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REPLI-g Single Cell Kit

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

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.

Cat No./ID: 150343
REPLI-g Single Cell Kit (24)
€512.00
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REPLI-g sc Polymerase, Buffers, and Reagents for 24 whole genome amplification reactions (yields up to 40 µg/reaction)
Cat No./ID: 150345
REPLI-g Single Cell Kit (96)
€1,737.00
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REPLI-g sc Polymerase, Buffers, and Reagents for 96 whole genome amplification reactions (yields up to 40 µg/reaction)
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.

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0

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 Phi 29 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.
1

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").
2

Innovative decontamination procedure.

Bacterial DNA (2000 copies) was spiked into REPLI-g sc Reaction Buffer, which was then irradiated with UV using the standard procedure for all buffers and reagents provided with the REPLI-g Single Cell Kit. In subsequent real-time PCR, no bacterial DNA was detectable following the UV decontamination procedure.

3

Comparable NGS results.

Whole genome sequencing of the Bacillus subtilis genome was performed on the Illumina MiSeq instrument. For analysis, 2 μg of genomic DNA or DNA amplified from a single cell (three different single cell experiments) and 103 cells, using the REPLI-g Single Cell Kit, was sheared into 300 bp fragments and 1 μg of each was used for library preparation. [A] Comparable sequence coverage was observed for gDNA and REPLI-g Single Cell amplified DNA*. [B] Comparison of nonamplified and REPLI-g amplified DNA revealed error rates in a similar, very low, percentage range.

* Aligned using the Burrows-Wheeler Alignment program (cut-off: 10x coverage): bio-bwa.sourceforge.net.
Comparison on non-amplified and REPLI-g Single Cell amplified DNA also revealed that sequences mapped to the genome with high percentage rates (data not shown).

4

Complete genome coverage.

Comprehensive analysis of 267 loci across the entire genome was performed using RT2 qPCR Primer Assays (QIAGEN) and real-time PCR following DNA amplification with the REPLI-g Single Cell Kit from 3 different single-cell experiments. Low and consistent CT values, with no dropout from any marker, indicate that DNA was successfully amplified from all areas of the genome and is highly suited for single-cell genomics.
5

Unbiased DNA amplification from a single cell.

The REPLI-g Single Cell Kit or kits from Suppliers G, N, and S were used to individually amplify 5 human cells. [A] Real-time PCR was used to analyze 3 markers to identify loss or variability in the amount of genomic loci. Unlike kits from other suppliers, the REPLI-g Single Cell WGA delivered unbiased amplification of DNA in each of the 5 cells, indicated by equivalent CT values for each marker. [B] Unlike with the REPLI-g Single Cell Kit, DNA amplified using the kits from Suppliers G and N demonstrated high dropout rates. For both kits, genomic marker X54 was not amplified in 2 of the 5 cells tested, and the kit from Supplier G did not amplify marker 99 in 1 of the 5 cells, indicating incomplete genome coverage and biased amplification that makes these kits unsuitable for reliable single cell research.
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REPLI-g Single Cell Kit 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.

7

Next-generation sequencing using REPLI-g amplified DNA requires less hands-on time and generates more sequence information than PCR-based methods.

PCR-based whole genome amplification (WGA) and library preparation for next-generation sequencing requires a purification step prior to library preparation that can result in ~3 times more hands-on time than REPLI-g based WGA and library preparation. Additionally, unlike REPLI-g amplified DNA, PCR-based methods also include PCR primer binding sites (indicated in red) on the WGA amplification product. Since next-generation sequencing read-lengths are between 50–200 bp, the resulting genome coverage could be strongly reduced using a PCR-based WGA method.

8

Unbiased amplification with Phi29 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 Phi29 polymerase, which displaces secondary structures enabling accurate and highly uniform amplification of the entire genome.
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Multiple Displacement Amplification (MDA) technology.

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.

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”).

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.

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
Applications

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

  • Next-generation sequencing
  • SNP genotyping with TaqMan® primer/probe sets
  • qPCR- and PCR-based mutation detection
  • STR/microsatellite analysis
  • Sanger sequencing
  • Pyrosequencing
  • Array technologies, such as comparative genomic hybridization (aCGH)
Learn more about our single cell products by visiting our Single Cell Resource.

 

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Brochures & Guides (2)
Kit Handbooks (1)
For whole genome amplification from single cells, limited samples, or purified genomic DNA
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Supplementary Protocols (12)

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This protocol is optimized for whole genome amplification from biopsies using the REPLI-g Single Cell Kit.
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This protocol is optimized for whole genome amplification from buccal cells using the REPLI-g Single Cell Kit.
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This protocol is optimized for whole genome amplification from flash-frozen tissue samples using the REPLI-g Single Cell Kit.
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