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

Isolate the desired peptide hormone gene :

Isolation of desired gene involves restriction nucleases, which can be purified from bacteria. This enzyme first cut the DNA double helix at specific sites defined by the local nucleotide sequence, and then cleaves a long double-stranded DNA molecule into fragments of strictly defined sizes.

Different restriction nucleases have different sequence specificities, and it is relatively simple to find an enzyme that can create a DNA fragment that includes a particular gene. The size of the DNA fragment can then be used as a basis for partial purification of the gene from a mixture.

Identifying the desired peptide hormone gene :

The technique that identifies a specific gene in a DNA fragment is Southern blotting. The presence of a particular gene in a fragment is detected with the help of a radioactive probe containing the DNA of that gene. The probe is prepared from a cloned DNA segment of the gene which is desired to be isolated.

Producing multiple copies of this gene :

Multiple copies of this gene are produced by a technique known as PCR (Polymerase Chain Reaction). In this technique sample of DNA can be enormously amplified within a short time without resorting to gene cloning.

In the PCR technique, small fragments of DNA having the size of an average gene can be multiplied. The technique essentially consists of mixing the ingredients such as primer, Taq-polymerase etc. and heating this mixture alternately to the melting temperature of the DNA and cooling to a temperature at which new DNA strands can be generated. Each strand of the sample DNA serves as a template for new strand synthesis.

In this way multiple copies of this gene has produced.

Produce large quantities of this hormone :

For producing large quantities of this hormone, at first, we have to find out our gene of interest that produce this hormone. Then paste this DNA into a helpful bacterium, and produce enormous amount of this hormone.

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