The substitution of a T for an A in the protein coding sequence of the hemoglobi

Question

The substitution of a T for an A in the protein coding sequence of the hemoglobin beta chain gene introduced a SNP. This SNP changed the amino acid incorporated into the hemoglobin beta chain, resulting in the disease symptoms of sickle cell anemia. Unaffected individuals have the DNA sequence GAGGAG, a BseR I restriction enzyme site, in their hemoglobin beta chain gene. The T substitution (GTGGAG) eliminates this restriction enzyme site.

Explain how you would use this information to design an RFLP screening test for sickle cell anemia – make sure to include the FOUR STEPS (DNA isolation, PCR, restriction enzyme digest, gel) that were outlined for you in the Module 3 lab exercise, but add details for each step that are relevant to each step (for example, for step 1, which cells would you get the DNA from? For step 2, what gene are you targeting with PCR, etc.). Give a 2-3 sentence explanation of how each of the four steps of the analysis works.

Explanation / Answer

step1) isolate the red blood cells (sickle as well as normal) and from the cells isolate the DNA. this DNA contains the Hb gene.

step 2) perform the PCR reaction using Hb gene forward and reverse primer and amplify the Hb gene. this PCR reaction will amplify both the gene normal as well as sickle cell Hb gene in different tubes.

step 3) after PCR digest the both PCR amplified gene with the RE BseRI in different tubes. the Hb gene having normal gene will cleve by the RE but the sickle cell gene will not cut by the RE.

step4) after RE digestion when we ran a gel of the digestion reaction we will get two DNA bands in Hb normal gene while in sickle cell gene Re will not be able to cut the DNA hence we will get a single band of the DNA.

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