Question

2) Recently the locus that controls fur color in cats has been identified. Two alleles for this locus (black and white) exist with the white allele being recessive. Upon sequencing the mucleotides of these alleles it was found they differed in a single nucleotide (T to G transition) To identify the region in which this mutation occurred, scientists performed a Southern blotting from pure-bred black (B) and white (W) cats. After digesting genomic DNA with three restriction enzymes (Hind Il. EcoRI and PMEI) and hybridized the resulting blot to a labelled cDNA from the fur locus. Their results are illustrated below. Based on this image answer the following questions (12)

Answer 1

ANSWER A and B :-

1. For identifying the fragments that are being formed from the restriction digestion, it is very important to know the genetic sequence on the basis of which the restriction sites can be identified and analysed.
2. Restriction fragment length polymorphism analysis can be used to detect single nucleotide transversions as can be seen in terms of replacement of Thymine to Guanine. The three different restriction sites for the enzymes may show variations in terms of formation of different sized bands.
3. In case of HindIII, two bands are formed of size 8kb and 5kb respectively. In case of EcoRI, 3.5kb fragment is formed and in case of PmeI, a approximate 9.5kb fragment is formed. The difference in terms of band sizes may occur due to alteration of the restriction sites due to base transversion event which may inhibit recognition and thus cleavage of the sequence. The latter two restriction enzymes leads to formation of only single bands indicating a single DNA sequence which should have a size larger than that of the combined total of the two bands digested by HindIII. But, the single bands formed by digestion with EcoRI and PmeI lead to formation of a combined total size of around 13kb although the restriction site varies for both sequences. Three separate lanes indicates specific cleavage with three restriction enzymes otherwise they would have been marked in a single lane.
4. HindIII recognizes the site A-AGCTT/TTCGA-A, EcoRI recognizes the site G-AATTC/CTTAA-G and PmeI indicates the site G-TTTAAAC/CAAATTT-G. The transversion specifically in case of the restriction sites of enzymes EcoRI and PmeI shows changes at the cleavage nucleotide sequence where the Thymine nucleotide is located. If the sequence of the alleles for the gene loci is known, then this can be used to spot the single nucleotide polymorphism. Hybridization with probe DNA leads to recognition of the sequence associated with the locus. Sites with alterations would show less specificity towards the probe which can be reflected in case of restriction enzymes EcoRi and PmeI.

Note :- Respected Sir, Its very important for the DNA sequence to be mentioned to identify the changes happening and at the same time to identify the probe sequence. The relation of polymorphism in terms of transcribed and untranscribed region can be derived through the phenotypic appearance. Taking into consideration there are changes associated with the DNA recognition sites for EcoRI and PmeI, there would be phenotypic changes detection in terms of the fur appearance. Please provide more details if possible and provide an upvote if the answer seems satisfactory.