(L17) Molecular Diagnostics

Question 1

What are southern and northern blots?

L17 S11

Answer 1

Southern blot:
-hybridization to detect specific DNA sequence

Northern blot:
-hybridization to detect specific RNA sequence

DNA/RNA sequence must be known in order to detect

Question 2

What is PCR and what is it used for?

L17 S12

Answer 2

dsDNA is taken and denatured

Primer (required sequence must be know) binds to original DNA

Taq polymerase is used to replicate new DNA from primer using original DNA as a template

Amplifies DNA

Question 3

What is qPCR and what is it used for?

L17 S15

Answer 3

Quantitative PCR

Same as normal PCR but includes a probe that fluoresce in presence of PCR product

Quantifies copy number

Question 4

What is RFLP and what is it used for?

L17 S21-22

Answer 4

Restriction fragment length polymorphism

Uses PCR to amplify sample followed by restriction enzymes to cleave DNA in a very precise manner

Due to differences everybody has in SNPs in restriction sites, every individual will have a unique way that the restriction enzymes will cleave their DNA

Used as a genetic finger print.
Can be used in paternity testing or disease detection (ie. sickle cell has 2 restriction sites where normal has 3).

Question 5

What is VNTR and how is it used?

L16 S23-24

Answer 5

Variable number of tandem repeats

There are several locations in the genome where there are short tandem repeats (STR).

Several diseases occur due to increased numbers of the STRs (Huntington disease, fragile X, etc.)

The number of STRs can be used to determine likelihood of developing one of these diseases (technical term is anticipation)

Question 6

How are recombinant proteins produced?

L16 S27-29

Answer 6

Gene for desired protein is isolated and introduced into a plasmid which is then introduced to bacteria.

Plasmid is designed so that the bacteria will produce the gene product which can then be isolated and used.

Question 7

How are antibodies mass produced?

L16 S33

Answer 7

Mouse is immunized with desired Ab target.

B cells that produce desired Abs are isolated

B cells are fused with immortalized tumor cells

Hybrid cell is now immortal and produced desired Ab.

Cell is cultured and Ab is isolated

Question 8

What does -mab at the end of a medication name mean?

L17 S38

Answer 8

Indicates that the drug product is a monoclonal antibody

Question 9

What is ELISA?
What types are there and what is each used for

L17 S38-40

Answer 9

Enzyme-linked immunosorbent assay

Indirect:

• immobilized Ag is on plate
• sample is added to plate and if Ab specific fo Ag is present it binds
• labeled anti-Ab Ab is added which will bind to and indicate if Ag specific Ab bound

Sandwich:

• immobilized Ab is on plate
• sample is added to plate and if Ag that the Ab is specific for is present it binds
• Ab specific for Ag is added which will bind Ag if it was present
• labeled anti-Ab Ab is added which will bind to and indicate if Ag-specific Ab bound

Question 10

What is Western blotting?

L16 S45

Answer 10

Protein mixture is isolated via SDS-PAGE and transferred to nitro cellulose membrane

Ab specific for desired protein is added and if protein is present it will bind.

Labeled anti-Ab Ab is added which will bind to and indicate if Ag-specific Ab bound