Hi, I am unable to find data on how single nucleotide insertion or deletion-like mismatch inside a primer affects PCR. I would be interested even in anecdotal empirical evidence. Have you ever used such primer ? What was the position of insertion or deletion from the 3 end ? Did you get amplification ? If the format was real-time PCR, how much was Cq delayed ? If you never worked with such primer, but saw it in an article, can you give me a reference ? (I want to emphasize, that I am not asking about substitution-like mismatches, I have a lot of literature on those).
Subquestion: how short can a primer be, if it contains insertion or deletion mismatch in the middle ?
How come you are unable to find any information? Here are two hits from doing simple google search for "mismatch primers"
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797725/
http://genome.cshlp.org/content/3/4/S39.full.pdf+html
As I say, I am interested in single nucleotide insertions and deletions (like deleting or inserting a nucleotide), NOT single nucleotide substitutions (NOT like replacing A with G etc). The first link you give is virtually all about substitutions. I knew about this work before, I mention in my question I have a lot of literature on substitutions.
The second link, Kwok et al. 1994 does, indeed, mention insertions and deletions, but incredibly briefly. (I saw this one before, too.) He says they can be put into mutagenesis primers. Then he says generally about point mutation, that they should be put in the center of 24- to 36-base oligonucleotide. Does not give citations on who used it for deletions or insertions.
I would like something more specific about insertions and deletions. Or empirical examples.
I used a primer with 2 deletion in the middle of that and finally I had sufficient PCR product with good Sequencing results, but unfortunately I don't have same experience in real- time.
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