Suppose you would like to study a gene that codes for a peptide hormone H secret

Question

Suppose you would like to study a gene that codes for a peptide hormone H secreted by human neurons. You know the primary sequence of this peptide hormone. Explain how you might be able to identify or isolate the gene that codes for this peptide hormone. How will you produce multiple copies of this gene for study? How will you produce large quantities of this hormone for evaluation as a potential therapeutic agent? Provide a detailed outline of steps you will use for each of these objectives explaining why that step is needed.

Explanation / Answer

1) identify/ isolate the gene

generate the transcript (mRNA) sequence from the primary sequence of the peptide hormone -> align it using BLAST tool -> select the maximum identity hit (NOTE: you may not get 100% identity due to intronic sequences)

2) producing multiple copies of the gene

Once the gene is identified as above, design forward and reverse primers preferrentially across exons(set appropriate parameters eg Tm, length, check foe homo- and hetero-dimers etc.) -> set up PCR (reagents required = PCR buffer, Mg2+ ions, Taq pol, dNTPs, forward and reverse primers and H2O to make up the volume) -> run on agarose gel

3) Producing large quantities of this hormone

Use the above designed primers -> develope cDNA for your gene -> clone it in an appropriate expression vector -> transform it in an appropriate bacterial competent cells (eg. BL21) -> Isolate your protein of interest (affinity chromatography)

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