Description
The REPLI-g Mitochondrial DNA Kit enables selective amplification of mitochondrial DNA from total DNA samples, without the need for prior mitochondrial DNA isolation. This kit provides DNA polymerase, buffers, and reagents for specific and uniform whole genome amplification from small samples of human and non-human mitochondrial DNA in total DNA samples. For amplification of non-human mtDNA, the REPLI-g Human mt Primer Mix needs to be substituted with an appropriate primer mix suitable for that species (not provided with the kit). The REPLI-g Mitochondrial DNA Kit is based on the multiple displacement amplification technology (MDA), that 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.
Total DNA preparations typically comprise approximately 0.1% of mitochondrial DNA (e.g., 0.1 ng of total human DNA contains 0.1 pg of mitochondrial DNA). The REPLI-g Mitochondrial DNA Kit provides specific amplification of the whole human mitochondrial genome, and yields approximately 4 µg of amplified mitochondrial DNA per reaction. This corresponds to an enrichment of mitochondrial DNA of up to 40 million-fold, depending on the starting amount (see figure "Enrichment of mitochondrial DNA"). Residual amounts of amplified genomic DNA are minimized and do not interfere with mitochondrial-specific downstream methods such as qPCR or direct Sanger sequencing.
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. 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 processive 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 with PCR-based amplification methods.
Amplification of mitochondrial DNA using the REPLI-g Mitochondrial DNA Kit involves just two basic steps (see flowchart "Purified mitochondrial DNA procedure"). First, the total human DNA sample is denatured by incubation in REPLI-g mt Reaction Buffer and REPLI-g Human mt Primer Mix for 5 minutes at 75°C and then cooled to stop the denaturation. However, to denature DNA sample from non-human species, the REPLI-g Human mt Primer Mix should be substituted with the appropriate primer mix for that species (not included in the kit). Next, REPLI-g DNA Polymerase is added and the isothermal amplification reaction proceeds for 8 hours at 33°C.
