A double-stranded fragment of viral DNA, one of whose strands is shown below, en

Question

A double-stranded fragment of viral DNA, one of whose strands is shown below, encodes two polypeptides called and vir-1 and vir-2. Adding this double stranded DNA fragment to an in vitro transcription and translation system yields peptides of 10 residues (vir-1) and 5 residues (vir-2). 5'-AGATCGGATGCTCAACTATATGTGATTAACAGAGCCTGCGGCATA AACT-3' (a) What are the nucleotide sequences of the open reading frames (start to stop codons, inclusively) within the mRNA transcripts coding for vir-1 and vir-2? Write them out and designate the 5' and 3' ends. (b) What is the amino acid sequence of each polypeptide? Use one letter symbols to denote amino acids. (c) In a mutant viral strain, the T at position 23 (counting from the left) on the DNA strand shown above has been replaced with G. Determine the amino acid sequences of the two peptides encoded by the mutant virus. (d) What would be the sequences of the oligonucleotide primers (each 10 bp in length) required to amplify by polymerase chain reaction the DNA open reading frame (start to stop codons, inclusively) corresponding to the vir-1 polypeptide? Be sure to designate the 5' and 3' ends of the primers.

Explanation / Answer

DNA SEQUENCE:

5' AGATCGGATGCTCAACTATATGTGATTAACAGAGCCTGCGGCATAAACT 3'

3' TCTAGCCTACGAGTTGATATACACTAATTGTCTCGGACGCCGTATTTGA 5'

a) From the above sequence:

For vir1 (10 residue):

5' ATG CCG CAG GCT CTG TTA ATC ACA TAT AGT TGA 3'

For vir2 (5 residue):

5' ATG CTC AAC TAT ATG TGA 3'

b) The amino acid sequence of the peptides are:

For vir1: M P Q A L L I T Y S Stop

For vir2: M L N Y M Stop

c)The mutation ahs been shown below in bold:

5'- AGATCGGATGCTCAACTATATGTGATTAACAGAGCCTGCGGCATAAACT -3'

5'- AGATCGGATGCTCAACTATATGGGATTAACAGAGCCTGCGGCATAAACT- 3'

3'- TCTAGCCTACGAGTTGATATACCCTAATTGTCTCGGACGCCGTATTTGA-5'

For the amino acid sequence of the peptides:

For vir1-

DNA sequence: 5'- ATG CCG CAG GCT CTG TTA ATC CCA TAT AGT TGA-3'

Amino acid sequence: M P Q A L L I P Y S Stop

For vir2-

DNA sequence: 5'- ATGCTCAACTATATGGGATTAACAGAGCCTGCGGCATAA-3'

Amino acid sequence: M L N Y M G L T E P A A Stop

d) For the synthesis of primer (GIVEN IN BOLD):

5'- ATG CCG CAG GCT CTG TTA ATC ACA TAT AGT TGA -3'

3'- TAC GGC GTC CGA GAC AAT TAG TGT ATA TCA ACT-5'

Forward primer: 5'- ATGCCGCAGG -3'

Reverse primer: 3'- TATATCAACT-5'

                        5'- TCAACTATAT-3'

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