Question

(3) Polymorphism analysis of the sickle cell gene fragment (317 bp) that helps to make hemoglobin. A lab technician is conducting the polymorphism analysis of the sickle cell gene fragment (317 bp) using PCR and restriction digestion by Taq 1 on six individuals. A) Schematic representation of restriction sites of Taq l in the normal gene fragment. Two normal S1 and S2 restriction sites produce DNA fragments of 174, 78, and 65 bp. The sickle cell mutation removes the S2 restriction site resulting in an abnormal fragment of 252 bp. B) The picture of a 4% agarose gel loaded with restriction fragments of six individuals numbered from 1 to 6. U stands for undigested DNA fragment. DNA marker Undigested DNA A A) Normal gene: 2 Restriction sites (S1 and 52) 65 bp 78 bp 174 bp B) Ana B) popr 13 221 222 224 225 226 70 hp - 317 -252 S1 S2 174 65 bp 252 hp Mutant gene: 1 Restriction site (S1) A) What is the expected fragment profile by Taq 1 digestion for the normal gene? B) What is the expected fragment profile by Taq 1 digestion for the sickle cell mutant gene?

Answer 1

A) According to figure 'A' S1 and S2 are the two restriction sites for the enzyme Taq 1 in normal gene. So, the expected profile for the bands in gel would be- CASE 1: for complete digestion- three bands of size 65, 78 and 174 bp would be seen. CASE 2: for partial digestion- maximum six bands can be seen that of size- 65, 78, 174, 317, 252 and 143 bp.

B) According to figure 'A' S1 and S2 are the two restriction sites for the enzyme Taq 1 in normal gene but for sickle cell mutated gene S2 site is disturbed and no longer be cut at this position by Taq 1. So, the expected profile for the bands in gel would be- CASE 1: for complete digestion- two bands of size 65 and 252 bp. CASE 2: for partial digestion- maximum three bands can be visualized of size- 65, 252 and 317 bp.

C) Band profile for patient 1 shows 4 band of size- 252,174, 78 and 65. Sickle cell anemia is an autosomal recessive disorder. This type of disease can have three possibility for individual - Normal, Carrier or Affected. Normal person will have both the gene in normal condition but affected person will have both the genes mutated while carrier person will have one normal gene and other is mutated. So the conclution is that here for patient 1- 252 bp band is from mutated gene and bands of size 78 and 174 bp are from normal copy of the gene and band size 65 is commen in both.

So, as discussed above patient 1 is carrier for sickle cell anemic.

D) Band profile for patient 2 to 6 shows 3 band of size-174, 78 and 65. As explaned in A and C question explanation these patients are normal because on digestion three bands are obtained that are for normal gene.