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Kun:LabNotes/CpgSeq/2007-10-12
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===Fri.Exp.Design: Design padlock probes to capture CpG islands=== I'm starting from the primers used in Taylor et al. Cancer Research, 2007, 67:8511-8. To convert these sequencing into padlock probes, I need to do the followning: *Remove the barcodes at the end of the primers; *Convert the right primers to their reverse complementary sequences, add Gs to the end so that the total length of the forward and reverse sequences are the same for all targets; *Add common linkers to the two capturing arms. *It turned out that the padlock probes are pretty long, getting 24 probes cost > $1000. Decided to make 10 shorter ones only. I also manually remove 4 bps from the PCDGHA12 primers to make them shorter. *Synthesizing 10 padlock probes in tubes are not cheap either. After confirm the discount pricing with IDT, I decided to get 24 probes in the plate format. *[[Media:PMID17875690_supp_table3.xls | Starting primer file]] and [[Media: PMID17875690_CpG_primers.txt| the actual input file.]] *[[Media:CpG_primer2padlock.txt | Perl script to convert primers to padlocks.]] *[[Media:CpG_padlock_set1.txt | The first set of padlock probes.]] *Actually the IDT discount price is still quite expensive. I decided to design additional 12 probes to meet the minimal plate requirement at Bioneer. I received another batch of 48 primer pairs from Huidong Shi. The format is slightly different. I designed twelve 85 mers based on these primer pairs. *[[Media: huidong_48_primers.txt| The input file.]] *[[Media:CpG_primer2padlock2.txt | Perl script to convert primers to padlocks.]] *[[Media:CpG_padlock_set2.txt | Additional 12 padlock probes.]] *I've received a reference DNA from Huidong Shi, and the EpiTect Bisulfite kit from Qiagen. I also ordered Jurkat DNA from NEB. I think everything is ready to go as soon as the padlock probes come in.
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