REPLI-g Single Cell Kit

For highly uniform whole genome amplification (WGA) from single cells or limited sample material

Features

  • WGA from single cell material with complete genome coverage
  • Unbiased amplification of genomic loci due to MDA technology
  • Optimized for use with new technologies, including NGS
  • Consistent yields of up to 40 µg (average product length >10 kb)
  • Novel tool for cancer research, stem cell research, or metagenomics
REPLI-g Single Cell Kit (24)

Cat. No. / ID: 150343

REPLI-g sc Polymerase, Buffers, and Reagents for 24 whole genome amplification reactions (yields up to 40 µg/reaction)
€597.00
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The REPLI-g Single Cell Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

DNA sequence analysis and genotyping of biological samples using innovative instrumentation, such as next-generation sequencing (NGS) platforms,  is often limited by the small amount of sample available. The REPLI-g Single Cell Kit is specially designed to uniformly amplify genomic DNA from single cells (1 to <1000 bacterial or tumor cells) or purified genomic DNA with complete genome coverage. Additional protocols are also available for use with fresh or dried blood or fresh or frozen tissue. Dedicated buffers and reagents undergo a unique, controlled decontamination procedure to avoid amplification of contaminating DNA, ensuring highly reliable results every time. Accurate amplification of genomes with negligible sequence bias and no genomic drop-outs is achieved with innovative Multiple Displacement Amplification (MDA) technology. In contrast to PCR-based WGA technologies, high fidelity rates are increased up to 1000-fold, avoiding costly false positive or negative results.

Performance

Complete genome coverage, highly suited for NGS and other downstream applications

DNA amplified using the REPLI-g Single Cell Kit has an average product length of >10 kb and maximized genome coverage. It has been tested with, and is highly suited for, numerous downstream analyses including next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and real-time PCR-based applications (see Table 2). Since there is no requirement for a separate PCR-based amplification step, REPLI-g whole genome amplification and library preparation requires less hands-on time and results in longer read-lengths than PCR-based methods (see figure " Next-generation sequencing using REPLI-g amplified DNA requires less hands-on time and generates more sequence information than PCR-based methods"). High-quality, comparable NGS results showing a high percentage of sequence coverage and very low error rates are achieved with both purified genomic DNA or REPLI-g Single Cell amplified DNA, including when starting from just a single bacterial cell (see figure “ Comparable NGS results”). These finding are underscored by a comprehensive analysis of a wide range of markers covering all human autosomal chromosomes and the X chromosome, with 3 different independent experiments demonstrating that DNA is successfully amplified from all areas of the genome without a single drop-out (see figure “ Complete genome coverage”).

Table 1. Range of sample material and research areas
Sample material (cells/DNA) Research area
Human/animal Biomarker research (SNPs, mutations, CNVs)
Stem cell research
Analysis of circulating fetal cells
Mosaicism studies
Genetic predisposition studies
Typing of transgenic animals
Cancer Somatic genetic variant analysis
Tumor progression
Tumor stem cells/evolution
Analysis of circulating tumor cells
Bacteria Metagenomic studies
Pathogen analysis
Microbial genotyping
Plants* Stomata research
Pollen analysis
* Cells without cell walls or purified genomic DNA.
The REPLI-g Single Cell Kit outperforms kits from other suppliers

PCR-based WGA methods, as generally used by other suppliers, result in short fragments terminated with PCR primer sequences that may affect downstream processes (e.g., next-generation sequencing; see figure " Next-generation sequencing using REPLI-g amplified DNA requires less hands-on time and generates more sequence information than PCR-based methods"). PCR-based WGA can lead to error-prone amplification that results in, for example, single base-pair mutations, STR contractions, and expansions, and also leads to biased and underrepresented loci due to the use of the low-fidelity enzyme Taq DNA polymerase. In contrast, the REPLI-g Single Cell Kit provides highly uniform amplification across the entire genome, with minimal locus bias during amplification. Four WGA kits, 2 utilizing MDA technology, including the REPLI-g Single Cell Kit, and 2 PCR-based methods, were tested for sequence representation and locus dropout using the single cell amplification protocols specific for each kit. Unlike with the REPLI-g Single Cell Kit, single cells analyzed using kits from other suppliers often failed in complete and unbiased sequence representation (see figure “ Unbiased DNA amplification from a single cell”).

See figures

Principle

The REPLI-g Single Cell Kit includes REPLI-g sc Polymerase, an optimized formulation of the innovative, high-fidelity enzyme Phi 29 polymerase, to amplify complex genomic DNA using Multiple Displacement Amplification (MDA) technology, along with gentle alkaline incubation to ensure very low DNA fragmentation or generation of abasic sites. It is specifically designed to provide high yields of amplified DNA from single cells, such as isolated tumor cells or bacteria (see Table 1). In addition, it can be used with various clinical and non-clinical research samples and with purified genomic DNA, while additional protocols are available for use with fresh or dried blood and fresh or frozen tissue. Typical DNA yields consistently reach 40 µg, regardless of the starting quantity of template, meaning subsequent genetic analyses can proceed without additional measurement of DNA concentration. The average product length of over 10 kb and complete genome coverage ensures that DNA amplified with the REPLI-g Single Cell Kit is highly suited for numerous downstream applications, including next-generation sequencing (NGS), array-based comparative genomic hybridization (array CGH), Pyrosequencing, and real-time PCR analysis (Table 2).

Table 2. Downstream applications and instrumentation
Application Instrumentation
Whole genome sequencing Next-generation sequencing platforms
Exome sequencing
SNP genotyping arrays Array platforms
Array CGH
qPCR/PCR technologies Real-time PCR/PCR cyclers
Sanger sequencing Capillary sequencers
Pyrosequencing PyroMark (QIAGEN)
Available from various suppliers
Amplification principle

The REPLI-g Single Cell Kit 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 an optimized form of the enzyme Phi 29 polymerase, which has exceptionally strong strand displacement properties. Additional priming events occur on each displaced strand that serve as a template, enabling generation of high yields of amplified DNA (see figure “ Multiple Displacement Amplification (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 Single Cell 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”).

Cell lysis and 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 Single Cell Kit uses gentle alkaline incubation, allowing cell lysis and uniform DNA denaturation of gDNA 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 (see figure " Effect of heat and alkaline denaturation on loci representation").

Effective elimination of detectable DNA contamination

All REPLI-g Single Cell Kit components undergo a unique, controlled decontamination procedure to ensure elimination of all REPLI-g amplifiable contaminating DNA. Buffers and reagents are exposed to UV radiation by an innovative and standardized procedure to ensure the absence of any detectable residual contaminating DNA (see figure “ Innovative decontamination procedure”). Following UV treatment, the kits undergo stringent quality control to ensure complete functionality.

See figures

Procedure

Simple, one tube procedure

The REPLI-g Single Cell Kit uses a simple and reliable method to achieve accurate genome amplification from single cells or limited samples. The easy reaction set-up and very low handling time of approximately 15 minutes makes this a straightforward and reliable method (see figure “ REPLI-g Single Cell Kit procedure”). Dedicated buffers and reagents have been developed to deliver high yields of DNA from single cells, limited tissue material, and purified DNA, with complete sequence representation and unbiased amplification (Table 3). REPLI-g amplified DNA can be stored long-term at –20°C with no negative effects (see figure " Consistent long-term stability").

Table 3. REPLI-g Single Cell Kit components
Kit component Advantages
REPLI-g sc Polymerase Long fragments up to 70 kb
1000-fold higher fidelity than Taq
Complete sequence representation
Homogeneous amplification for all loci
REPLI-g sc Reaction Buffer Optimized for unbiased amplification and representation of all loci
Buffer DLB (lysis and denaturation) Efficient preparation for amplification
Non-DNA damaging process
UV decontamination procedure Ensures elimination of detectable residual DNA contamination
Contains salt, primers, and dNTPs
See figures

Applications

DNA amplified with the REPLI-g Single Cell Kit can be used in a variety downstream applications, including:

  1. Next-generation sequencing
  2. SNP genotyping with TaqMan primer/probe sets
  3. qPCR- and PCR-based mutation detection
  4. STR/microsatellite analysis
  5. Sanger sequencing
  6. Pyrosequencing
  7. Array technologies, such as comparative genomic hybridization (aCGH)

Learn more about our single cell products by visiting our Single Cell Resource.


Supporting data and figures

Resources

Brochures & Guides (3)
Accelerate your NGS performance through Sample to Insight solutions
Introducing QIAseq
PDF (450KB)
Accelerate your NGS performance through Sample to Insight solutions
Supplementary Protocols (13)
This protocol is optimized for whole genome amplification from biopsies using the REPLI-g Single Cell Kit.
This protocol is optimized for whole genome amplification from flash-frozen tissue samples using the REPLI-g Single Cell Kit.
This protocol is optimized for whole genome amplification from buccal cells using the REPLI-g Single Cell Kit.
Purification of DNA after whole Genome amplification using the REPLI-g Single Cell Kit
Kit Handbooks (1)
For whole genome amplification from single cells, limited samples, or purified genomic DNA
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
Are there important considerations for plasma generation and urine handling?

It is strongly advised to follow the recommendations for preparing sample material provided in the corresponding Protocol Sheet to ensure reliable results.

Plasma: It is recommended to perform plasma separation immediately after blood collection when using EDTA or citrate as anticoagulant to prevent the release of genomic DNA into the plasma fraction.

Urine: Because circulating cell-free DNA in non-stabilized urine samples is rapidly degraded after sample collection due to high nuclease activity, eluates may contain no DNA or exhibit low DNA concentration. Therefore, it is recommended to stabilize urine samples. Even when using stabilized urine, it is recommended to perform a centrifugation step immediately after stabilization to prevent the release of genomic DNA from cells. Alternatively, non-stabilized urine samples can be processed immediately after collection and centrifugation using ATL-pretreatment and automated DNA extraction as described in the corresponding Protocol Sheet.

FAQ ID - 3699
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
3330 - Is there any contamination from E. coli genomic DNA in the polymerase provided with the Repli-G Single Cell Kit?

A DNA decontamination process is performed on the REPLI-g sc polymerase and REPLI-g buffers.

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
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
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
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