Gene Mutations query

Genetics Genetic Engineering
1

Hi

A mutation has been induced in the gene encoding a protein using the mutagen. The resulting mutant protein can be purified and its amino acid sequence determined. Using the amino acid sequences, one can determine the DNA sequence of this protein. Question: how do you then generate a probe to identify the gene within a genomic library??

Question 2: suppose you found more than one gene, how would you distinguish a functioning gene from a pseudogene?

2

Dear Daniel

You can Back-translate the Amino Acid Sequence:

  • Use the amino acid sequence to infer possible nucleotide sequences (considering the degeneracy of the genetic code).
  • Focus on conserved regions (ideally 8–15 amino acids) with low codon degeneracy (e.g., Met, Trp, or codons with fewer alternatives).
  • Design a Degenerate Oligonucleotide Probe:
  • Due to multiple possible codons per amino acid, your probe will be a degenerate
  • Label your probe as per your resources

Regarding your second query:

  • Check for Transcription (Expression):
  • Use RT-PCR or Northern blot to see if the gene is transcribed.
  • Use RNA-seq data if available.
  • Check for Functional ORF (Open Reading Frame):
  • A pseudogene often has:
    • Premature stop codons
    • Frameshift mutations
    • No proper promoter
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