The overall goal of the following experiment is to analyze and quantitate five methyl cytosine and five hydroxyethyl cytosine within a specific locus of genomic DNA. This is achieved by gating the hydroxyl group of five Hydroxyethyl Aine, which converts a Cleavable MSP one site into a non cleavable site. As a second step, the genomic DNA is digested by the restriction enzymes, MSP one and HPA two, which utilize their differential methylation sensitivities to identify gluc glucose related five hydroxyethyl cytosine sites.
Next PCR is used in order to interrogate the locus. Results are obtained that show the presence of five hydroxyethyl cytosine in a specific locus. QPCR can be used to quantitate the amount of five methyl cytosine and five hydroxyethyl cytosine.
The primary advantage of this technique over other methods such as bi sulfite sequencing, is that you can discriminate between five methyl aine and five hydroxyethyl aine.Performing. The experiments today will be Romas Vice Villa Romas was a primary developer of this kid at New England Biolabs Prior to the start of this protocol purified the genomic DNA from B Sea brain tissue as described in the written procedure in a 1.5 milliliter reaction tube. Mix the genomic D-N-A-U-D-P glucose NEB buffer four and enough nuclease free water to bring the total volume to 310 microliters.
Split this reaction mixture into two tubes of 155 microliters each. Then add 30 units or three microliters of T four beta gluc cosal transferase to one tube. Mix well by gently pipetting up and down.
Do not add the T four BGT to the second tube as it is the control reaction, but be sure to add three microliters of water. Incubate both tubes at 37 degrees Celsius for 12 to 18 hours during which time the BGT will add glucose to the hydroxyl group of five hydroxyethyl cytosine groups in the sample. To begin the restriction enzyme digestion aliquot 50 microliters of the reaction mixture into each of three 0.2 milliliter PCR strip tubes labeled one through three.
Then aliquot 50 microliters of the control mixture into each of three PCR strip tubes labeled four through six. Add 100 units of msp, one into tubes one and four. Next, add 50 units of HPA two into tubes two and five and mix well by gently pipetting up and down.
Do not add anything to tubes three and six as they are controls. Incubate all six tubes at 37 degrees Celsius for at least four hours. Then add one microliter of proteinase K into each tube and incubate at 40 degrees Celsius for 30 minutes.
Finally, inactivate the proteinase K by incubating at 95 degrees Celsius for 10 minutes. This procedure is demonstrated using NE B'S long amp TAC for endpoint PCR. However, other PCR reagents and procedures can be substituted to a 0.2 milliliter PCR reaction tube on ice.
Add five x long amp TAC reaction buffer DN NTPs forward and reverse primers the previously obtained template, DNA long AMP TDNA polymerase and enough nuclease free water to achieve a final volume of 50 microliters. Gently mix the reaction if necessary, collect all liquid to the bottom of the tube by a quick spin. Transfer the PCR tubes from ice to a thermocycler with the block preheated to 94 degrees Celsius and start the cycling program for a routine three step PCR.
There should be one initial denaturation step at 94 degrees Celsius for 30 seconds, followed by 30 cycles of 94 degrees Celsius denaturation for 15 seconds, 55 to 65 degrees Celsius, a kneeling for 30 seconds and a 65 degrees Celsius extension for 20 seconds or 50 seconds per kb. This should be followed by one final extension at 65 degrees Celsius for five minutes. Realtime PCR can also be performed to quantitate amounts of five methyl cytosine and five hydroxyethyl toine.
Please refer to the product manual found on nb. com for specific information. A comparison of five hydroxyethyl aine amounts at Locus 12 was performed using endpoint PCR and real-time PCR four different mouse BSY tissue samples were analyzed including brain, liver, heart, and spleen.
The variation in five hydroxyethyl cytosine is apparent in the boxed gel lane of the endpoint PCR samples for comparative purposes. Real-time PCR data was normalized to uncut DNA and a standard curve was used to determine copy number. The samples were normalized by dividing the copy number of samples by the copy number of the undigested control.
Here, the variation in five hydroxyethyl cytosine can be seen by comparing the yellow bars, A few notes on performing the technique. It's critical that all the enzymatic reactions are carried to completion. For the BGT, it's important that you go overnight so that you can cocos isolate all the DNA sites for Ms.P one and HEPA two, it's important that you go at least four hours with those reactions to make sure that you reach completion.
And finally, we provide DNA controls and primer controls so that you can carry out control reactions side by side.
