プライマー伸長キャプチャ：重縮退DNAソースからターゲットシーケンスの取得

This video explains primer extension capture or PECA technique that allows the targeted retrieval of DNA sequences from extremely low copy number highly fragmented DNA sources. We have used this technique to sequence complete mitochondrial genomes from neanderthal bones over 40, 000 years old, and the method can be used for capture of any small target regions or single nucleotide polymorphisms and from many individuals. The main steps in this procedure are PCR amplification of a primary DNA library, quantification of the amplified library by quantitative PCR target primary extension on the amplified library bead capture of primary extension products to remove non-target fragments and elution and sequencing of the captured and wash targets.

Hello, I'm Adrian Briggs from Shanti Payable's Lab in the Department of Genetics at the Max Plank Institute for Evolutionary Anthropology in Leipzig. Today we're going to show you a procedure for isolating specific DNA sequences of interest from a complex highly degraded DNA source such as ancient bones. We have used this procedure to study the mitochondrial genetic diversity of Homo Neal lenses or Neanderthal man.

Let's get started. The DNA used in our experiment comes from Neanderthal bones between 40, 000 and 70, 000 years old, which were found at fore sites across the Neanderthal geographic range. A bone is first exposed overnight to UV light to decontaminate the surface of any extraneous DNA.

A fine drill is used to produce between 100 and 500 milligrams of bone powder DNA is extracted from the bone powder using a procedure described in the 2007 article comparison and optimization of ancient DNA extraction by Nadine Rockland and Michael Hof.Writer. The DNA extract is then converted to a sequencing library by the addition of universal adapter priming sites to the end of all DNA fragments because a 4 5 4 library generated from a Neanderthal bone contains only tiny amounts of DNA, sometimes just a few picograms. This primary DNA library is first amplified by PCR using emulsion PCR primers corresponding to the universal library primers.

To prepare the PCR mix for library amplification, combine the following reagents in A PCR tube, use 10 microliters of 10 X GAMP PCR buffer, two 10 microliters of magnesium chloride, two microliters of BSA one microliter of DNTP mix three microliters each of 4 54 MPCR forward and reverse primers, one microliter of amply gold DNA polymerase 25 microliters of library template. And finally, add 45 microliters of PCR grade water to yield a total volume of 100 microliters amply. Tech gold DNA polymerase is stable at room temperature, so it's not necessary to work on ice.

After preparing the PCR reaction. Place the PCR tube in a thermocycler starting with a denaturation step of 95 degrees Celsius for 12 minutes, followed by 14 cycles of 95 degrees Celsius for 30 seconds, 60 degrees Celsius for one minute and 72 degrees Celsius for one minute. At the end of these 14 cycles, set a 72 degrees Celsius five minute final step after which the PCR tube can be set indefinitely on 10 degrees Celsius until ready to use after PCR amplification is complete, followed with purification of the PCR product using a Kyogen MinLu spin column according to manufacturer's instructions, elute DNA from the column with 50 microliters of EB buffer supplied with the MinLu kit before primary extension and product capture.

Quantitative PCR should be performed on aliquots of the unamplified and amplified libraries in order to quantify and check the quality of amplified library product. The 14 cycle amplified library should contain several thousand times more DNA than the primary library. If it does not, amplification conditions should be checked and repeated.

Primary extension capture primers, which will be used to capture the sequences of interest from the amplified library, should be designed like normal PCR primers to match a desired target region with an ideal melting temperature of 60 degrees Celsius to the five prime end of each of these target sequences at this 12 base tail sequence and include a biotin residue at the five prime end up to 150 PEC primers can be used together in a single multiplex mix made from 200 micromolar stocks. For the primary extension reaction, prepare a PCR mix in a suitable PCR tube by adding five microliters of 10 X GAMP PCR buffer two five microliters of magnesium chloride one microliter of BSA 0.5 microliters of DNTP. Mix five microliters of the primary extension capture primer mix 0.5 microliters of tack gold DNA polymerase, two microliters of the 14 cycle amplified library DNA, and finally 31 microliters of PCR grade water to yield a total volume of 50 microliters.

If the amplified library template contains more than one times 10 to the 12 copies per microliter, the template should first be diluted with EB buffer so that not more than two times 10 to the 12 copies are added to the reaction mix. After preparing the PCR tube, place it in the thermocycler and set it for a single reaction run. Starting with a 12 minute denaturation at 95 degrees Celsius, followed by 60 degrees Celsius for one minute, five minutes at 72 degrees Celsius for primary extension.

And when the single cycle is finished, 72 degrees Celsius to hold the PCR product until the next step. In order to avoid non-specific primer kneeling and capture directly PEPPE 150 microliters of PBI or PB buffer from Kyogen Ute purification kit into the PCR reaction tubes, while the reaction mixture remains in the thermocycler held at 72 degrees Celsius. Doing this, inactivates the polymerase while the temperature is too high for non-specific extension of the target primers, close the tubes vortex briefly and then spin the liquid down with a benchtop centrifuge.

Use only PBI or PB buffer in the step as other binding buffers like Qiagen PM Buffer can interfere with the downstream capture process. Next, remove the mix from the PCR tube and transfer to a Rogen MinLu spin column for purification according to manufacturer's instructions as previously done elute DNA from the column with 30 microliters of EB buffer supplied with a MinLu kit. In order to remove all fragments that have not a kneeled to PEC primers, a bead based capture step is performed.

First, resus suspend a stock solution of M 270 strip divide in beads by vortexing and remove an amount of beads equivalent to 25 microliters per sample. Wash the beads twice with 500 microliters of two times bew buffer and resuspend the same volume of two times BW as the volume of beads originally taken. Prepare 25 microliters of the suspension.

For each sample add 25 microliters of the purified eluted DNA to 25 microliters of the bead suspension, saving the remaining five microliters for later quantification. Mix the DNA and bead suspension by pipetting up and down and stirring with the perpet tip and then rotate slowly on a benchtop rotator for 15 minutes at room temperature. After mixing, transfer the contents to a fresh 1.5 milliliter PCR tube.

During the previous rotation incubation step, some non-target DNA may have become stuck to the tube wall. So transferring the bead suspension to a new tube avoids the possibility of this non-target DNA being released into the final products. During the heat elution step, using the magnetic particle collector pellet the beads, after 10 to 20 seconds, all of the beads will have migrated to the wall, leaving a clear SUP natant that should be removed to wash the beads, add 500 microliters of one times BW buffer to the tube and vortex for about three seconds at slow speed.

Once with the tube upright and again with the tube inverted, no beads should remain sticking to the tube wall. Using a tabletop centrifuge, spin down the suspension for one to three seconds, just long enough to draw all liquid from the walls of the tube. Return the tube to the MPC and wait 10 to 20 seconds until all the beads are collected at the side of the tube.

Then remove the SUP natant. The first wash is now complete. Repeat this procedure of further four times following the last one times BW wash and supernatant removal.

Add 500 microliters of one times hot wash buffer. Mix the suspension by briefly vortexing and spinning down as before, and then shake the tube for two minutes at 65 degrees Celsius on a thermal block shaker at 1000 RPM following a brief spin removal traces of the supernatant within one minute of removal from a thermal block. This step will remove fragments that may be binding to PEC primers, but were not extended on in the primer extension step resuspend the pellet in 30 microliters of EB buffer and transfer the mix to a fresh 1.5 milliliter PCR tube.

Incubate the tube in the thermal cycler at 95 degrees Celsius for three minutes. Pellet the beads in the magnetic particle collector. It should take 10 to 20 seconds for the supernatant to clear completely and remove the S supernatant to a fresh labeled tube.

Taking care not to transfer any beads. Keep the supernatant, which contains the captured product of DNA fragments. The beads can be discarded at this stage.

To amplify the captured product, prepare a PCR mix to a total volume of 100 microliters as done in part two and perform the same PCR cycling conditions. Again, after PCR amplification of the captured product is complete, follow it with purification using a Qiagen Ute spin column as previously done in section two elute DNA from the column with 50 microliters of Eeb Buffer, the product is now ready for high throughput sequencing. We designed 574 PEC primers to tile across the whole Neanderthal mitochondrial genome.

We split these into four multiplex mixes and two serial rounds of primary extension capture on libraries from five Neanderthal fossils from four sites across Eurasia that are dated between 40, 000 and 70, 000 years old, generating between 170, 000 and 500, 000 sequences per individual and aligning the results to a mitochondrial reference sequence. We found that the PEC procedure had massively enriched for mitochondrial DNA achieving complete coverage across the mitochondrial DNA for each individual at an average of between 18 and 56.3 fold coverage. With these results, we could generate a high quality complete mitochondrial genome sequence for each individual.

We then performed a phylogenetic comparison of these sequences with the worldwide sample of present day humans and our closest living relatives, the chimpanzee and bonneau. This confirmed previous findings that Neanderthal mitochondrial DNA is closely related, but clearly distinct from that of modern humans. Furthermore, comparison between the Neanderthal sequences themselves revealed extremely low genetic diversity in Neanderthals lower than among humans worldwide today, and similar to present day Europeans.

This evidence combined with an analysis of protein evolution in the Neanderthal sequences suggest that Neanderthals had a long-term low population size, which is consistent with archeological evidence, suggesting that Neanderthal population density never reached the levels achieved by modern humans. We've just shown you how to perform primate extension capture to amplify and capture target sequences in preparation for high throughput sequencing. When following this procedure, it's important to follow all the temperature specifications on the thermocycler, but be aware that primers can have different eeling temperatures, so you may have to adjust the eeling temperature to suit your particular primers and take care during all the washing steps to remove as much liquid as possible.

So thank you for watching and good luck with your experiments.