CB15: Molecular tools & diagnostics 2 Flashcards
Why is the antibiotic gene of a plasmid vector useful?
It allows the selection of cells containing the vector.
Why is the tag of a plasmid vector useful?
It is used in the purification, visualisation & detection of the desired protein.
Why is the selectable marker of a plasmid vector useful?
It helps to control expression of a gene.
How does gel electrophoresis separate proteins?
By molecular weight.
What is the gel used for protein resolution?
Polyacrylamide
What dye is used for protein resolution?
Coomassie dye
What detergent is used for protein resolution?
SDS PAGE
What does SDS PAGE do?
It unfolds polypeptides & gives them a uniform negative charge.
How are proteins resolved by gel electrophoresis?
1) Proteins are heated to be denatured.
2) Detergent is added.
3) Reducing agent is added to break the disulphide bond within & between protein.
How does immunoblot work?
1) Proteins are prepared with SDS PAGE.
2) One antibody detects the protein, and another antibody detects the first antibody with the protein.
3) The second antibody has chemiluminescence/bioluminescence so it can be seen.
What does ELISA stand for?
Enzyme-Linked ImmunoAbsorbent Assay
How does ELISA work?
This works similar to an immunoblot but it’s done in solution.
The first antibody detects the protein & it’s washed. Second antibody will change colour when it encounters its substrate.
What is ELISA used for?
To find out the protein concentration.
How does a lateral flow test work?
1) An antibody is attached to an area and it is linked to an enzyme.
2) Antibody captures the protein.
3) A metabolic reaction between enzyme & protein leads to a colour change in the dye.
4) Solution is drawn across the strip by capillary action.
5) Excess protein is captured in the control zone.
What is an advantage of a lateral flow test?
Very quick
What is a lateral flow test used for?
covid, distemper, feline FIV, parvovirus
What is PCR used to detect?
- covid
- porcine respiratory & reproductive syndrome virus
- detection of Hap I gene (involved in Leptospira infection)
What are PCR & restriction enzymes followed by DNA sequencing used to detect?
- canine degenerative myelopathy
- breast cancer (human)
What are short tandem repeats followed by PCR used for?
paternity testing
What does STR stand for?
Short Tandem Repeats
Why are STR used for paternity tests?
One is inherited from the mother & one from the father.
How is PCR used in STR analysis & paternity tests?
- PCR amplifies the STR section
- Different number of STR results in different size of PCR product so it’s identifiable
What are STR used for apart from paternity tests?
Used by govt as identification service to prevent smuggling, poaching & fraudulent activities in disease control & maintenance of food safety standard.
