How can I design and oligonucleotide probe to identify a clone containing a newly purified enzyme. Given a specific amino terminal sequence pf the enzyme MNDHYWIK?
Indicate redundancy by putting all possible nucleotides for a particular position in parenthesis.
I need help please!
Hi there,
You just have to use the genetic code degeneration and introduce in your primer any triplet coding for each aa of the sequence:
ATGAAYGAYCAYTAYTGGATHAAR
Y is C or T; H is A, C or T; R is A or G
From your amino acid sequence, the only 100% match in the NCBI protein database is to a histidine kinase from Gramella Portivictorea. The whole genome of this bacteria is available and you can download the nucleotide sequence so that specific PCR primers can be made to amplify the whole protein coding region. If this is not your organism of interest, then try searching the genome of your organisms if it is avaialble.
Dominique's answer is not incorrect but annealing such a degenerate primer of only 24 bp is not favorable and what will you do with it? For PCR you need a second primer, for hybridization you will likely need a longer oligo.
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