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REPLI-g Screening Kit

For high-throughput manual or automated whole genome amplification from small or precious samples
  • Process 96 samples with just 20 minutes of handling time
  • Parallel WGA of multiple samples
  • Highly-stable DNA suitable for long-term storage
  • Room temperature setup allows easy liquid handling
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). The REPLI‑g Screening Kit provides 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, blood, and cultured cells. The streamlined, room-temperature procedure enables straightforward liquid handling that requires just 20 minutes. The kit is designed for rapid mutation screening using a range of genotyping, PCR, sequencing, or microarray assays. Amplified DNA can be used directly in most downstream applications without purification or quantification.
Cat No./ID: 150126
REPLI-g Screening Kit (200)
$2,166.00
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For 200 x 40 µl whole genome amplification reactions:
2 x 100 µl REPLI-g Mini DNA Polymerase, 2 x 1.7 ml
REPLI-g Buffer SB1, 2 x 1.7 ml REPLI-g Buffer SB2
The REPLI-g Screening 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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REPLI-g screening procedure.
1
Uniform yield of high-molecular-weight DNA.

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.

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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
Typical DNA yields from a REPLI-g Screening Kit reaction are up to 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).
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 Screening Kit technology delivers highly uniform DNA amplification across the entire genome with minimal sequence bias and is combined with a straightforward and fast procedure to enable processing of multiple samples. 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. Amplified DNA can be used directly without purification or quantification in most downstream applications, including genotyping (see figure Reliable SNP genotyping), end-point PCR, quantitative real-time PCR, and sequencing.
Procedure
The very fast, simple, and accurate method is capable of parallel amplification of several genomic samples, as decribed 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").
Applications

REPLI-g amplified DNA has been successfully used on all common genotyping platforms, including high-throughput genotyping platforms such as:

  • Genotyping (e.g., SNP, deletions, insertions)
  • End-point PCR, quantitative real-time PCR
  • Sequence analysis
  • Microarray
Features
Specifications
Amplification Whole genomic DNA
Applications Genotyping, sequencing
Denaturation step Heat
Handling time for 96 samples null
Maximum input volume 10 ng DNA, 0.5 µl whole blood, >600 cells/µl
Minimal pipetting volume needed 1 µl
Quality assessment No
Reaction time 10–16 hours (overnight)
Reaction volume 40 µl
Samples per run; throughput High (1–96 samples)
Starting amount of DNA >10 ng purified genomic DNA
Starting material Genomic DNA, blood, cells
Technology Multiple Displacement Amplification (MDA)
Yield ~8 µg

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Kit Handbooks (1)
For whole genome amplification from purified genomic DNA, blood, and cells
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