For highly uniform whole genome amplification and quality assessment from small or precious samples
  • Reproducible amplification from a variety of starting materials
  • Scalable service, with 100 µg and 500 µg standard scales
  • Extensive quality assessment including detailed report
  • Highly experienced team providing the service you Need
The REPLI-g service — based on proven REPLI-g whole genome amplification technology — allows the amplification of large amounts of DNA from limited samples with minimal sequence bias. A variety of samples can be used, including genomic DNA, whole blood, dried blood cards, buffy coat, buccal swabs, fresh or frozen tissue, and tissue culture cells. A stringent quality control assay provides information on the quality of the amplified DNA, enabling reliable predictions for the success of your downstream assays. REPLI-g Service is available in 100 µg or 500 µg scales.
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Product Cat. no. List price:
REPLI-g Service, Single Tube (100 µg)
Whole Genome Amplification Service from single tubes, 100 µg scale
805923
107,00 €
REPLI-g Service, Single Tube (500 µg)
Whole Genome Amplification Service from single tubes, 500 µg scale
805925
217,00 €
REPLI-g Service (100 µg)
Whole Genome Amplification Service from microtitre plates, 100 µg scale
805943
84,40 €
REPLI-g Service (500 µg)
Whole Genome Amplification Service from microtitre plates, 500 µg scale
805945
168,00 €
The REPLI-g Service is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Accurate genotyping.|Uniform yield of high-molecular-weight DNA.|Schematic representation of REPLI-g amplification.|Detailed quality assessment report.|Highly representative amplification.|Consistent and accurate whole genome amplification.|
Twenty DNA samples amplified using REPLI-g technology, without subsequent DNA purification, were subjected to genotyping analysis using 3 STR loci (CSF1PO, TPOX, and THOI). Results were compared with those obtained for unamplified genomic DNA. The DNA was separated by polyacrylamide gel electrophoresis, and visualized by silver staining. A lane with one band represents a homozygote, while a lane with two bands represents a heterozygote for the specific STR locus.|Agarose gel (1%) electrophoresis of 40 DNA samples amplified using REPLI-g whole genome amplification and visualized by SYBR® Green staining. The average product length is typically greater than 10 kb.|DNA polymerase moves along the DNA template strand displacing the complementary strand. The displaced strand becomes a template for replication, enabling high yields of high-molecular-weight DNA to be generated.|A detailed quality assessment report providing [A] an overview of sample DNA quality with predicted success of downstream applications and [B] a breakdown of the quality of each amplified sample, including the amount of gDNA received and the amplification yield. Row H is used for in-house quality assessment using control DNA.|The relative representation of eight loci was determined using real-time quantitative PCR for DNA amplified using [A] REPLI-g technology, [B] DOP-PCR, and [C] PEP. Locus representation was determined by comparison to 1 µg of unamplified control DNA. (Dean, F.B. et al. (2002). Proc. Natl. Acad. Sci. U.S.A. 99, 5261. © 2002 National Academy of Sciences, U.S.A.) 
|Real-time PCR was performed on 47 human loci (2 loci on each autosomal pair, 2 loci on the X chromosome(s), and 1 locus on the Y chromosome) from 44 different samples amplified using REPLI-g technology. Each sample was amplified approximately 10,000-fold with a maximum bias of representation between the loci being only 6-fold.|
Performance

Whole genome amplification may be achieved by two different methods: PCR-based amplification or multiple displacement amplification (MDA). PCR-based methods can generate nonspecific amplification artifacts, give incomplete coverage of loci, and generate DNA less than 1 kb long that cannot be used in many downstream applications. In contrast, REPLI-g using MDA provides highly uniform amplification across the entire genome, with minimal sequence bias. See figures “Highly representative amplification” and “Consistent and accurate whole genome amplification”. 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 length of the REPLI-g amplified DNA enables complex restriction enzyme analysis and long-range PCR reactions.

 

Principle

REPLI-g technology provides highly uniform DNA amplification across the entire genome, with minimal sequence bias. The method is based on Multiple Displacement Amplification (MDA) technology (see figure "Schematic representation of REPLI-g amplification"), which carries out isothermal genome amplification utilizing a uniquely processive DNA polymerase capable of replicating 100 kb without dissociating from the genomic DNA template. The DNA polymerase has a 3'5' exonuclease proofreading activity to maintain high fidelity during replication and is used in the presence of exonuclease-resistant primers to achieve high yields of DNA product. The amplified DNA can be directly used in a variety of downstream applications, including, genotyping (e.g., SNP, STR [see figure “Accurate genotyping”], microarray analysis), end-point PCR, quantitative real-time PCR, and sequencing. 

 

Procedure

Ideal results are obtained using 100 ng starting template. However, a uniform concentration of amplified DNA is usually achieved regardless of the quantity of template DNA. Sample material is lysed and the DNA is denatured by adding lysis buffer (heavily degraded genomic DNA may not serve as a good template for amplification). This gentle alkaline denaturation allows uniform amplification across the whole genome with minimal sequence bias. After neutralization, a master mix-containing buffer (including exonuclease resistant primers, DNA polymerase, and distilled water) is added. The isothermal amplification reaction proceeds overnight at 30°C. This simple and robust method is capable of accurate amplification of entire genomes.

Following DNA amplification, sequence bias is assessed using a proprietary assay of specific loci that are highly sensitive to template DNA quality. A stringent quality control assay provides information on the quality of the amplified DNA, enabling reliable predictions for the success of your downstream assay to be made (see figure “Detailed quality assessment report”).


Applications

The performance of REPLI-g amplified genomic DNA has been validated for a variety of downstream applications, including:

  • Genotyping (e.g., SNP, STR, microarray)
  • End-point PCR, quantitative real-time PCR
  • Sequence analysis 
Feature
Specifications
Amplification Whole genomic DNA
Applications Genotyping, hybridization, RFLP
Quality assessment Yes
Samples per run; throughput Mid, high
Starting amount of DNA 100–500 ng genomic DNA
Starting material Genomic DNA, blood, cells, buffy coat
Technology Multiple Displacement Amplification (MDA)

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