(b) You have discovered a protein that performs an interesting function. After p
ID: 213347 • Letter: #
Question
(b) You have discovered a protein that performs an interesting function. After purifying the protein and performing "Edman degradation," you have determined that the following sequence of amino acids is present within the sequence of your purified protein: MVSKGEELFT Where is this sequence located within your protein? (check one) N-terminus C-terminus Cannot be determined (c) Using the amino acid sequence provided in (b), you set out to design a set of degenerate primers in order to amplify the cDNA sequence corresponding to your protein of interest from a cDNA library. In order to do so, you need to synthesize all the oligonucleotide primers that may possess complementary to the 5' and 3' ends of your targeted gene. Assuming you chose a length of 20 bases for your "forward" primer, how many possible degenerate sequences would you need to create in order to cover all possible sequences corresponding to your protein? List the nucleotide sequence of one of your designed primers below. Be sure to label the 5' and 3' ends. (d)
Explanation / Answer
b. Given protein sequence: MVSKGEELFT
AUG is the start codon during translation. It encodes Met. It is the first amino acid to be incorporated into the polypeptide chain.
Hence the given sequence is H2N-MVSKGEELFT-COOH
c.
Given protein sequence: MVSKGEELFT
Number of codons for M = 1
Number of codons for V = 4
Number of codons for S = 6
Number of codons for K = 2
Number of codons for G = 4
Number of codons for E = 2
Number of codons for F = 2
Number of codons for T = 4
Number of codons for L = 6
Each amino acid can be encoded by more than one codon except for Met and Trp.
So, the overall probability is the product of all the probabilities of individual amino acids.
Number of probabilities for the degenerate primer = 1 X 4 X 6 X 2 X 4 X 2 X 2 X 2 X 4 X 6
= 36864 different combinations.
d. Sample sequence: 5'-AUGGUUUCUAAAGGGGAAGAAUUAUUUACC-3'
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