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Probe design:
==Probe design==
===Column synthesized probes (36)===
*[[Kun:LabNotes/CpgSeq/2007-10-12]]: probe = revcomp(RP) + linker + FP
*[[Kun:LabNotes/CpgSeq/2007-10-12]]: probe = revcomp(RP) + linker + FP
*Just realized that there was a mistake in probe design (11-20-2007). I assumed that after bisulfite treatment, the two strands are still complementary, which is not true. The probes have to be designed based on one strand only. If the forward PCR primers contain no "C" and the reverse primers contain no "G", that means the target strand is the forward strand. In that case, the padlock probes should be: revcomp(FP) + linker + RP. Here are the revised perl scripts: [[Media: CpG_primer2padlock_v2.txt | first 24 primers]]; [[Media: CpG_primer2padlock2_v2.txt | second 12 primers]]. Here is the new [[Media: CpG_probes_v2.txt | probe file]].
*Just realized that there was a mistake in probe design (11-20-2007). I assumed that after bisulfite treatment, the two strands are still complementary, which is not true. The probes have to be designed based on one strand only. If the forward PCR primers contain no "C" and the reverse primers contain no "G", that means the target strand is the forward strand. In that case, the padlock probes should be: revcomp(FP) + linker + RP. Here are the revised perl scripts: [[Media: CpG_primer2padlock_v2.txt | first 24 primers]]; [[Media: CpG_primer2padlock2_v2.txt | second 12 primers]]. Here is the new [[Media: CpG_probes_v2.txt | probe file]].
===Agilent probes, V1===
*Several considerations in this design include:
**Degenerate bases: I allowed at most two degenerate bases per primer. To convert PCR primers to padlock probes, multiple probes will be designed for one primer pair in order to cover all combinations.
**Gap size: The range of amplicon size is 175-225bp, so the gap size is approximately 135-185bp.
**Amplification adaptors and linker sequences: I will use the same design as the one for exonome probes.
    AP1: TTGGGTCATATCGGTCACTGTT
    AP2: GATCAGGATACACACTACCCGTG
    Linker: GTTGGAGGCTCATCGTTCCTATTCAGCTGCAGATGTTATCGAGGTCCGAC
            ------                  ----              ------
            Mme I                    Alu I              Mme I
   
**Conversion of PCR primers to padlock probes: AP1 + revcomp(FP) + linker + RP + AP2.
**Here is the [[Media: getCpGPrimers.txt |perl code for primer design]]. The input file is cpgIslandExt.txt downloaded from UCSC GoldenPath database.

Revision as of 19:30, 12 December 2007

<calendar> name=Kun:LabNotes/CpgSeq format=%name/%year-%month-%day date=2007/11/01 view=threemonths </calendar>


Probe design

Column synthesized probes (36)

  • Kun:LabNotes/CpgSeq/2007-10-12: probe = revcomp(RP) + linker + FP
  • Just realized that there was a mistake in probe design (11-20-2007). I assumed that after bisulfite treatment, the two strands are still complementary, which is not true. The probes have to be designed based on one strand only. If the forward PCR primers contain no "C" and the reverse primers contain no "G", that means the target strand is the forward strand. In that case, the padlock probes should be: revcomp(FP) + linker + RP. Here are the revised perl scripts: first 24 primers; second 12 primers. Here is the new probe file.

Agilent probes, V1

  • Several considerations in this design include:
    • Degenerate bases: I allowed at most two degenerate bases per primer. To convert PCR primers to padlock probes, multiple probes will be designed for one primer pair in order to cover all combinations.
    • Gap size: The range of amplicon size is 175-225bp, so the gap size is approximately 135-185bp.
    • Amplification adaptors and linker sequences: I will use the same design as the one for exonome probes.
   AP1: TTGGGTCATATCGGTCACTGTT
   AP2: GATCAGGATACACACTACCCGTG 
   Linker: GTTGGAGGCTCATCGTTCCTATTCAGCTGCAGATGTTATCGAGGTCCGAC
           ------                   ----               ------
           Mme I                    Alu I               Mme I
   
    • Conversion of PCR primers to padlock probes: AP1 + revcomp(FP) + linker + RP + AP2.
    • Here is the perl code for primer design. The input file is cpgIslandExt.txt downloaded from UCSC GoldenPath database.