You are studying an enzyme and wish to use PCR to amplify the gene encoding this

Question

You are studying an enzyme and wish to use PCR to amplify the gene encoding this enzyme. Here is the amino acid sequence of the enzyme (in single letter code):

MMMCWCDQFLAAAIDAAKKAGQIIRKGFYETKHVEHKGQVDLVTETDKGCEELVFNHLKQLFPNHKFIGEETTAAFGVTELTDEPTWIVDPLDGTTNFVHGFPFVCVSIGLTIGKVPVVGVVYNPIMEELFTGVQGKGAFLNGKRIKVSAQSELLTALLVTEAGTKRDKATLDDTTNRINSLLTKVRSLRMSGSCALDLCGVACGRVDIFYELGFGGPWDIAAGIVIVKEAGGLIFDPSGKDLDITSQRIAASNASLKELFAEAMMAAMM

Design two degenerate primer sequences that will amplify the entire Open reading frame (ORF) of the corresponding gene in a PCR reaction. To facilitate the cloning of the gene after PCR amplification, place a BamH1 restriction endonuclease site at each end of your primers. Hints: restriction endonuclease sites are cleaved more efficiently when 1-2 extra nucleotides are included at each end; these extra nucleotides are usually Gs or Cs. You need a forward and a reverse primer. In general, primers that are 18-30 nucleotides long are suitable for PCR amplification. Write out your primer sequences 5’ to 3’.

1. Forward primer sequence:

2. Reverse primer sequence:

Explanation / Answer

Nucleotide sequence of the given protien is -

atgatgatgtgctggtgcgatcagtttctggcggcggcgattgatgcggcgaaaaaagcg ggccagattattcgcaaaggcttttatgaaaccaaacatgtggaacataaaggccaggtg gatctggtgaccgaaaccgataaaggctgcgaagaactggtgtttaaccatctgaaacag ctgtttccgaaccataaatttattggcgaagaaaccaccgcggcgtttggcgtgaccgaa ctgaccgatgaaccgacctggattgtggatccgctggatggcaccaccaactttgtgcat ggctttccgtttgtgtgcgtgagcattggcctgaccattggcaaagtgccggtggtgggc gtggtgtataacccgattatggaagaactgtttaccggcgtgcagggcaaaggcgcgttt ctgaacggcaaacgcattaaagtgagcgcgcagagcgaactgctgaccgcgctgctggtg accgaagcgggcaccaaacgcgataaagcgaccctggatgataccaccaaccgcattaac agcctgctgaccaaagtgcgcagcctgcgcatgagcggcagctgcgcgctggatctgtgc ggcgtggcgtgcggccgcgtggatattttttatgaactgggctttggcggcccgtgggat attgcggcgggcattgtgattgtgaaagaagcgggcggcctgatttttgatccgagcggc aaagatctggatattaccagccagcgcattgcggcgagcaacgcgagcctgaaagaactgtttgc ggaagcgatgatggcggcgatgatgtaa

BamHI restriction site is - GGATCC

Forward primer -

gcggatccatgatgatgtgctggtgcga

Reverse primer -

gcggtaccttacatcatcgccgccat

Since we need to end the protein expression so we need to add stop codon in the reverse primer.

Related Questions

Navigate

• Browse (All)
• Browse Y
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.