID -695
Are there components in biological samples that can inhibit the REPLI-g reaction?
Some components present in biological samples may inhibit the REPLI-g reaction. For example, if too much blood is added, excess heme present will have an inhibitory effect. For this reason, we limit the volume of blood lysates to use in the WGA reaction. Buccal swab samples and mouthwash specimens should be processed taking care to pellet and wash the cells, as this will produce cleaner lysates and better amplification product.
FAQ ID -667
Do you have an off-the shelf diagnostics kit for WGA?
The REPLI-g Mini/Midi kits are intended as general-purpose reagent for laboratory use. No claim or representation is intended or made with respect to clinical use (diagnostic, prognostic, therapeutic, or blood banking) of the REPLI-g technology.
FAQ ID -678
Why do I get amplification in a negative control DNA tube using the REPLI-g Kit for WGA?

Phi29 DNA Polymerase has an extremely high processivity and will extend primer-dimers that may be present in the reaction, leading to unspecific amplification products. Furthermore, the REPLI-g reaction is highly sensitive to any traces of DNA. Even minute quantities of contaminating DNA from other sources are eventually amplified over the long duration of the reaction (6-16h). However, these non-specific products will not generate specific results in downstream genetic analysis.

FAQ ID -675
Will TA cloning work with REPLI-g WGA product?
No. MDA (Multiple Displacement Amplification) does not generate a TA overhang, so REPLI-g whole genome amplification product will not work with a TA cloning kit.
FAQ ID -672
Can I do whole genome amplification from brain tissue using REPLI-g?

We have successfully amplified DNA from frozen brain tissue sections. A 2 mm section of frozen tissue should be used. Please follow the Supplementary protocol “Whole genome amplification from flash-frozen tissue sections using the REPLI-g® Midi Kit

Alternatively, use a QIAGEN product for isolating genomic DNA from your specific starting material first, and then use the purified gDNA for whole genome amplification with the REPLI-g Mini/Midi, or take advantage of our REPLI-g WGA Services.

FAQ ID -669
How should I store the REPLI-g amplification product?

We recommend that you treat the amplified DNA exactly as you would treat genomic DNA. The best way to store gDNA is frozen at high concentration at -20ºC in 1x TE. The worst way to store gDNA or the amplification product is at low concentration (less than 10 ng/ul) at 4ºC in water.

We have observed significant degradation of genomic DNA stored at 2-3ng/ul at 4ºC in as little as 2-3 weeks. Frozen samples can be thawed and aliquoted. Keep a working stock to use directly in any downstream application. This will decrease the risk for degradation of higher concentrated stocks.

Repeated freeze-thaws, vortexing, rapid pipetting up and down, storage in water and heating to over 75ºC should be avoided as these processes can shear gDNA or MDA amplified DNA, and/or contribute to rapid DNA degradation.

FAQ ID -692
Can FTA paper for dried blood spots be used with REPLI-g?
We have done limited testing on FTA paper. These initial studies have been successful. We have done more extensive testing on the S&S 903 and Isocode papers and achieved excellent results, even with samples 8 years old.
FAQ ID -685
Do you have data on the DNA yield at different incubation time for the Repli-G Kits?
Yes.  We have data for Repli-G Midi Kit and Repli-G Ultrafast Mini Kits. Click here to see the data.
FAQ ID -3113
Do you know of anything in the buffers/DNA sample that can cause bias in REPLI-g WGA?
The main cause for bias in the REPLI-g amplification product is too little input gDNA, or gDNA that is degraded.
FAQ ID -684
Can cDNA prepared with the QuantiTect Whole Transcriptome Kit be reampliied using REPLI-g Kits?

We recommend amplification of the original starting material with the QuantiTect Whole Transcriptome Kit. If the original starting material is no longer available, the cDNA can be reamplified at least once using REPLI-g Kits.

We have not validated more than one round of reamplification.

 

FAQ ID -1611
Do you synthesize thiophosphate modified random hexamers?
We do not synthesize our own hexamers.
FAQ ID -711
Can I re-amplify whole genome amplification product?
We recommend amplification from the original starting material. If a large amount of amplified DNA will be needed, we recommend to increase the REPLI-g Kit reaction volume accordingly. Since the yield is determined by the reaction components, the final concentration of amplified DNA will be the same. Therefore, increasing the volume will increase the yield. However, if the original starting material is unavailable, the REPLI-g amplified DNA can be re-amplified at least once. We have not validated more than one round of re-amplification.
FAQ ID -670
Can I amplify from plasma or serum samples using the REPLI-g Mini/Midi kits?

Plasma and serum will work, but these samples typically contain small numbers of cells, and small amounts of free DNA. Therefore, sufficient sample volume needs to be added to get enough cells to amplify. For our REPLI-g Whole Genome Amplification Services, we need about 50 ul of this sample type for successful amplification.

We suggest to concentrate plasma and serum samples to a final volume of 100-200 ul using centrifugal micro-concentrators. The Millipore 5 micron filter Ultrafree-CL SV will work fine for this purpose. Use 2.5 ul of the concentrate in the REPLI-g Mini/Midi kit reaction.  If you use a minimum of 200-300 ul of sample for spin column concentration, you can remove 2.5 ul of the retained 10 ul for amplification using the kit. Please see the article Retention of leukocytes in capillaries: role of cell size and deformability by G. P. Downey et al. in the Journal of Applied Physiology 69(5), p. 1767-1778. It demonstrates that white cells will be retained on a 5 micron filter while red cells and platelets will filter through.

You can also try our Supplementary protocol “Whole genome amplification from plasma and serum using the REPLI-g® Midi Kit

FAQ ID -671
What is the smallest amount of DNA I can put into a 50 ul REPLI-g reaction?

The minimum amount of DNA for successful WGA with the REPLI-g Kit is 10 ng. Optimal amplification is achieved with 100 ng or more of genomic DNA template. Comprehensive and accurate amplification of the human genome can be accomplished directly from 300 or more human cells, from tissue culture, or from 1.0 ul whole blood without prior DNA purification. Amplification is dependent upon the quality of input DNA.

FAQ ID -663
Any data on the fidelity of the REPLI-g MDA technique?

Phi29 DNA polymerase is a high fidelity proofreading enzyme and assures a very low replication error rate. It has an error rate of 1 x 10-6 - 10-7 nucleotides both in its intrinsic enzymatic activity and during the amplification reaction.

In contrast, Taq DNA polymerase has an intrinsic error rate of approximately 2 x 10-5 nucleotides, with an accumulation of about one mutation per 900 bases after 20 PCR cycles.

FAQ ID -707
What is the stability of the REPLI-g MDA product?
We have been conducting an ongoing stability study for more than a year without observing breakdown of the amplified product. There is nothing in the amplification product that indicates that it would not be stable for a number of years.
FAQ ID -693
What is the enzyme used in the REPLI-g reaction?
The enzyme used in the REPLI-g reaction is Phi29 DNA Polymerase.
FAQ ID -704
Can I do whole genome amplification from mitochondrial DNA using REPLI-g?
Yes, we offer the REPLI-g Mitochondrial DNA Kit for this application.
FAQ ID -668
Is 2 kb the minimal gDNA fragment size for REPLI-g Whole Genome Amplification?

Highly degraded samples tend not to be amplified evenly across the genome. In general, an average fragment size of 2 kb in a DNA sample is the lower limit, assuming no portions of the genome are degraded to the point where information will be missing. Often, when the largest fragment in a gDNA sample is 2 kb, other fragments are much smaller and some regions of the genome may have been lost due to degradation. At least a small portion of 10 kb fragments or larger need to be present in the gDNA sample for even amplification of the entire genome.

Further information on working with fragmented DNA can be found in the WGA Tutorial on our Whole Genome Amplification Resource Page.

 

Note: If you are interested in amplifying DNA from paraffin-embedded samples we recommend to use the REPLI-g FFPE Kit.

FAQ ID -682
What are the differences between MDA and DOP/PEP methods of Whole Genome Amplification?

DOP (Degenerate Oligonucleotide-primed PCR) and PEP (Primer Extension Preamplification) are PCR-based whole genome amplification (WGA) methods. REPLI-g amplification uses MDA (Multiple Displacement Amplification) which is not a PCR-based method. MDA is scalable with yields adjustable from ug to mg quantities, whereas DOP typically yields 2-3 ug of DNA per reaction. DOP also generates a shorter product which is not suitable for certain downstream applications (e.g. Southern blot and sub-cloning).

DOP and PEP products are different from REPLI-g MDA products for a number of reasons. First, after amplification is complete, PEP products have active thermostable polymerase that will degrade the amplification product over time, because the polymerase cannot be inactivated. Second, PEP reactions consist of PCR amplicons which have the potential to contaminate other reactions as 'runaway amplicons' (e.g., amplicons in the aerosol that may be co-amplified if they accidentally get into other reactions).

REPLI-g product has neither of these issues. The polymerase is heat-inactivated after the REPLI-g reaction is complete, so it cannot digest the amplified product. There are no issues with 'runaway amplicons', because the reaction is performed at constant temperature by a hyper-branching amplification mechanism, amplifying the genome randomly without generating specific amplicons.

FAQ ID -665
Do you have a protocol for cleanup of REPLI-g amplified DNA?

Yes, please follow the Supplementary Protocol 'Purification of REPLI-g amplified DNA using the QIAamp DNA Mini Kit' (RG14).

 

 

 

FAQ ID -1545