I don\'t need the \"answer\" to this question, per se-- I am simply confused as
ID: 52289 • Letter: I
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I don't need the "answer" to this question, per se-- I am simply confused as to what it's asking. How can I ligate an amplicon into the EcoR1 site, when I don't have the DNA sequence? If there is extra information this problem would require, I would appreciate it if you would let me know what information would be needed. I've been stuck on this for a while.
Make an amplicon that can be cloned into the EcoR1 site of the expression vector shown below:
1.) Sequence of Forward primer (5'-3'): ?
2.) Sequence of Reverse primer (5'-3'): ?
myc epitope | | 6 × His | | Term BGH SV40 ori & enhancer pUC ori Figure 5-23 Human Molecular Genetics, 3/e. (© Garland Science 2004)Explanation / Answer
You don't need to know the sequence at the ends of the amplicon to ligate it in a EcoR1 site. The vector should be treated with EcoR1 to generate sticky ends.
5' GAATTC 3' 5' G 5' AATTC 3'
3' CTTAAG 5' ------------------ (treatment with EcoR1)--------> 3' CTTAA 5' G 5'
Then the DNA you are to ligate can be -
1. treated with EcoR1 to generate similar sticky ends ( this will lead to ligation in presence of DNA ligase by complementary base pairing)
2. or you can treat your DNA with Exonuclease to generate blunt ends, then add adapters specific for EcoR1 to the ends of the DNA and then ligate it to the EcoR1 treated vector. In this case, the sequence of the adapters will give you the necessary information to construct the forward and reverse primers.
So, you can give the end (partial) sequence of the primers accordingly.
(since the DNA amplicon sequence is not given, I think this is what the question seeks)
P.S.- You can refer to PRINCIPLES OF GENE MANUPULATION AND GENOMICS BY PRIMROSE AND TWYMAN - Chapter 3- Introduction of Restriction Endonuclease site in primers.
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