The overall goal of this technique is to systematically investigate the genome-wide DNA methylation landscape in large patient cohorts. This method can help answer key questions in the epigenetic field, especially with regards to which regions in the DNA methylation landscape get deregulated in patients. The main advantage of this technique is large number of the patient can be investigated in a cost effective manner.
This technology can have positive implications into the therapy and diagnosis of the patient because we can investigate existing biomarkers and also identify novel biomarkers. Though this method can provide insight into patient cohort, it can also be applied to other systems such as DNA methylation in cells derived from cultured cell lines from mouse-derived samples. Demonstrating this procedure along with Dr.Ya-Ting Hsu will be Mr.Joseph Liu, a technician in our lab.
The sequencing part will be demonstrated by Ms.Dawn Garcia who is a technician in our sequencing core. After isolating genomic DNA from tumor or normal tissue according to the text protocol, add 1.2 micrograms of total genomic DNA to 96 microliters of TE buffer and 24 microliters of 5X bind wash buffer to make a 120 microliter solution. Sonicate the DNA solution for 30 seconds, then allow it to rest for 30 seconds.
Repeat the sonication a total of 20 to 25 times. Use a bioanalyzer system with a commercial high-sensitivity DNA kit according to the manufacturer's protocol to run one microliter of sonicated DNA solution to check the size of the fragments. They should be in the range of 150 to 350 base pairs.
To carry out single fraction elution, prepare elution buffer by making a one-to-one solution of low-salt buffer and high-salt buffer. Use 200 microliters of the elution buffer to resuspend the methyl-binding DNA or MBD beads and incubate the suspension on a rotating mixer for three minutes. After the incubation, place the tube on the magnetic rack for one minute, then carefully pipette the liquid without touching the tip of the pipette on the beads and transfer it to a clean DNase-free 1.5 milliliter microcentrifuge tube.
Store this sample on ice. Repeat the elution for the second sample and collect it in the same tube as the first sample. To each non-captured wash and dilution fraction, add one microliter of glycogen, one-tenth the volume of three molar sodium acetate, pH 5.2, and two sample volumes of 100%ethanol.
After mixing the solution well, incubate at negative 80 degrees Celsius for at least two hours. Then centrifuge the reaction at 11, 363 times g and four degrees Celsius for 15 minutes. Carefully discard the supernatant without disturbing the pellet and add 500 microliters of cold 70%ethanol.
Spin the tube again before repeating the ethanol wash. Air dry the pellet for five minutes. Then use DNase-free water or buffer to resuspend it.
Check that the total amount of DNA is over 20 nanograms according to the text protocol. Then place the DNA on ice or store at negative 20 degrees Celsius until further use. For DNA sequencing library preparation, use a fully automated library construction system and follow the standard protocol supplied by the manufacturer.
Select DNA fragments of 200 to 400 base pairs in length for library preparation. After preparing the DNA sequencing library, remove the reaction and use a fluorometric quantitation system to quantify the DNA sequencing library. To set up a PCR amplification reaction, in a PCR tube add 15 microliters of DNA sequencing library, 25 microliters of PCR master mix, two microliters of PCR primer mix, and eight microliters of water.
Use the program outlined in the text protocol, making adjustments as necessary based on the recommendations of the PCR mix manufacturer. Carry out enough cycles to produce two to 10 millimolar of library DNA. After cleaning up the PCR reactions, barcode each sample and pull four samples together into one lane of a flow cell.
Use the standard 50 base pair single-read protocol of our sequencing. Carry out bioinformatics according to the text protocol. Using MBD cap seek to identify CpG islands across different genomic regions that are differentially methylated in tumor with respect to normal tissue, this study revealed that out of the total CpG islands located in gene promoters, 19.5%showed differential methylation in tumors compared to normal.
Similarly, 55.2%of intragenic CpG islands investigated showed differential methylation. Intragenic promoters showed 28.1%And for gene promoters without CpG islands, 1.8%showed differential methylation. This heat map clearly depicts differentially methylated regions across 77 breast tumors, 10 breast normal tissues, and 38 breast cancer cell lines.
In this figure, methylation is quantified in two endometrial cancer subgroups, non-recurrent vs. recurrent, in two patients at different CpG sites in the promoter CpG island of the identified target gene SFRP1. Once mastered, this technique can be done in four to five days if it is performed properly.
While attempting this procedure, it is important to remember to first test the quality of a genomic DNA being tested. Following this procedure, additional methods like pyrosequencing can be performed in order to answer questions like, which are the exact CpGs in the identified differentially enriched regions that have been altered? After its development, this technique paved the way for researchers in the field of epigenetics to explore DNA methylation in large patient cohorts.
After watching this video, you should have a good understanding of how to investigate genome-wide DNA methylation in patient samples using the MBD cap sequencing technique. Keep this in mind, sometimes it can be difficult to catch the high quality of the DNA from such as FFP and DNA samples, so proper assessment of the genomic DNA is always taken when we perform the procedure.
