For reliable whole genome amplification from human mitochondrial DNA in total DNA samples
The REPLI-g Mitochondrial DNA Kit provides specific and uniform whole genome amplification from human mitochondrial DNA in total DNA samples. The simple procedure enriches for mitochondrial DNA with minimal contamination from nuclear DNA, thus avoiding the need for time-consuming isolation of mitochondrial DNA and increasing the sensitivity of downstream assays.
Benefits of the REPLI-g Mitochondrial DNA Kit include:
High sensitivity of downstream assays due to enrichment of mitochondrial DNA
Accurate and reproducible results even from small total DNA samples
Versatility suitable for human DNA isolated from various starting materials, including hair
Fast and easy protocol with all reagents included in the kit
Working with mitochondrial DNA
One limitation to the use of mitochondrial DNA has been the need to isolate it from nuclear DNA, particularly in cases where the sensitivity of mitochondrial DNA marker assays needs to be increased. The isolation procedure involves many time-consuming steps and leads to substantial losses of mitochondrial DNA. The REPLI-g Mitochondrial DNA Kit overcomes this limitation by enriching for mitochondrial DNA sequences in total DNA samples.
High fidelity mitochondrial specific amplification
REPLI-g Mitochondrial DNA technology provides fast and highly uniform DNA amplification across the entire mitochondrial genome. The method is based on Multiple Displacement Amplification (MDA) technology, which carries out isothermal genome amplification utilizing a uniquely process ive DNA polymerase. Due to its high processivity and strand displacement activity, REPLI-g DNA Polymerase is capable of amplifying up to 100 kb without dissociating from the DNA template and minimizes unequal sequence and locus representation compared to PCR-based amplification methods.
Simple one-tube procedure
Amplification of mitochondrial DNA using the REPLI-g Mitochondrial DNA Kit involves just two basic steps (see flowchart). First, the total DNA sample is denatured by incubation in REPLI-g mt Reaction Buffer for 5 minutes at 75C then cooled to stop the denaturation. Next, REPLI-g DNA Polymerase is added and the isothermal amplification reaction proceeds for 8 hours at 33C.
High yields and enrichment of mitochondrial DNA
Total DNA preparations typically comprise approximately 0.1% of mitochondrial DNA (e.g., 1 ng of total human DNA contains 1 pg of mitochondrial DNA). The REPLI-g Mitochondrial DNA Kit provides specific amplification of the whole human mitochondrial genome and yields approximately 4 g of high-purity amplified mitochondrial DNA per reaction. This corresponds to an enrichment of mitochondrial DNA of up to 40 million fold, depending on the starting amount (Figure 1).
REPLI-g amplified mitochondrial DNA can be used in a variety of downstream applications, including:
SNP and STR genotyping
RFLP and Southern blot analysis
Visit www.qiagen.com/goto/REPLI-g to discover the benefits of REPLI-g technology today!
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