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REPLI-g Mitochondrial DNA Kit

For highly uniform whole genome amplification from human and non-human mitochondrial DNA
  • Sensitive amplification from varying sample types
  • Suitable for amplification of human and non-human mtDNA
  • Overcomes the need for time-consuming isolation of mtDNA
  • Informative results from samples with degraded nuclear DNA
  • DNA amplification from blood cards and hair

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.

Cat No./ID: 151023
REPLI-g Mitochondrial DNA Kit (25)
€251.00
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DNA Polymerase, Buffers, and Reagents for 25 x 50 μl mitochondrial DNA specific whole genome amplification reactions
The REPLI-g Mitochondrial DNA 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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Enrichment of mitochondrial DNA.
0.1 ng total DNA (representing approximately 15 cells) containing approximately 100 fg of mitochondrial DNA was amplified using the REPLI-g Mitochondrial DNA Kit. The total yield of mitochondrial DNA after amplification was up to 4 µg, corresponding to a 4 x 107-fold increase in mitochondrial DNA.
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Purified mitochondrial DNA procedure.

Amplification of mitochondrial DNA using the REPLI-g Mitochondrial DNA Kit involves two basic steps. First, total DNA sample is denatured at 75°C in REPLI-g mt Reaction Buffer, then cooled. Next, REPLI-g DNA Polymerase is added for the isothermal amplification reaction.

Performance
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.
Principle

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.

Procedure
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.
Applications

REPLI-g amplified mitochondrial DNA can be used in a variety of downstream applications, including:

  • Genotyping (e.g., SNP, deletions, insertions)
  • Endpoint-PCR, quantitative real-time PCR 
  • Sequence analysis
Features
Specifications
Amplification Whole genomic DNA
Applications Genotyping, sequencing, RFLP
Denaturation step Heat
Maximum input volume 10 µl template DNA
Minimal pipetting volume needed 1 µl
Quality assessment No
Reaction time ~8 hours (overnight)
Reaction volume 50 µl
Samples per run; throughput Mid
Starting amount of DNA ~10 ng purified total DNA
Starting material Genomic human DNA
Technology Multiple Displacement Amplification (MDA)
Yield ~4 µg

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Handbooks & Protocols (1)
For specific whole genome amplification of human mitochondria from total DNA samples
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Kit Handbooks (2)
For specific whole genome amplification of human mitochondria from total DNA samples
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