Molecular Diagnostics

Question 1

In order to effectively use Hybridization and PCR, what must you know?

Answer 1

The sequence of the pathogen or the sequence of the gene must be known in order to perform these tests

Question 2

Hybridization

1. Function
2. How

Answer 2

1. Detection and quantification of target DNA or RNA
2. • target gene converted to single strand DNA and immobilized a membrane
   • probes complimentary to the sequence are added to the sample
   • measure fluorescence

Question 3

Blotting techniques, target and purpose of

Southern

Norther

Western

Eastern

Answer 3

Southern—target is DNA, to determine with fragments are associated with a gene

Northern—target is RNA, measure the size and quantities of mRNA and answer questions about gene expression

Western—target is protein, measures amount of protein or antibody

Eastern—target is lipid, carb, phosphorylation;detects post translational modifications.

Question 4

What is different about qPCR?

Answer 4

Has a fluorescent probe that can be measured.

Probe is activated when degraded

Question 5

Restriction Fragment Length Polymorphism (RFLP) is also called what?

How does it work

Answer 5

DNA fingerprinting.

DNA is chopped by enzymes at specific sites.
If that site has a mutation, it will be longer or shorter.

Fragments are run through gel electrophoresis, and bands of DNA can be visualized

Question 6

In normal beta-globulin, what is considered normal in terms of RFLP?

How is this different than sickle cell?

Answer 6

Have 3 Ddel restriction site and will get two fragments

In sickle cell there will only be 2 restriction cites, and will result in 1 fragment.

Question 7

What are variable number tandem repeats (VNTR) useful for?

Answer 7

The identification and severity of inherited disease such as Huntington disease.

Question 8

How are recombinant proteins synthesized

Answer 8

Gene for desired product is spliced into bacterial plasmid.

Plasmid is re-inserted into bacteria.

Bacteria produce product.

Question 9

What are examples of large scale production of recombinant proteins

Answer 9

Insulin
Growth hormone
Erythropoietin
Clotting factors
Vaccines.

Question 10

What are the important residues in insulin?

What happens when they are modified?

Answer 10

Proline at position 28
Lysine at position 29

Changing this changes the absorption rate

Question 11

What is a monoclonal antibody?

Answer 11

Antibody specific for a single epitope on antigen, many are used as drugs

Question 12

Enzyme linked immunosorbent assay (ELISA) tests what?

Answer 12

Test for the level of specific antigen or antibody concentrations

Question 13

Two types of ELISA and how to they work?

Answer 13

Indirect—measures antibody in sample.
Must line well with antigen, add specific antibody, add second flagging antibody to measure.

Sandwich—measures antigen
Line well with monoclonal antibody, add antigen, and flagging antibody for measuremen.

Question 14

ELISA is used in what 3 common tests?

Answer 14

Diagnosing HIV (indirect)—can give false positive and false negative, so must be confirmed with WESTERN BLOTTING.

MI detection (sandwich)

Pregnancy test (sandwich)