Genetic Disorders 1

Question 1

what are the basic requirements for PCR?

Answer 1

DNA template
Oligonucleotide Primers
Deoxynucleotide triphosphates (dNTPs)
DNA polymerase enzyme
PCR buffer

Question 2

what does PCR buffer contain?

Answer 2

contains magnesium as cofactor for enzymes an pH compatible with optimal enzyme activity

Question 3

what are the 3 steps of PCR?

Answer 3

Denaturation
Annealing
Extension

Steps repeated multiple times

Question 4

what causes cystic fibrosis?

Answer 4

defective transport of chloride ions across the membranes of epithelial cells.
Caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR)

Question 5

what type of mutation is cystic fibrosis?

Answer 5

autosomal recessive disorder, Both genes need to be defective

Question 6

what are the components of the PCR master mix?

Answer 6

PCR buffer
MgCl2
dNTPs
CFTR-F Primer
CFTR-R Primer
Platinum Taq Polymerase
MilliQ H2O

Question 7

what are the steps in PCR?

Answer 7

Initial denature- 95C for 2mins
Denature- 95C for 30sec
Anneal- 60C for 30 sec
Extend- 72C for 30sec

Repeat from step 2 for 30 cycles
Final extension- 72C for 5mins

Question 8

what is the purpose of a molecular weight marker?

Answer 8

So we can have a reference to measure the length of the DNA fragments in the gel

Question 9

what is the purpose of a gel negative control?

Answer 9

to detect if the master mix has contamination

Question 10

why would acrylamide gels be used instead of agarose gel?

Answer 10

Acrylamide is better at detecting small nucleotide changes in the DNA. The DNA changes we are trying to detect are small.

Question 11

what is an oligonucleotide ligation assay?

Answer 11

Uses sets of fluorescently labelled primers, with pairs specific for the normal or mutated sequence at each mutation site

Question 12

what are the steps in an oligonucleotide ligation assay?

Answer 12

1. Wild and Mutant Allele-Specific Primers (APSs) are annealed to their respective sequences. If there is no mutation, the mutant ASP will not bind. Each ASP has a slightly different length so that they can be separated. A common Probe anneals at the sequence following each ASP.
2. Any ASPs that have bound are ligated with their neighboring Common Probe. This produces products of a known length for the Wild vs. Mutant alleles
3. The ligated products are separated by electrophoresis, and detected by their fluorescent signal. As the product lengths are known, we can identify which ASPs are bound to the patient’s DNA sequence as an indicator of their mutation status.

Question 13

where is the cystic fibrosis mutation site?

Answer 13

F508

Question 14

what is Tay-Sachs disease?

Answer 14

An autosomal recessive genetic disorder resulting from various mutations in the hexosaminidase subunit alpha (HEXA) gene.

Question 15

what is Huntington disease?

Answer 15

An autosomal dominant condition resulting from mutations in the HTT gene, specifically in the CAG codon triplicate region.