Is Melanin a PCR Inhibitor?

Question

I have been growing B16F10 Mouse Melanoma cells. I need to extract the genomic DNA and do PCR to amplify a specific region. However, no matter what temperature or magnesium concentration I use, I have no luck.

I obtained mouse DNA from someone who was genotyping mice. I tested the PCR oligos with that DNA, and the reaction worked, so it's not an oligo problem.

The genomic DNA had poor 260/230, about 0.6. I thought the EDTA levels may have been high, so I did an isopropanol precipitation to clean it up. This made the 260/230 ratios about 2.1. But the PCR still didn't work.

What I had noticed during the precipitation however was that the DNA pellets were gray to black. I assume this is melanin, because the cells are melanoma cells, and turn black.

Could this melanin be a problem in PCR?

Answer 1

Melanin is a potent inhibitor of PCR - when you use B16 cells (or any other cell line that produces melanin) you have to purify your sample from it. Unfortunately a simple Phenol-Chloroform extraction or an ethanol precipation won't do the magic, since melanin co-precipitates with the nucleic acids.

I recommend following the following paper: "CTAB–Urea Method Purifies RNA from Melanin for cDNA Microarray Analysis".

The protocol derived from the paper can also be found here.

Answer 2

I don't have much expertise in PCR (so please do not ask me technicalities). A research has proven that melanin is a PCR inhibitor as it forms complexes with thermostable DNA polymerase and inhibits its activity, thus inhibiting PCR. See this paragraph below:

We found that both RNA and cDNA preparations derived from melanocytes contain a RT-PCR inhibitor that copurified with nucleic acids. Investigation of the candidate inhibitor melanin revealed that it potently blocks PCR at concentrations below 200 ng/ml, whereas 100 microg/ml melanin was required to inhibit reverse transcription. Melanin and thermostable DNA polymerase preferentially formed a distinct complex with reduced migration velocity as compared to pure polymerase in nondenaturating polyacrylamide gel electrophoresis. Our findings demonstrate that melanin is a potent inhibitor of thermostable DNA polymerase in vitro and that the inhibitory effect is conferred by a direct and reversible polymerase-melanin interaction.

The same paper also gives a solution to your problem as:

The inhibition of the enzyme by melanin could be reversed by diluting solutions of preformed complexes or by adding excess amounts of other proteins such as bovine serum albumin or dry milk.

Reference: http://www.ncbi.nlm.nih.gov/pubmed/10814530

Answer 3

There are many suggestions how to avoid melanin-caused PCR inhibition. Some we have found helpfull (like using smaller amount of DNA sample + increasing number of cycles or adding BSA to PCR reaction), other (like trying different DNA isolation and PCR kits, purification columns etc.) are good only for profits of biotech companies. The solution is in fact very simple: using proper procedure for DNA isolation. Rutinely we prepare DNA using high-salt lysis buffer (250 mM EDTA, pH 8,5, 1% SDS and 100 ug/ml proteaseK) and, after incubating at 50°C, single extraction step with phenol/chloroform, followed by precipitation with 1 volume of EtOH (not more, otherwise you precipitate EDTA). Under these conditions melanin remains with DNA. For samples containing melanin we include one extraction step with phenol only before phenol/chloroform extraction. Melanin gets extracted into water/phenol interphase and falls throught phenol during centrifugation; this does not happen when you extract with phenol/chlofoform mixture. For efficient melanin extraction the high salt is important. If you need to eliminate melanin from ready-made DNA samples, add LiCl to 2M concentration and extract with phenol.