You are working in a lab and have some free time to do extra research. You decid

Question

You are working in a lab and have some free time to do extra research. You decide to launch your own mini project studying Huntington's disease. In unaffected individuals, the Huntington gene has 26 or fewer CAG repeats (each CAG repeat codes for a glutamine). Individuals with Huntingtin's disease have 36+ CAG repeats. You want to determine the role of the glutamine repeats in a wildtype cell. To do so, you decide to delete all CAG repeats from the Huntington disease in a mouse using CRISPR. 2. You need to first check and see if you successfully removed all the CAG repeats with CRISPR. In 442, you learned 4 DNA-focused methods (PCR, ASO, PCR primer initiation, and restriction site analysis) that you could use. After obtaining a sample from the mouse and isolating genomic DNA, explain the steps required for 2 of the techniques (include the expected result if your CRISPR was successful and removed the CAG repeats in both allele of Huntington - label it "expected result"). The first result is provided for you:* i.Option 1 - PCR Amplify the region of interest using PCR with flanking primers (before & after the tandem region) Run the PCR product on a gel. Compare the length to the same reaction done with the wt gene (should be 78 bp shorter)» Expected result: if the CAG repeats were removed, the CRISPR PCR product should be 78 bp shorter than the wt a. b. c.

Explanation / Answer

Restriction site analysis using

a) Restriction digest of desired fragments using Restriction endonuclease. RE like EcoP15I, Bam H1 which will specifically digest CAG.

b) Run the digested fragments in agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresis to separate the fragments on the basis of length or size or molecular weight.

c) Expected result: If CAG repeats were removed, the restriction digestion product will not produce any digested fragments.

ASO ( Allele-specific oligonucleotide).

a) Make a string of glutamine repeats. 20 or 30 repeats for normal, >50 repeats late or onset of disease.

b) Amplify the region of interest using allelic specific primers and run the amplified the PCR in agarose gel electrophoresis along with positive and negative control.

c) Expected results: If CAG repeats were removed, ASO PCR should be shorter than the expected size or nil product should be obtained.

Related Questions

Navigate

• Browse (All)
• Browse Y
• Subjects

---

---

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