Extracting good quality mtDNA from human hair is tough. It took me months of testing tens of different protocols, wash concentrations and methods to be able to get clean extractions with low amounts of contamination. Hair is super tough, due to the keratin fibers that exist there, and to get mtDNA out, you actually have to break it down (the mtDNA circles are trapped inside). The DNA part is actually pretty easy (just get the hair at the root). It’s a meticulous process, and you need a LOT of starting material. MtDNA exists in such small quantities that you need quite a bit to even get 1 ng of it. Subsequently, if you’re doing any next-gen sequencing, using a library preparation kit that works with low input amounts (i.e Nextera) will be our best bet. I created a small protocol to help with the wash and extraction procedure. The protocol is adapted from this paper (Protocol adapted from : Assessing Deep Sequencing Technology for Human Forensic Mitochondrial DNA Analysis, Mark R. Wilson. Ph.D.)
Obtain 2cm of hair shaft (get a few of these pieces). For 1ng need around 100+ 2cm pieces.
Make 5% terg-a-zyme mixture (i.e: 2g in 40 mL)
suspend hair in 5% terg-a-zyme (1 mL) in an eppendorf tube, place in incubator at 37˚C , 900 rpm for 20 minutes.
Remove the 1 mL of 5% terg-a-zyme and add 1 mL of nuclease-free H2O, incubate for 10 minutes. Flick the tube every 2-3 minutes.
Remove 1mL of H2O, and add 100% ethanol. Incubate for 10 minutes. Flick the tube every 2-3 minutes.
Remove the 100% ethanol and let the hair dry. Use a P10 or P20 to remove any remaining drops. All ethanol must be removed.
Place clean hair fragment/s in a solution of 300 uL Qiagen Buffer ATL, 20 uL 0.6 U/uL proteinase K (12U of prot. K) and 40 uL of 0.5M DTT. 6 ul of 50mg/ml proteinase K. for 100 hair pieces, add 600 uL buffer atl, 12 ul of proteinase K and 80 uL of 0.5M DTT.
Digest for 1-2 hour at 56˚C, 900 rpm on a thermal shaker.
Add 300 uL of Qiagen Buffer AL to the digestion solution and incubate at 70˚C, 900 rpm for 10 minutes. Add 600 uL for 100 hair pieces.
4)Proceed to the magnetic bead step (addition of 15 uL prepfiler magnetic particles) in the Prepfiler forensic DNA extraction Protocol.PrepFiler Kit
Vortex the PrepFiler® Magnetic Particles tube for approximately 5 seconds, invert the tube to confirm that no visible pellet remains in the bottom of the tube, then centrifuge briefly. For multiple samples, vortex the magnetic particles tube every 5 minutes during the next step.
Pipet 15 µL of magnetic particles into the tube containing the sample lysate.
Cap the sample lysate tube, vortex it at low speed (approximately 500 – 1200 rpm) for 10 seconds, then centrifuge it briefly.
After centrifuging, add 180 µL of isopropanol to the sample lysate tube
Cap the sample lysate tube, vortex it at low speed (approximately 500 – 1200 rpm) for 5 seconds, then centrifuge it briefly
Put the sample lysate tube in a shaker or on a vortexer, then mix at room temperature at 1000 rpm for 10 minutes
7)Vortex the sample DNA tube
*If magnetic particles are present on the sides of the sample DNA tube above the meniscus, invert the tube to resuspend the particles.
*Vortex the sample DNA tube at maximum speed (approximately 10,000 rpm) for 10 seconds, then centrifuge briefly.
Note: It is acceptable to have magnetic particle aggregates suspended in the solution or on the side of the tube below the meniscus
10) Place the sample DNA tube in the magnetic stand and wait until the size of the pellet of magnetic particles on the back of the tube stops increasing (approximately 1 – 2 minutes). 11) With the sample DNA tube remaining in the magnetic stand, use a pipette to carefully remove and discard all visible liquid phases. Do not aspirate magnetic particles or disturb the magnetic particle pellet. You can use a size P200 or P1000 pipettor to remove most of the liquid, then use a size P20 pipettor to remove the remaining liquid.
With the sample DNA tube remaining in the magnetic stand, open the tube, then allow the magnetic particles-bound DNA to air-dry for 7–10 minutes (air-drying for more than 10 minutes may reduce DNA yield). If the room temperature is >25°C, reduce drying time to 5 minutes.
Bring the thermal shaker temperature to 70°C.
Add 50 µL of PrepFiler® Elution Buffer to the sample DNA tube. Do not use water. You can use low-TE buffer.
Cap the sample DNA tube, vortex it at maximum speed (approximately 10,000 rpm) until there is no visible magnetic particle pellet on the side of the tube (approximately 5 seconds), then centrifuge it briefly
Place the sample DNA tube in a thermal shaker (or heat block), then incubate at 70°C and 900 rpm for 5 minutes. If you use a heat block, briefly vortex and centrifuge the tube every 2 – 3 minutes.
Vortex the sample DNA tube at maximum speed (approximately 10,000 rpm) until there is no visible magnetic particle pellet on the side of the tube (approximately 2 seconds), then centrifuge briefly
Place the sample DNA tube in the magnetic stand, then wait until the size of the pellet at the side of the tube stops increasing (at least 1 minute).
Pipet the liquid in the sample DNA tube (which contains the isolated genomic DNA) to a new spin tube or 1.5-mL microcentrifuge tube for storage. Do not aspirate magnetic particles or disturb the magnetic particle pellet.
If the eluted DNA extract is turbid, centrifuge the tube for 5 – 7 minutes at maximum speed (approximately 10,000 rpm), then transfer the clear supernatant to a new 1.5-mL microcentrifuge tube.
The isolated DNA can be stored at 4°C for up to one week, or at –20°C for longer storage.
Check the sample using mitochondrial genome selecting primers and ones for some NUMT sequence. That is, run a PCR reaction with a set of mitochondrial primers on one amount, and nuclear primers on another amount. The nuclear primer is to check for nuclear DNA contamination, and the mtDNA set is to see if you have mtDNA Present.
Lane 1: Optimized Qiagen/Prepfiler extraction on hairsample1 (20 2cm pieces); nuclearprimer1 and mtprimer1 30 cycles, loaded 9uL x2 on gel Lane 2: Optimized Qiagen/Prepfiler extraction on hairsample1 (100 2cm pieces); nuclearprimer1 and mtprimer1 30 cycles, loaded 9uL x2 on gel Lane 3: PCR negative control nuclearprimer1 and mtprimer1 30 cycles, loaded 9uL x2 on gel
Note: The lower band is the nuclear primer and the upper band is the mtDNA one, the lowermost band is just runoff primers.