primer RE over-hang nucleotides
Posted 28 December 2009 - 02:26 AM
Are there common or recommended nucleotide sequences for these over-hangs? or it depends on the individual and the GC content?
Posted 28 December 2009 - 04:17 AM
Posted 28 December 2009 - 10:02 AM
can you please elaborate more? How can an 1-8 mer over-hang cause hairpin? or how can I notice it and avoid it?
Posted 28 December 2009 - 10:34 AM
5'NNNNNNNNNNN ||||||| N 3'NNNNNNN
where the 3' end of the primer matches a portion of the primer leaving a 5' overhang. Extension of hte primer will occur (it will self-prime) destroying the primer.
Also you need to avoid this situation:
5' NNNNNNNNNNNNNNNNNNN 3' |||||||||| 3' NNNNNNNNNNNNNNNNNNNNNN 5'
where the two primers anneal to each other leaving a 5' overhang. These will again extend, destroying the primers.
For these to be a problem, there needs to be a 5' overhang and exact matches in the last few 3' bases of the hairpin or dimer.
The tools at IDTDNA.com will determine hairpins and primer-dimers, but fail to distinguish the 3' vs. 5' overhangs, and list many with poor matching at the 3' end, leading to many false warnings of problems.