I'm having difficulty finding a possible solution to my problem: I have a plasmid encoding a protein. In the nucleotide sequence of this protein, I need to add a nucleotide sequence (highlighted in yellow) at a specific position (blue arrow). The problem is that downstream of that specific position there is a sequence that is identical to the motif of the DNA fragment to be added (the motif is CCGCCGTACCCGCCG and I've underlined it in black). As a result, when I try to generate primers containing the sequence to be added at their 5' end, they all have a high risk of annealing at the wrong position, a high risk of forming secondary structures, a high CG percentage, and consequently a high Tm. How would you suggest to proceed?
Assuming that you do not want to get the whole sequence synthesised then if your 15 base motif is represented by S15 could you get a primer synthesised sequence s15,s15,s15then 25 bases specific to your template atthe 3' end ( fplc or hplc purified on synthesis). Then choose a high annealing temp reverseprimer ( the rules for primer design are more flexible if the template genome is small as in plasmid dna. There is a high risk of secondary structure as you say so the pcr should be done in 8%dmso and a final concentration of 1Molar betaine
Paul Rutland Sorry, I did not clearly understand the first part of your answer. Did you suggest designing primers containing the motif CCGCCGTACCCGCCG 3 times at their 5' end followed by 25 bp of the vector sequence?
Frw: 5' - CCGCCGTACCCGCCGCCGCCGTACCCGCCGCCGCCGTACCCGCCGNNNNNNNNNNNNNNNNNNNNNNNNN - 3'
Rev: 5' - NNNNNNNNNNNNNNNNNNNNNNNNNCGGCGGGTACGGCGGCGGCGGGTACGGCGGCGGCGGGTACGGCGG - 3'
If yes, I obtain 70bp long primers with a very high Tm temperature (98 C) and 64% GC. I fear the Tm is too high.
Yes that is what I am suggesting. Tm calculations do not work at very high GC or length but any temperature below the Tm should give a pcr product so long as the secondary structure does not interfere too much with the binding which is why dmso and betaine are used. In the first pcr cycle the entire length of the primer does not anneal because the sequence does not exist completely until after cycle 2. These primers do not look any worse than GC tailed DGGE primers which are also high Gc but still seem to work well
- Badges
- Science topic
- Similar topics
- Filing