Hi All,
I am trying to test how my mutated protein affect the methylation of repeat sequences in genome (ex. LINE-1 and Alu). However, I found it quite hard to get the full length sequence of LINE-1 from NCBI, because it is not a gene.
I have found these following primer pairs in a paper:
LINE-1: forward: 5'-ATTTTATATTTGGTTTAGAGGG-3'
reverse: 5'-ATCAAAAATCAAAAACCCACTT-3'
Alu: forward: 5'-TTTGTAATTTTAGTACTTTGGGAGGT-3'
reverse: 5'-TTTAAAACRAAATCTCRCTCTATCRCCCAAAC-3'
(Source: http://www.nature.com/onc/journal/v26/n17/fig_tab/1210039f2.html#figure-title)
However, when I do a BLAST with those sequence, I cannot get a clear cut answer telling me that its targeting the right region, because those LINE-1 are interspersed between genes so only show up as chromatin region so and so..
Should I just trust the paper & order the primers?
If anyone can give me a link of full length LINE-1 or Alu sequence I would be super grateful!
Also, if any of you have sets of LINE-1 or Alu bisulfite sequencing primers that are proven to work great, could you please share them with me?
Thanks many!
Lyra
Found some patent application with bisulfite sequencing primers:
https://www.google.com/patents/WO2012097903A1?cl=en
If you download the repeatmasker (rmsk) table from the UCSC browser, you'll see that Alu and LINE repeats describe large families of repeats. They are also of variable sizes, with most copies only partial. They also differ in evolutionary age, with older repeat families having less CpG sites as they have been mutated over time. Methylated CpG is very mutagenic as after oxidative conversion to a thymine the base-excision DNA repair system doesn't know which is the damaged base to cut out.
My colleagues developed ES-PCR for detecting hypomethylation of repeat sequences. It's a very sound method. See: http://www.ncbi.nlm.nih.gov/pubmed/20964632
You might also find our review useful.
Article Hypomethylation of repeated DNA sequences in cancer
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