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REPLI-g Advanced DNA Single Cell Kit

For whole genome amplification (WGA) from single eukaryotic cells, limited samples or purified genomic DNA
  • WGA with complete genome coverage from single eukaryotic cells or 1 ng genomic DNA 
  • Simple 3-step protocol requires only 2 hours and 20 minutes, start to finish
  • Consistent yields of up to 25–35 µg DNA from 1 eukaryotic cell
  • Includes cryo-protect reagent to minimize DNA damage during single cell capture, storage or shipping

DNA sequence analysis and genotyping of biological samples using next-generation sequencing (NGS) platforms is often limited by the amount of available sample. The REPLI-g Advanced DNA Single Cell Kit has been specifically optimized for use in sensitive microarray and NGS applications, providing high uniformity, with minimal allelic dropout, when starting with single cells or purified genomic DNA. Buffers and reagents undergo a unique, controlled decontamination procedure to minimize contaminating DNA and ensure highly reliable results. Accurate amplification of genomes with negligible sequence bias and minimal genomic dropouts is achieved with innovative Multiple Displacement Amplification (MDA) technology.

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Cat No./ID: 150363
REPLI-g Advanced DNA Single Cell Kit (24)
€626.00
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Buffers, enzymes and single cell storage solution for 24 WGA reactions
Cat No./ID: 150365
REPLI-g Advanced DNA Single Cell Kit (96)
€1,982.00
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Buffers, enzymes and single cell storage solution for 96 WGA reactions
Cat No./ID: 150370
REPLI-g Single Cell Cryo-Protect Reagent (15 ml)
€305.00
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Single cell reagent protects cell integrity during isolation, storage and shipping
The REPLI-g Advanced DNA 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

0
REPLI-g Advanced DNA Single Cell workflow.
The REPLI-g Advanced DNA Single Cell Kit features a simplified protocol of just 3 steps with only 20 minutes hands-on time for reaction setup and 2 hours for reaction time. The typical yield from a single cell is 25–35 µg of amplified DNA – sufficient for downstream applications involving qPCR and next-generation sequencing. The REPLI-g Advanced DNA Single Cell protocol saves at least 1 hour of time versus the first-generation REPLI-g Single Cell Kit (cat. nos. 150343 and 150345).
1
Unbiased amplification with Phi 29 polymerase.
[A] Upon encountering secondary DNA structures, Taq polymerase may pause synthesis, slip, or dissociate from the template. This can result in inaccurate DNA amplification, incomplete loci coverage, and short fragment sizes. [B] REPLI-g Kits utilize Phi 29 polymerase, which displaces secondary structures enabling accurate and highly uniform amplification of the entire genome.
2
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.

3
REPLI-g Advanced DNA Single Cell Kit provides consistent results (single cells).
REPLI-g Advanced DNA Single Cell Kits were tested in multiple experiments by 2 operators, using 4 different lots of reagents, with 4 different types of eukaryotic cells. Single cells were isolated with the QIAGEN QIAscout.

For each experiment, 6000 cells were loaded onto the QIAscout array and left overnight in a standard cell incubator. For suspension cells, the QIAscout array was first treated with Cell-Tak to facilitate adherence of the cells. On the day of cell collection, QIAscout was installed on a 10x objective of an inverted microscope (Axio Vert.A1, Zeiss [Carl Zeiss AG]) and the array was screened using brightfield microscopy. Microrafts containing single cells were dislodged and transferred to a secondary vessel containing 4 µl REPLI-g Advanced sc Storage Buffer prior to starting the REPLI-g Advanced DNA Single Cell protocol for single cells.

Data presented is from 7 separate experiments (4 experiments with Jurkat cells and 3 experiments with additional cell lines) and 4 separate lots of reagents (A–D). Final DNA yield was quantified using PicoGreen (Thermo Fisher Scientific). Other quantification methods that are not specific for dsDNA might overestimate the yield.
4
REPLI-g Advanced DNA Single Cell Kit provides consistent results (gDNA, 1 ng).
REPLI-g Advanced DNA Single Cell Kits were used to amplify 1 ng human genomic DNA. Two different operators (DU and AJ) and different lots of Reaction Buffer (A–E) were used in 7 separate experiments to confirm protocol and reagent reproducibility.

Final DNA yield was quantified using PicoGreen (Thermo Fisher Scientific). Other quantification methods that are not specific for dsDNA might overestimate the yield because REPLI-g Advanced DNA Single Cell amplification products might contain unused reaction primers and nucleotides.
5
Reproducibility of REPLI-g Advanced DNA Single Cell Kit using single Jurkat cells.
REPLI-g–amplified DNA from isolated single Jurkat cells using the first generation REPLI-g Single Cell Kit (i.e., Original REPLI-g Single Cell Kit) and the new second generation REPLI-g Advanced DNA Single Cell Kit was analyzed by qPCR using both single (DOLK, C4:00, C4:30, C4:40, C4:60, C4:80 and 3q) and multi-copy (Multi) loci. The multi-copy gene provides information regarding the overall success of the WGA (whole-genome amplification) while the single-copy genes give an estimation about uniformity of WGA. A major increase in the CT value for single copy genes is indicative of potential allelic dropout.

The results show improved robustness and reproducibility with the REPLI-g Advanced DNA Single Cell Kit. The simple 2 hour amplification reaction is adequate for obtaining sufficient amplified DNA for qPCR applications, while the improved reaction conditions limit allelic dropout.
6
Reproducibility of REPLI-g Advanced DNA Single Cell Kit using single LoVo cells.
REPLI-g–amplified DNA from isolated single LoVo cells using the first generation REPLI-g Single Cell Kit (i.e., Original REPLI-g Single Cell Kit) and the new second generation REPLI-g Advanced DNA Single Cell Kit was analyzed by qPCR using both single (DOLK, C4:00 and 3q) and multi-copy (Multi) loci. The multi-copy gene provides information regarding the overall success of the WGA (whole-genome amplification) while the single-copy genes give an estimation about uniformity of WGA. A major increase in the CT value for single copy genes is indicative of potential allelic dropout.

The results show improved robustness and reproducibility with the REPLI-g Advanced DNA Single Cell Kit. The simple 2 hour amplification reaction is adequate for obtaining sufficient amplified DNA for qPCR applications, while the improved reaction conditions limit allelic dropout.
7
Reproducibility of REPLI-g Advanced DNA Single Cell Kit using single MCF7 cells.
REPLI-g–amplified DNA from isolated single MCF7 cells using the first generation REPLI-g Single Cell Kit (i.e., Original REPLI-g Single Cell Kit) and the new second generation REPLI-g Advanced DNA Single Cell Kit was analyzed by qPCR using both single (DOLK, C4:00 and 3q) and multi-copy (Multi) loci. The multi-copy gene provides information regarding the overall success of the WGA (whole-genome amplification) while the single-copy genes give an estimation about uniformity of WGA. A major increase in the CT value for single copy genes is indicative of potential allelic dropout.

The results show improved robustness and reproducibility with the REPLI-g Advanced DNA Single Cell Kit. The simple 2 hour amplification reaction is adequate for obtaining sufficient amplified DNA for qPCR applications, while the improved reaction conditions limit allelic dropout.
8
Reproducibility of REPLI-g Advanced DNA Single Cell Kit using gDNA.
REPLI-g Advanced DNA Single Cell Kits were used to amplify 1 ng human genomic DNA. Two operators (DU and AJ), different DNA Polymerase lots (2–5) and different lots of Reaction Buffer (A–C) were used to confirm protocol and reagent reproducibility. REPLI-g–amplified gDNA was analyzed by qPCR using both single (DOLK and C4:00) and multi-copy (Multi) loci. The multi-copy gene provides information regarding the overall success of the WGA (whole-genome amplification) while the single-copy genes give an estimation about uniformity of WGA. A major increase in the CT value for single copy genes, when compared between WGA reactions, is indicative of potential allelic dropout.

The results show REPLI-g Advanced DNA Single Cell Kits have a high degree of reproducibility regardless of the operator or lots of reagents used. The simple 2 hour amplification reaction is adequate for obtaining sufficient amplified gDNA for qPCR applications, while the improved reaction conditions limit allelic dropout.
9
Reproducibility of REPLI-g Advanced DNA Single Cell Kit yield (gDNA).
REPLI-g Advanced DNA Single Cell Kits were used to amplify 1 ng human genomic DNA. Two operators (DU and AJ), different DNA Polymerase lots (2–5) and different lots of Reaction Buffer (A–C) were used to confirm protocol and reagent reproducibility. Yield of WGA was measured using PicoGreen (Thermo Fisher Scientific) reagent in conjunction with a fluorometer. PicoGreen reagent displays enhanced binding to double-stranded DNA and is therefore suitable to quantify the double-stranded DNA product. Other quantification methods that are not specific for dsDNA might overestimate the yield because REPLI-g Advanced DNA Single Cell amplification products might contain unused reaction primers and nucleotides. The average yield across all experiments is 41 µg, which is an amplification of ~40,000-fold.

For PicoGreen measurement, the protocol described in the Appendix of the REPLI-g Advanced DNA Single Cell Kit Handbook was used.
10
Improved uniformity of REPLI-g Advanced DNA Single Cell Kit.
Single Jurkat cells were isolated using the QIAscout and transferred to a secondary vessel containing 4 µl REPLI-g Advanced sc Storage Buffer or 4 µl PBS. REPLI-g Advanced sc Storage Buffer is a feature of the new kit. It preserves cell integrity prior to cell lysis and helps reduce DNA damage.

Single cells in 4 µl of REPLI-g Advanced sc Storage Buffer were processed with the REPLI-g Advanced DNA Single Cell Kit (REPLI-g Adv) while single cells in 4 µl of PBS were processed with the REPLI-g Single Cell Kit (REPLI-g Orig). Amplification yields were quantified using PicoGreen (Thermo Fisher Scientific). 40 ng of amplified DNA from single cells, as well as unamplified gDNA from bulk Jurkat cells, was used to build targeted NGS libraries using the QIAseq Targeted DNA Human Comprehensive Cancer Panel (DHS-3501Z) covering 275 genes (837 kb). Sequencing was performed on the MiSeq (Illumina).

To evaluate uniformity, calculations were made for how many target bases reach 5–30% (at minimum) of the mean read depth. The new REPLI-g Advanced DNA Single Cell Kit resulted in higher uniformity than placing cells in PBS and using the original REPLI-g Single Cell Kit.
11
REPLI-g Advanced DNA Single Cell Kit reduced allelic dropout and improved consistency.
Single allelic dropout (ADO) for REPLI-g–amplified DNA from single cells was assessed using libraries generated using the QIAseq Targeted DNA Human Comprehensive Cancer Panel and sequenced using a MiSeq (Illumina). A comparison between 6 single Jurkat cells using the REPLI-g Advanced DNA Single Cell kit and 6 single Jurkat cells using the original REPLI-g Single Cell Kit is shown. Unamplified gDNA from bulk Jurkat cells was used as reference. NGS results were analyzed using the GeneGlobe Data Analysis Center (www.qiagen.com/shop/genes-and-pathways/data-analysis-center-overview-page/).

Each library is based on a single cell after WGA, with 40 ng amplified DNA used as starting material, and a mean read depth of 127. To compare the ADO rates for both kits, we analyzed the single ADO for SNPs that are heterozygous (frequency of SNP allele: 25 – 75 %) in the unamplified bulk DNA. Calculation of ADO rate is: (# of heterozygous loci in gDNA – # of heterozygous loci in single cell after WGA)/(# of heterozygous loci in gDNA).

The REPLI-g Advanced DNA Single Cell Kit shows a lower rate of ADO and improved inter-sample consistency.
12
Comparison of REPLI-g Advanced DNA Single Cell Kit to other methods.
Single Jurkat cells captured using the QIAscout were used in MALBAC Single Cell WGA Kit (Yikon Genomics), PicoPLEX WGA Kit (Rubicon Genomics, Inc.) and REPLI-g Advanced DNA Single Cell Kit (QIAGEN) amplifications according to the protocol provided with each kit. Amplified DNA was analyzed using qPCR for single (DOLK, C4:00 and 3q) and multi-copy (Multi) loci. The multi-copy gene provides information regarding the overall success of the WGA (whole-genome amplification) while the single-copy genes give an estimation about uniformity of WGA. A major increase in the CT value for single copy genes is indicative of potential allelic dropout.

The results show improved robustness and reproducibility with the REPLI-g Advanced DNA Single Cell Kits versus other commercially-available kits. The simple 2 hour amplification reaction is adequate for obtaining sufficient amplified gDNA for qPCR applications, while the improved reaction conditions limit allelic dropout.
13
Improved uniformity of REPLI-g Advanced DNA Single Cell Kits using NGS.
Single Jurkat cells were collected using the QIAscout. Isolated single cells on QIAscout microrafts were transferred to a secondary vessel containing 4 μl REPLI-g Advanced sc Storage Buffer (REPLI-g Advanced DNA Single Cell Kit), 5 µl Extraction Cocktail (PicoPLEX WGA Kit [Rubicon Genomics]) or 5 µl Lysis Reaction Mix (MALBAC Single Cell WGA Kit [Yikon Genomics]).

40 ng of amplified DNA, as well as unamplified gDNA (isolated from bulk Jurkat cells), was used to build targeted NGS libraries using the QIAseq Targeted DNA Human Comprehensive Cancer Panel (DHS-3501Z). This panel has full exon coverage of 275 genes with coverage of 837 kb. Sequencing was performed on the MiSeq (Illumina).

To evaluate uniformity, calculations were made for how many target bases reach 5–30% (at minimum) of the mean read depth. The combination of the REPLI-g Advanced sc Storage Buffer and the improved chemistry of the REPLI-g Advanced DNA Single Cell Kit results in higher uniformity compared to alternative methods.
Performance
Genotyping and DNA sequence analysis of biological samples can be limited by a small amount of available sample. The REPLI-g Advanced DNA Single Cell Kit allows uniform amplification of whole genomic DNA from limited sample amounts and enables a greater variety and number of analyses to be performed. The average product length of amplified DNA is typically more than 10 kb, with a range between 2 kb and 100 kb, enabling all downstream applications such as complex genetic analysis, including long-range copy number variations, to be performed (1). DNA amplified with the REPLI-g Advanced DNA Single Cell Kit is highly suited for next-generation sequencing (NGS), array CGH genotyping applications or qPCR analysis.

Typical DNA yields from REPLI-g Advanced DNA Single Cell Kit amplifications are approximately 25–35 µg per 50 µl reaction. Depending on the quality of the input cell and its DNA, the resulting amount of amplified DNA may be less (fragmented or damaged gDNA should not be used). For best amplification results, we recommend collecting or storing cells in REPLI-g Advanced sc Storage Buffer (included with kit or available separately).
Principle
The REPLI-g Advanced DNA Single Cell Kit contains an optimized Phi 29 polymerase formulation, as well as buffers and reagents, for whole genome amplification (WGA) from single eukaryotic cells, very small amounts of sample or purified genomic DNA using Multiple Displacement Amplification (MDA). Phi 29 polymerase, a phage-derived enzyme, is a DNA polymerase with 3'→5' exonuclease activity (proofreading activity) that delivers up to 1000-fold higher fidelity compared to Taq DNA polymerase. Supported by the unique, optimized REPLI-g Advanced DNA 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. The REPLI-g Advanced DNA Single Cell Kit is an improved version of the REPLI-g Single Cell Kit and includes an optimized reaction chemistry, a new single cell storage buffer and an improved protocol to increase the uniformity of amplification and reduce potential amplification bias. In addition, the MDA reaction time has been shortened to 2 hours.
Procedure
The REPLI-g Advanced DNA Single Cell Kit provides highly uniform amplification across the entire genome, with negligible sequence bias. The method is based on Multiple Displacement Amplification (MDA) technology, which carries out isothermal genome amplification utilizing a uniquely processive DNA polymerase capable of replicating up to 100 kb without dissociating from the genomic DNA template. In contrast to PCR-based methods, Phi 29 polymerase has a 3'–5' exonuclease proofreading activity to maintain 1000-fold higher fidelity than Taq Polymerase during replication. MDA technology is used in the presence of exonuclease-resistant primers to achieve high yields of DNA product from all kinds of eukaryotic tissues.

Genetic analyses often require substantial amounts of genomic DNA. Whole genome amplification solves the problem of low DNA quantity when starting with 1–1000 cells. The REPLI-g Advanced DNA Single Cell Kit overcomes these limitations by using a simple and reliable method to achieve accurate genome amplification. In addition, all components provided with the REPLI-g Advanced DNA Single Cell kit are exposed to UV radiation in a standardized procedure to minimize contaminating DNA. Reaction setup takes only 20 minutes and 3 steps, making the REPLI-g Advanced DNA Single Cell Kit protocol easy and reliable.

In the first step of the procedure, the cell sample is lysed under isothermal alkaline conditions and the cell is denatured. Cell lysis at room temperature reduces the number of possible DNA breaks and therefore improves uniformity of whole genome amplification. After denaturation has been stopped by the addition of neutralization buffer, a master mix containing buffer and DNA polymerase is added. The isothermal amplification reaction proceeds for 2 hours at 30°C. Because of the shortened MDA reaction time, the REPLI-g Advanced DNA Single Cell Kit enables single cell collection, WGA and even downstream analysis to be performed in a single day.
Applications
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.


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


References

1. Hosono, S. et al. (2003) Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13, 954.

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