Hi!
We are sequencing exon 5-6 from the BEST1 gene, we have multiple patients afected, with the explaining mutation, but in this cohort we found that in our forward sequences we can identify positively the mutation, but not in the reverse sequence.
Yes, we sequenced them multiple times, with alternative PCR products, primers and looked for pseudogenes regions, but no answer to this phenomena is found.
somebody has some insights?
Do your primers map to any repetitive regions or ones with hair pin secondary structures?
I suspect that your reverse primer is sitting with its 3' end covering a SNP. So under pcr conditions we get both alleles amplifying and in the case of a heterozygote the forward primer anneals to both alleles and will sequence both alleles for the known mutation you are looking for. Possibly under the sequencing conditions the mismatch of the reverse primer to the second snp lying under the reverse primer means that only one allele sequences because the other allele has both snps and this means that you are really sequencing a hemizygote. You can get round the problem by using a reverse sequencing primer inside of your current one . A better result would be to move your reverse primer further out by 50 bases or more then you should be able to sequence both the expected base change and the second snp which may be of interest . Also it might be worth checking for snp at dbsnp to see if any are already known in this region
Hi Paul, always a pleasure to read you, we also checked for SNP´s on both primers and didn´t find any, so it seems that this is not the problem...
About the hairpins and secodnary structures; as you can see:
gaggctgag gcaggaggat cacttgagcc cagcagttcc aggctgcagt 61956600 gcgctaagat cgcaccgctg cactccaacc tcggtgacag agccagaccc 61956650 tttctctgga aataaataaa taccctgccc acatgctcag cccagaacag 61956700 cacctagtag gtgctcagaa atttttttgt tgttgaaaga aagaggatgg 61956750 caaaggagtg ctgaggttcc tataggtcag caggtgccgg ccatcccttc 61956800 tgcaggttct cccacccacc gccttcttca ctccactctg cagGCTTTAT 61956850 GACTCCGGCA GAACACAAGC AGTTGGAGAA ACTGAGCCTA CCACACAACA 61956900 TGTTCTGGGT GCCCTGGGTG TGGTTTGCCA ACCTGTCAAT GAAGGCGTGG 61956950 CTTGGAGGTC GAATCCGGGA CCCTATCCTG CTCCAGAGCC TGCTGAACgt 61957000 gagcccactg tacagacagg gctgccgcag agtgggaagg gctgtggtcc 61957050 acaggaaaca aggtttccta caaagagaag ccttgggccc ctgagggtct 61957100 tccgagagcc tgaggtgggg ttgcagaatc ttttccaaca gcaatccaca 61957150 gcccgaggtg gtcccttctc agaggcccct ccctcttctc caagtctgtg 61957200 aggtcctggt tcccttttga tagatgagga agctgagaca caaagaggtt 61957250 tagtgagctt cccatggcca cacagccagg aatggaccat aggtaccagg 61957300 ccctggtacc tggagaagag gtgggggcga gcccagggtg ggggcaggtg 61957350 gtgttcagaa ccccatcccc ctcttctgcc ccccagGAGA TGAACACCTT 61957400 GCGTACTCAG TGTGGACACC TGTATGCCTA CGACTGGATT AGTATCCCAC 61957450 TGGTGTATAC ACAGgtgagg actaggctgg tgaggctgcc cttttgggaa 61957500 actgaggcta gaaggaccaa ggaagcagct ggggtgggaa gggctcacct 61957550 agaggctaag tggctcccct gggagttggg tccacacttt gaagttgggt 61957600 ctggactttg aagtgccaag ttctaagagt ccaggctcct gcctggccca 61957650 gtccagtaga ggcaatgtga ttatccccat attaaagaga ggttggccgg 61957700 gtgcagtggc tcatgcctgt aatcccagca ctttgggaag ctgaggcagg 61957750 tggatcacct gaggtcagga gttcgagacc agcctggcca acatggtgaa 61957800 accccatctc tactgaaaat acagaattag ctgtgtggtg gtgcacgcct 61957850 gtaatcccag ctacttggga ggctgaggca ggagaatcgc ttgaacccgg 61957900 gaggtggagg ttgcagtgag ctgagatcat gccactgcac tccagcctgg 61957950 gcgacacagc aagactctgt ctcaaacaaa caaacaaaca aacaaacaaa 61958000 ggggttaaca gagcccctaa gtcacataag tgtgcaagtc agaacaaggc 61958050 cttggtctcc tgtctcagac tcccagcccc tggagcatcc tgatttcagg 61958100 gttcccacct agccctttgc taccacatcc tcctcctcct cccagGTGGT 61958150 GACTGTGGCG GTGTACAGCT TCTTCCTGAC TTGTCTAGTT GGGCGGCAGT 61958200 TTCTGAACCC AGCCAAGGCC TACCCTGGCC ATGAGCTGGA CCTCGTTGTG 61958250 CCCGTCTTCA CGTTCCTGCA GTTCTTCTTC TATGTTGGCT GGCTGAAGgt 61958300 gggcctctcc agggccctgc tgggctggag gcatggccag aggggtcatg 61958350
As you can see there´s plenty of possibilities for secondary structures on this amplicon so the answer is "probably yes".
thanks for your answers and your time.
Thank you Oscar but even dbsnp may be of little value if this is an isolated or bottlenecked cohort in which case there is a possibility that this is a very localised variation in linkage disequilibrium with the proposed snp. If you are thinking that secondary structure is and issue you can ensure better pcr amplification by cycling in 1M betaine and up to 8%dmso either individually or together and run the reverse sequence reaction also in either dmso or betaine. If it is a secondary structure issue I would expect that the intensity of the electropherogram peaks would drop suddenly at the blockage. This might not be immediately obvious as the analysis software tends to pull up the peak heights for better visualisation but can be checked in Snaapgene or ABIs free base calling software
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