Mutations in the CFTR gene result in cystic fibrosis in humans, a condition in w

Question

Mutations in the CFTR gene result in cystic fibrosis in humans, a condition in which abnormal secretions are present in the lungs, pancreas, and sweat glands. In the effort to positionally clone the CFTR gene, the gene was mapped to a region of 500 kb on chromosome 7 containing 3 candidate genes.

a) Using your knowledge of the disease symptoms, how would you distinguish between the candidate genes to decide which is more likely to encode the CFTR gene?

b) How would you prove that your chosen candidate is the CFTR gene?

This is from a chapter covering: Forward genetic screens, Recombinant DNA technology, DNA sequencing technologies, Gene libraries (Genomic and cDNA), and Gene cloning using recombinant DNA technology. So I imagine, they are looking for us to design an experiment using some of these to determine the information asked in parts a and b.

Explanation / Answer

There is a old simple but cumbersome technique for this which is comparatively cost effective called LOH or Loss of Hetrozygosity.

1) Isolate DNA

2) amplify with primers for CFTR. One of the Primers should be labelled on 5' with radioiosotope or fluorescent tagged

3) Now run it on 6 % sequencing gel

4) Develope an autoradiogram

4) Neglect the common bands. Look for a set of allelic distribution. Acoording to disease condition, among the set of amplified alleles where there is a loss of an allele due to deletion mutations (more than 40%) that set corresponds to the CFTR and others do not.

Other techniques include "comparative genomic hybridization where LOH and gain of function both can be traced.

Modern techniques include:

(i) amplification of target

(ii) Fragmentation of amplified target

(iii) linearize and use it as a probe for hybridization (Micro-array experiment)

(iv) Select the hybridized target and go for sequencing to identify mutation

(v) Do the sequence analysis to identify which candidate gene it was from the group of 3 using bioinformatics tools

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