Different sequencing result of PCR products from the same cDNA - (May/24/2007 )
Hi guys, I need to PCR amplify a gene from Sea Urchin cDNA for protein expression. This gene is ~1.9kb, GC%~41%. I sequenced the PCR products directly several weeks ago, and sent another tube of PCR products (from the same cDNA library from the same tube, but different enzyme, Ex Taq vs Phusion) for sequencing this week, but the results are quite different: though their size are the same, only 94% bases are identical. I think these two sequencing result might come from polymorphism, I searched the net and one paper said: "The sea-urchin genome is highly polymorphic, meaning that the two copies of the genome in the diploid organism vary from each other by about 4 percent, or one difference in spelling every 25 letters."
However, neither of the chromotogram of these two sequencing result shows double peaks (I think if it were polymorphism, then two copies of the diploid chromosome should have equal chance to be amplified). All the places where polymorphism occur only perfect single peak. I cannot interpret this result. I need this gene fragment to be ligated to another fragment, but now I am at a loss which fragment to use.
Does anyone think it possible that, during first cycles in PCR, only 1 copy in the diploid chromosomes have the priority to be amplified, and the other copy is not amplified at all (so that the PCR sequencing result only reveal one peak)?
The differences in the two PCR reactions are the emzyme (Ex Taq, phusion) and buffers used, different denaturing temperature (94 or 98?C), different extension time (2 min, 45s), and different reaction set up date. The cDNA are the same. Can different enzyme lead to different chromosome copy to use?
The above two are the only reasons I can think out so far, but they don't make sense to me either. I am thinking hard where else could be wrond.. Can someone help me solve this problem? thanks!
However, neither of the chromotogram of these two sequencing result shows double peaks (I think if it were polymorphism, then two copies of the diploid chromosome should have equal chance to be amplified). All the places where polymorphism occur only perfect single peak. I cannot interpret this result. I need this gene fragment to be ligated to another fragment, but now I am at a loss which fragment to use.
Does anyone think it possible that, during first cycles in PCR, only 1 copy in the diploid chromosomes have the priority to be amplified, and the other copy is not amplified at all (so that the PCR sequencing result only reveal one peak)?
The differences in the two PCR reactions are the emzyme (Ex Taq, phusion) and buffers used, different denaturing temperature (94 or 98?C), different extension time (2 min, 45s), and different reaction set up date. The cDNA are the same. Can different enzyme lead to different chromosome copy to use?
The above two are the only reasons I can think out so far, but they don't make sense to me either. I am thinking hard where else could be wrond.. Can someone help me solve this problem? thanks!
It is indeed strange that you don't see double peaks. It looks that the two reactions each have their own preference.
Are there major changes in the protein if you translate the DNA?
Because you could present both the fragments in a ligation reaction and then it will choose its preferred fragment itself. But off course you need to be sure then that both fragments will work as being the protein.
However, neither of the chromotogram of these two sequencing result shows double peaks (I think if it were polymorphism, then two copies of the diploid chromosome should have equal chance to be amplified). All the places where polymorphism occur only perfect single peak. I cannot interpret this result. I need this gene fragment to be ligated to another fragment, but now I am at a loss which fragment to use.
Does anyone think it possible that, during first cycles in PCR, only 1 copy in the diploid chromosomes have the priority to be amplified, and the other copy is not amplified at all (so that the PCR sequencing result only reveal one peak)?
The differences in the two PCR reactions are the emzyme (Ex Taq, phusion) and buffers used, different denaturing temperature (94 or 98?C), different extension time (2 min, 45s), and different reaction set up date. The cDNA are the same. Can different enzyme lead to different chromosome copy to use?
The above two are the only reasons I can think out so far, but they don't make sense to me either. I am thinking hard where else could be wrond.. Can someone help me solve this problem? thanks!
It is indeed strange that you don't see double peaks. It looks that the two reactions each have their own preference.
Are there major changes in the protein if you translate the DNA?
Because you could present both the fragments in a ligation reaction and then it will choose its preferred fragment itself. But off course you need to be sure then that both fragments will work as being the protein.
the translated proteins in most polymorphism places have been changed. I cannot test the protein expression because it is not a whole gene, only part of it...