진핵 세포에서 염색체 외 원형 DNA의 게놈 차원의 정화

Genome-wide purification of extrachromosomal circular DNA from Eurkaryotic cells. Hi, I'm Rasmus. Here I will present the whole outline for the method for circular DNA purification.

In the first part, yeast cells are cultured and harvested. Then circular DNA is purified and amplified in three steps. First, by column separation where DNA circles are purified and enriched.

Then, by treatment of DNA with exonuclease to digest all remaining linear DNA, and finally by amplification of DNA circles using the phi-29 polymerase. In the fifth part of this protocol, DNA circles are sequenced and reads are mapped to the Saccharomyces cerevisiae reference genome. In the end of this video, we'll show you some representative results.

We will discuss the perspectives for future studies and the advantages for using this method. Part one, cell harvesting. The starter culture is grown at 30 degrees with agitation to maximum density, which is approximately 200 million cells per milliliter and is reached after 24 to 48 hours.

The out grown culture is transferred to a Falcon tube and pelleted by centrifugation for three minutes. The supernatant is removed. The large pellet is vortexed in TE buffer and repelleted by centrifugation.

Part two, column purification. In this step, we use a commercially available column purification kit that yields highly purified plasmids from genomic bacterial DNA. Before using this kit with yeast, the cell wall needs to be disrupted.

This is done by resuspending the washed cell pellet in L-1 buffer from the kit. The cell suspension is transferred to a microcentrifuge tube and glass beads are added to one third of the total volume. The sample is vortexed at maximum speed for 10 minutes and beads are removed by centrifugation at 2, 000 revolutions per minute for 30 seconds.

The supernatant is transferred to a new tube. In this method we add highly diluted plasmids to the supernatant in three different ratios. We propose to include highly diluted plasmid to the cycle.

This can serve as internal reference guides. This you can use to quality check your DNA purification and amplification, as well, you can do a semi quantity estimation of the non-circular DNAs per cell using these spike in plasmids. Only highly diluted plasmids should be added since they will affect your sequence resolution of other circular DNAs.

The cell suspension containing spiked plasmids is treated with L-2 solution from the kit and the kit protocol is followed. After loading the column, proteins lipids and chromosomal DNA are removed by washing the column twice. Extra chromosomal circular DNA is highly enriched in the eluate fraction.

Part three, linear DNA digestion. In this step, we use an exonuclease to digest remaining linear DNA. An ATP dependent exonuclease specifically targets linear double stranded and single stranded DNA without digesting circular DNA.

The specificity of the exonuclease for linear DNA was confirmed by gel electrophoresis after 29 hours of DNase treatment. The results show that genomic DNA is digested while plasmid DNA remains as indicated by the red arrow. Yeast contains 16 chromosomes, up to 1, 500 kilobases in length.

So to facilitate exonuclease digestion, a rare cutting endonuclease is used to obtain an increased number of accessible DNA ends. If using the ate based restriction enzyme NotI, each chromosome is cleaved up to seven times as indicated. Column purified DNA is treated with NotI at 37 degrees in a heating block overnight.

After heat inactivation of NotI, the cleaved DNA is treated with exonucleases at 37 degrees according to the manufacturers protocol. After exonuclease treatment, the enzymes are heat inactivated at 70 degrees for 30 minutes. It can take several days to digest all linear chromosomal DNA by additional ATP and exonuclease To ensure that all linear DNA is completely digested, samples are analyzed by PCR or qPCR methods using an internal reference gene such as ACTI.

Part four, amplification of DNA. In this step, we enrich and amplify circular DNA using the phi-29 polymerase. The reaction takes place at 30 degrees using random hexamer oligonucleotides and phi-29.

The polymerase has high fidelity and proof reading activity, ensuring unbiased DNA amplification. Each polymerase can, by the rolling circle replication, synthesize up to 100 kilobases of DNA. The polymerase is also highly processive amplifying DNA up to 10 to the ninth fold.

Part five, sequencing. Following the protocol for a high throughput next generation sequencing platform, DNA is sonicated to an average size of 300 base pairs and sequenced as 141 nucleotide single end reads. Sequence reads are mapped to the Saccharomyces cerevisiae reference genome, using the Galaxy free database.

Sequenced data are uploaded to Galaxy and reads are mapped using the short read aligner Bowtie2, that allows mapping of partial reads. Part six, representative results. After sequencing and mapping to the Saccharomyces cerevisiae reference genome, reads can be visualized in the genome browser within the Galaxy software.

DNA of circular origin can, for instance, be counted as regions that contain coherent reads greater than one kilobase as indicated on the screen. An example of a region on chromosome four that contains coherent reads spanning greater than one kilobase is the region with the hexose transporter genes six and seven. When using this method on a population of yeast cells, mapped reads are frequently found at this locus and read coverage clearly suggests that a DNA circle formed between parralog hexose transporter genes.

Because of the high sequence homology between the hexose transporter genes, we propose that illegitimate homologous recombination leads to circularization of this DNA. In model for circular DNA mutation is when DNA repair process is run imperfectly. For instance, if a double strand break occurs in a repetitive element, then the homolog sequence on the same chromatid can be used as template for DNA repair by homologous recombination.

After resection, strand invasion, DNA synthesis, and resolution of all of the injunctions then a DNA circle can loop up from the genome causing an DNA deletion on the chromosome. The effects of treating DNA with exonucleases and phi-29 polymerase can be visualized using propidium iodide stain. Stain controls of genomic DNA appear as in the left image, while plasmid DNA appears as in the right image.

After 29 hours of exonuclease treatment 400 nanograms of genomic DNA, the majority of the DNA is digested as indicated in the left image. This result is confirmed by gel electrophoresis as shown on the gel image on the right. After 72 hours of exonuclease treatment followed by DA synthesis by the phi-29 polymerase, millions of copies of DNA can still be identified.

Likewise, after 29 hours of exonuclease treatment of 400 nanograms of genomic DNA, plus an additional 100 nanograms of plasmid DNA, the majority of the genomic DNA is digested. However, the plasmid DNA is not digested as indicated by the gel image. After phi-29 polymerase treatment of the 72 hour exonuclease treated sample, the majority of amplified DNA appears as stained plasmids.

So we first screened genes for circular DNA and we were really surprised to find several hundred different types of DNA samples. Yes, it was really surprised to find how common DNA circles are in the yeast population, and we believe that this method can be used for many types of studies. Obviously, one would be to look at human tissue.

We know that gene can be amplified on an acircular elements and yeast of course, and even tumor cells. I think in general it's going to be interesting to look at somatic cells and investigate what type of circular DNA we're going to find there and how we might this effect.Circularization. In aging cells we have circles that accumulate as we age and we expect more genes to be found on circular DNA.

And then additionally, we would like to look at the molecular mechanisms that DNA circularization and obvious thing to do is to look at yeast where we have mutants that are defective in homologous recombination and then nonhomologous In studying these mutants we expect that the DNA circularization profiled what different from the different types of mutants and hopefully from yeast studies we will be able to learn much more about DNA circularization. So compared to other methods, this method is much more sensitive. Might be actually able to find the DNA circle in approximately one out of 10, 000 cells.

And as far as I know from studies from the trial done at great length. Mutant alleles normally have to reach a proportion of one to 10%of the population before the can be detected. Right, and by this technique you don't have to have any prior knowledge of the DNA circles formed, unlike techniques Or the DNA circles have to be somewhat bonded and then you can easily detect them as amplifications by sequencing or by chromosome staining.

Yeah, so I guess really the main advantage is the sensitivity and I hope we can do a lot of interesting things with this method.