I'm trying to solve an assignment question where we are given a Amino Acid sequence and was asked to use this a basis to design probes and select clones?
The aminoacid sequence is S-N-W-D-Q-A-M-G-L-F-V-I-R-P-C-E-H-Y-K-T I used a online tool EMBOSS Backtranseq to backtranslate from protein to nucleic acid.
This is the sequnce which I got AGCAACTGGGACCAGGCCATGGGCCTGTTCGTGATCAGGCCCTGCGAGCACTACAAGACC
Can someone explain me why this is the mostly likely sequence of the all possible codons?
Then, when I start to design probes, I'm facing trouble with all the onlines which ask for minimum of about 100 bases to design the probe. Can someone help me please?
Use primer3 from Pubmed. Your problem might due to wrong parameter you gave the software. You first need to create a fake FASTA format then ask the produce to be between 20 and 120. I am able to find two pairs of primers.
Primer pair 1
Sequence (5'->3') Template strand Length Start Stop Tm GC% Self complementarity Self 3' complementarity
Forward primer:
CCATGGGCCTGTTCGTGAT
Reverse primer: GTCTTGTAGTGCTCGCAGGG
Product length 43
Primer pair 2 Sequence (5'->3') Template strand Length Start Stop Tm GC% Self complementarity Self 3' complementarity
Forward primer: CCATGGGCCTGTTCGTGA
Reverse primer GGTCTTGTAGTGCTCGCAGG
Product length 44
It is hard to say your strategy will serve you. It will be more likely to work if you design degenerated probe according to N-W-D-Q-A-M-G, or P-C-E-H-Y-K-T, while the first one contains W and M residue, which is better to degenerated probe. a fully possibility covered 18 bases from N-W-D-Q-A-M plus a G from aa G, making into 19 bases degenerated probes, which contains 1 right in 64 probes. If you use this degenerated probes, you will have a fair chance to get what you wish.
Thank you Ziguo..Yes, we were asked to design 2 degenerate probes to increase the chances of finding the clone.
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