You are studying disease X, a genetic disorder that arises from a trinucleotide

Question

You are studying disease X, a genetic disorder that arises from a trinucleotide repeat expansion mutation which is a mutation where entire codons are multiplied during the replication process. The mutation is found in the CaM gene, that is a 450-base pair (bp) sequence. The trinucleotide repeat expansion results in the addition of 10 codons (30 bp) at nucleotide 150 (ie in the centre of the gene, away from the 5’ and 3’ end). The sequence of the normal gene is shown below.

a) Design primers for the amplification of this gene

b) Design a PCR-based method to detect this disorder in patients.

c) Describe or draw your expected results for an individual with the disease vs. a healthy individual control.

d) A sample collected from a patient with similar symptoms to those suffering from Disease X has been brought to the lab. You use your PCR based test and determine that the CaM gene is the same size as that of a healthy individual. You suspect that the arises from a point mutation (a single nucleotide change that results in a defect in the protein.) Describe a how you could confirm this hypothesis.

Explanation / Answer

A. Forward primer- TACCGACTAG

Reverse primer- TTGCAGT

B. To detect it we should use RFLP PCR technique.restriction fragment length polymorphism (RFLP) is a technique that reveals variations in homologous DNA sequences. It refers to a differnce between samples of homologous DNA molecules from differing locations of restriction enzyme site, and to a related laboratory technique by which these segments can be illustrated. In RFLP analysis, the DNA sample is broken into pieces (and digested) by rertriction enzyme and the resulting restriction fragments are separated according to their lengths by gel electrophoresis.   

We will use a couple of restriction enzymes and mix with DNA molecule, then with the product we will add some random primers and run a PCR with taq polymerase enzyme. After that we will run gel electrophoresis and record our banding patterns.

if polymorphism present there in DNA the restriction endonuleases will not cut at the same site of normal DNA, we will get a different banding patterns from normal DNA.

D. With RFLP PCR we can easily detect Single Nucleotide Polymorphism .

Related Questions

Navigate

• Browse (All)
• Browse Y
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.