The overall goal of the following experiment is to discover and identify long range associated DNA partners. This is achieved by fixation, cell lysis and digestion of DNA by the first restriction enzyme followed by ligation to create a chimeric DNA composed of segments from the two associated genes. Next linkers are added to the chimeric DNA after the second restriction digestion To facilitate its amplification, PCR amplification is then performed using linker and bait DNA specific primers in order to obtain sufficient amounts of the associated DNA fragments Results are obtained that show the long range associated DNA partners with the bait DNA based on sequence analysis of the amplified DNA fragments.
The main advantage of this technique over existing methods like chromosome confirmation capture or three C, is that A-C-T-S-A can be used to identify a unknown long range associated DNA partner effectively. Whereas three C arose for verification of only previously suspected interactions. To begin this protocol culture, human cells in an incubator supplied with 5%carbon dioxide at 37 degrees Celsius until they're 80 to 90%confluent.
Human prom acidic leukemia cells are used for this demonstration. Collect the cells by pouring them into a 50 milliliter NOC tube and centrifuge at 1200 RPM for 15 minutes. Following centrifugation, remove the medium by aspiration.
Add five milliliters of culture medium to resuspend the cell pellets. After counting the cells with a hemo cytometer, take approximately one times 10 to the seventh cells to a volume of 40 milliliters with RPMI 1640, medium containing 10%FBS. Then add 1.7 milliliters of 37%formaldehyde to fix the dilute chromatin.
Incubate the sample at room temperature for 10 minutes with gentle shaking. Following incubation, quench the reaction with 2.4 milliliters of two molar glycine. Next, centrifuge the sample for 15 minutes at 1200 RPM and four degrees celsius.
After removing the super natin, wash the pellet once with 40 milliliters of ice cold PBS then centrifuge the sample to retrieve the washed cell pellet resuspend the resulting pellet in 40 milliliters of ice cold lysis buffer with freshly added protease inhibitors and 0.1 millimolar PMSF incubate the sample in a cold room with rotation for 90 minutes. Finally, centrifuge the sample at 2, 500 RPM for 15 minutes and remove the super natin and obtain the isolated nuclei. Re suspend the nuclei in 0.5 milliliters of one XNEB buffer three and add 15 microliters of 10%SDS.
Incubate the sample at 37 degrees Celsius for one hour with shaking. Then add 45 microliters of 20%Triton X 100 to sequester the SDS and incubate again at 37 degrees Celsius for one hour with shaking and aliquot of one times 10 to the sixth. The nuclei or about 15 micrograms is used for restriction enzyme digestion to 55 microliters of nuclei solution.
Add 433 microliters of one XNEB buffer three and 12 microliters of BGL two. To make a total digestion reaction volume of 500 liters, incubate the reaction at 37 degrees Celsius overnight. The next day inactivate the restriction enzyme by adding 95 microliters of 10%SDS and denature the enzyme by heating at 65 degrees Celsius for 20 minutes.
In a water bath, add seven milliliters of one x ligation buffer and 360 microliters of 20%Triton X 100. Incubate the sample at 37 degrees Celsius for one hour. Lower the temperature to 16 degrees Celsius and add 50 microliters of 400 units per microliter.
T four DNA Ligase. Allow the ligation reaction to incubate at 16 degrees Celsius for four hours and then at room temperature for 30 minutes. After the ligation is complete, add 300 micrograms of proteinase K and incubate at 65 degrees Celsius overnight.
Then add five micrograms of RNAA and incubate at 37 degrees Celsius for 30 minutes. Purify DNA by phenol chloroform extraction and precipitate DNA in isopropanol dissolve the precipitated DNA in 150 microliters of sterile distilled water. To add linkers to the chimeric DNA, first digest the DNA by incubating two micrograms of purified DNA with five units of msp, one at 37 degrees Celsius for four to six hours following digestion, inactivate MSP one at 65 degrees Celsius for 10 minutes.
Then precipitate the DNA in ethanol with one microliter of five milligrams per milliliter. Glycogen dissolve the resulting DNA palate in 50 microliters of sterile distilled water. Next mix 50 microliters of MSP one treated DNA with two microliters of a 20 micromolar linker oligonucleotide L and one microliter of a 20 micromolar oligonucleotide S then add one microliter of sterile distilled water and six microliters of 10 XT four DNA Ligase buffer.
Cover the mixture with liquid wax. Denature the oligonucleotides at 50 degrees Celsius for one minute and allow to cool down gradually to 10 degrees Celsius in a 0.5 degrees Celsius per minute gradient. Using a thermal cycler, add one microliter of 400 units per microliter T four DNA ligase and incubate the reaction at 15 degrees Celsius overnight.
The next day purify the linker ligated DNA using a kaya quick PCR purification kit and elute in 50 microliters of sterile distilled water. The choose a primer for the specific region of DNA to be investigated for long range interactions. In this example, Abel will be used as the primer or the bait.
Prepare the purified DNA for the first round PCR by combining one microliter of the purified DNA with one microliter of 20 micromolar able one specific primer, 46 56, and one microliter of 20 micromolar linker specific primer 29 63. Then add three microliters of three x ClinTech, DNA polymerase one cocktail that has been previously mixed with 32 P-D-C-T-P according to the written protocol, followed by three microliters of sterile distilled water. Perform PCR amplification using the hot start PCR thermal cycle described in the written protocol after the PCR cycle is complete.
Purify the first round PCR products using a kaya quick PCR purification kit Elute in 30 microliters of sterile distilled water. Use one microliter of the first PCR product diluted 100 times to perform a second round of PCR using nested primers by adding one microliter of 20 micromolar able one specific primer, 46 26, and one microliter of 20 micromolar linker specific primer. 29 61.
Again, add three microliters of three x ClinTech, DNA polymerase, one cocktail and three microliters of sterile distilled water. Execute the second round of PCR using a thermal cycling schedule of 25 cycles of 95 degrees Celsius for 20 seconds. 67 degrees Celsius for 40 seconds and 72 degrees Celsius for one minute, followed by an extension at 72 degrees Celsius for five minutes.
Visualize the PCR products by running a 5%urea page gel and then scanning the exposed screen in a phospho imager. Each PCR band can be recycled from the gel by dissolving the gel strips in an einor tube containing 60 microliters of sterile distilled water and incubating at 95 degrees Celsius for five minutes.Centrifuge. The dissolved gel products briefly at 10, 000 RPM for 10 seconds.
To collect all the samples. Remove one microliter to use as the DNA template for performing PCR with the prime repair. 29 61 46 26.
Using the same conditions as described above, after purification and sequencing analysis can be performed with an online tool to determine chromosomal location. Once at the genome bioinformatics site, click blatt to generate a new window and paste the DNA sequence. After submitting the DNA sequence, the BLATT search results will appear.
Click browser on the hit with 100%identity to reveal the location of the DNA sequence. The able one region was used as bait to determine its long range DNA interactions. Two BGL two sites and one BAM H one site were chosen for the A CT assay in the second round of PCR primer set, 46 26 29 61 was used to amplify able M1 46.30 29 61 was used for able M two and 46, 36 29 61 was used for able M three.
A typical gel pattern reveals one to several bands as is shown here for able M1, able M two, and able M three here. The DNA sequence of clone able M1 is shown to demonstrate that each fragment from an A CT assay consists of two combined DNA segments. One segment is derived from the bait DNA region while the second segment comes from the associated partner.
The segments are joined to each other by the first restriction enzyme recognition sequence. The second enzyme recognition sequence will appear at the end of the associated partner sequence. The cloned able M1 fragment contains DNA from the region of ABLE one located at chromosome nine Q 32.4, and the associated partner is located at chromosome three P three, which was identified as proc two.
Similarly, the able M two associated partner was localized to chromosome five Q 21.1 while clone able M three was identified as an intra chromosomal association near the ABLE one locus in contrast to three C assays. The methodology outlined in this video will select for the most prevalent long range interactions. However, by increasing the number of PCR R cycles, it is possible to identify additional less frequent associations as well using the imprinting and control region or ICR at the IGF two H 19 locus as bait DNA in mouse fiberblast cells, different cycling programs were applied in the first and second rounds of PCR in the A CT assay.
The A CT assay can also be used to identify differences in nuclear architecture and long range interactions between normal cells and cancer cells. Normal colon tissue and colon cancer tissue was homogenized and the A CT assay was performed following the procedures described herein. Shown here is the DNA structure of the ICR region at IGF two H 19 locus and the IGF two gene dpn.
Two sites for the A CT assay are labeled primers used in the second round PCR are labeled by arrows and numbers in different colors. The gel pattern of the A CT assay represents the PCR results using primer 29 61 with either primer pair 41, 61, 41 63, 51 45, and 51 51. Unique bands that appear only in normal colon tissue are labeled by yellow arrows, and those bands that appeared only in colon cancer tissue are labeled in red arrows After its development.
This technique pave the way for researchers in the field of market biology to explore nuclear architecture and gene regulation in the three dimensional manner.
