We are using REPLI-G kit method to extract DNA from COVID-19 infected human host cells to isolate mt-DNA of patients, to ensure that the isolation is pure and doesn't contain contaminants of nuclear DNA, what is the best approach that can be utilized ? Studies suggest differential centrifugation and affinity purification of the organelle as a preferred method. How efficient is differential centrifugation for mt DNA isolation ?
To enhance the purity of mtDNA isolation and minimize contamination with nuclear DNA, additional purification steps such as affinity purification or gradient centrifugation can be incorporated into the protocol. These methods specifically target mitochondria or mtDNA, further improving the purity of the isolated material.
It's essential to validate the efficiency and purity of mtDNA isolation using techniques such as PCR, qPCR, or sequencing to ensure that the isolated DNA meets the desired quality standards for downstream applications. Additionally, comparing the results obtained using differential centrifugation with other isolation methods can help determine the most suitable approach for your specific experimental requirements.
Hello Shazveen Saleem
The gold standard of mitochondrial DNA enrichment is through density gradient centrifugation. If you have an ultracentrifuge, you may load total DNA onto a caesium chloride density gradient and centrifuge for 10 hours at 450,000 x g to separate DNA by size.
The gradient is sufficient to separate types of DNA with slight differences in density due to differing GC content, or physical form (e.g., linear versus circular molecules). Two distinct bands, corresponding to sheared linear nuclear DNA above and circular mitochondrial DNA below, are visible under ultraviolet light. You may insert a needle into the tube at the appropriate location and remove the lower band enriched in mitochondrial DNA.
If you do not have an ultracentrifuge, you may use the method provided below which will give you circular double stranded mitochondrial DNA substantially free of genomic DNA.
(Protocol in brief):
1) Lyse cells to form a lysate,
2) Incubate said lysate with an amount of Proteinase K effective to digest proteins in said lysate,
3) Digest any RNA in said lysate,
4) Add a protein-precipitation reagent to said lysate to obtain precipitate and a supernatant,
5) Precipitate total DNA in said supernatant and form a pellet,
6) Suspend the supernatant pellet in a buffer,
7) Incubate the suspended pellet at about 70°C for about 30 minutes to form a solution,
8) Incubate the solution with an amount of an exonuclease for a time-effective to cleave non-circular double stranded DNA and obtain circular double stranded mitochondrial DNA,
9) Incubate said circular, double stranded mitochondrial DNA with an amount of Ampure beads effective to bind said circular double stranded mitochondrial DNA,
10) Wash said beads with ethanol and
11) Elute said mitochondrial DNA from the said beads.
For a detailed protocol, you may want to refer to the link below.
https://patents.google.com/patent/WO2014071178A1/en
There is also another approach to enrich mitochondrial DNA. The SageHLS system from Sage Science offers a more straightforward and automated method for the enrichment of mitochondrial DNA.
In this system, 1-1.5 million whole cells are loaded onto a proprietary agarose gel cassette alongside lysis buffer, which is transferred into the sample well by electrophoresis. Once cell lysis has occurred, the cell contents move through the agarose by electrophoresis. Cellular material, including proteins, RNA, and lipids, move rapidly through the agarose. Nuclear DNA, which remains mostly of chromosomal length, is retained in the sample well, while the smaller mitochondrial DNA migrates into the gel, allowing for their separation. Electro-elution is then used to recover the mitochondrial DNA.
This is a novel approach to mitochondrial DNA isolation which provides enrichment levels comparable to gradient centrifugation. You may want to refer to the link below for more information on SageHLS gel cassette for mitochondrial DNA extraction.
https://sagescience.com/targeted-mitochondrial-dna-extraction-and-enrichment-using-the-sagehls/
Best.
- Badges
- Science topic
- Similar topics
- Agricultural Science
- Agronomy
- Crop Science
- Crop