REPLI-g Screening Kit

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).

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.

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").

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