BSP in repetitive region - (Nov/11/2005 )
I am trying to amplify a fragment of DNA that is in a highly repetitive region of the genome. I had originally attempted to design nested primers, but have now moved to hemi-nested due to the lack of suitable primer sites. The region, in addition to being highly repetitive, is very CpG-rich. The entire stretch I'm looking at is about 1.5Kb, so I divided it into 3 regions/primer sets- I have been able to get product for one of these regions using a temperature gradient, but the product size is different in each lane. I get the expected size (450bp) at the lowest temp, then increasingly smaller products as temperature increases. Each lane also has a laddering effect. I'm thinking that this is due to the primer laying down at different repeats, and then the shortest fragment is preferentially amplified.
Does anyone have any suggestions how to get around this? I'm not sure if the solution lies in the PCR itself, or if I might have to digest the gDNA into individual repeats before bisulfite treatment. Any suggestions would be much appreciated.
I am interested to hear how you would be able to determine that the methylation status from your results are in fact the repeat element you are wanting to look at and not something in some other region of the genome?
This is something we are trying to get around also. with great difficulty. Unless you are able to select primers in unique sequence regions that span into your repeat, it is very very challenging!
Thanks for writing back last time. I'm still struggling with PCR in the terminal repeat area. I tried a touchdown PCR protocol, but it only seemed to eliminate the banding, not give any real products. I've read a couple papers suggesting that the difficulties in amplifying these repetitive regions may arise from strand reannealing, and that various conditions (i.e. urea, 95C modification, etc.) may help. I attached a picture of my gel this time to at least show you what I've been seeing. I'm trying to amplify ~500 bp products, but am seeing much smaller bands. The top row is first round, the third row is second (please ignore the 2nd and 4th rows). I'm using a 90s extension step- is this not long enough to preferentially amplify longer products?
Thanks in advance
the small bands you are encountering seems to look like possible primer dimer formation, is there are negative control lane on your gel?
The strand annealing problem can be derived from incomplete bisulfite treatment, as your DNA must be single stranded for modification to occur, the first elements to reanneal would be your repetitive elements, if your primers were designed to a specific element, I assue it's a SINE or LINE and are very abundant within the genome, you should get a product regardless of the conversion efficency.
I am not too sure if you are performing MSP or BSP however because of their abundance you should be getting a product of expected size, based on this, I suspect your primer design may not be optimal and you should look at them again.