I'm currently trying to amplify circularized DNA. Only a small sequence of this circularized DNA is known and was used for Primer design. The other part of the circularized DNA is unknown genomic DNA.
I tried to amplify this circular DNA with the aim to have one part of the known sequence on the left border and the other part of the known sequence on the right border of the genomic DNA. It is necessary to obtain these left and right borders for a following sequencing step.
Here arises the problem: I tried to amplify the DNA with Taq, but the 5'-3' exonuclease activity made it impossible to obtain the left and right borders. I also tried the same amplification with 5'-Biotin labelled Primer trusting that the Biotin stops the 5'-3' Exonuclease activity of Taq. But this was also not succesful. A third way I tried was an isothermal amplification with phi29 to obtain long repeating linear DNA, which I than used as template for a 2nd PCR with Taq. But somehow this idea also didn't work out. Has anyone of you some idea how I could modify the 5' end of the primer which stops the 5'-3' exonuclease activity of Taq. Or has anyone another idea how to get this linear DNA with the desired left and right border?
Thank you in advance
Hi Tobias,
I can think of 2-3 ways which may be feasible to apply here to resolve the issue
1) Is it possible to linearize (using restriction enzyme) the circular fragment in the region where sequence is known, linearising the moleculae followed by PCR may resolve this issue.
2) Use the primers containing extra non-complementary bases at the 5'end. These bases will not bind to the circular template (will keep on hanging) hence will not be accessible to the 5'-3' exonuclease activity of TAq polymerase
3) An alternative will be to design an Hairpin motif (using extra bases) at the 5'-end of the primers which will not restrict access of this end of the primer to the exonuclease activity. Such primers have been shown to work perfectily in PCR and are also known to enhance specificity.
see if the above suggestions are suitable to resolve your problem
bye
i suggest to retain to better results for example: the Use the extra 5-primers with non-complementary nucleotide that will not be accessible to the 5'-3' nuclease activity of polymerase
Thank you Ajay and Abbas for your suggestions.
Unfortunately I don't have a restriction site within this known sequence, so this will be no option.
Today I received some primers with non complementary nucleotides at the 5' end and hopefully this will solve my problem.
I will keep you informed whether or not we're successful.
If the two primers that you are using have a substantial overlap--you don't say how much that complementary overlap is--then isn't it possible that they don't work simply because they anneal with each other and not the template plasmid? And if most of your primers are in this duplex form, they can't serve as primers for extension on any template. If this type of primer complementarity is the problem, you might think about how to use single primer reactions to generate enough template to sequence. Is it possible to isolate enough of the circular plasmid for a sequencing reaction?
does rolling circle amplification work on this template or can you design 2 primers facing away from each other that do not overlap. These should then generate a normal pcr product and if you are not getting a product than try a longer extension time if this is an inverse pcr and you do not yet know the size of the particular circle ( of many) that you are trying to amplify
Just to keep you updated.
The usage of primers with 5'-overlaps with non-complementary nucleotide and Q5 polymerases solved the problem. Thank you all for your suggestions!
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