Zaidi Molecular Diagnostics

Question 1

Describe hybridization

Answer 1

A single stranded DNA will bind to either another DNA or RNA strand to form a hybrid

An single stranded oligonucleotide is used as a probe that will bind to a complimentary piece of DNA And allow for the DNA to be found

Question 2

Describe the difference between Northern and Southern blotting

Answer 2

In southern blotting, the probe and the target nucleic acids are DNA; determines which restriction fragments are associated with a gene

In northern blotting, the probe is single stranded DNA and the target is mRNA; measures the size and quantities of mRNA molecules

Question 3

Describe the differences between western and eastern blotting

Answer 3

Western: protein is the target; measures the amount of protein or antibody

Eastern: PTM (lipid, carb, phosphorylation) detects post-translational modifications

Question 4

What is the purpose of cell-free cloning PCR

Answer 4

To amplify isolated DNA regions

• earlier detection of microorganisms; HIV, bacterial and fungal infections that cause lyme disease
• gene mutation detection

Question 5

What is the purpose of qPCR?

Answer 5

used to quantify the copy number of a gene

includes a fluorescent probe that fluroesces only if the PCR product is present

• detect levels of an infectious agent
• determine levels of gene expression

Question 6

Describe the use of recombinant proteins clinically

Answer 6

The cDNAs can be inserted into expression vectors and allow replication, translation, and transcription

-can create a large scale production of insulin, growth hormone, erythropoietin, clotting factors, and vaccines

Question 7

Describe the production of genetically engineered insulin

Answer 7

The insulin producing gene is taken from a pancreases B cell

a DNA plasmid is extracted from a bacterium and cut out with a restriction enzyme, which forms a plasmid vector

tis is inserted into the bacterial plasmid vector to from recombinant DNA

this DNA is introduced to the bacterial cell

recombinant bacteria multiply in a fermentation tank and make insulin, wince it is in their genetic code

insulin is used to treat diabetic patients

Question 8

Describe the composition of normal insulin when compared to

1. Lispro
2. Insulin aspart

Answer 8

Normal insulin has a proline at 28 and a lysine at 29 at the C terminus of the B chain

1. Lispro has the reverse of these two a.a.s
2. Insulin aspart replaces proline on 28 with aspartic acid

Question 9

What is the clinical use of abciximab

Answer 9

inhibits platelet aggregation

Question 10

What is the clinical use of baciliximab

Answer 10

prevents rejection of a transplanted kidney

Question 11

What is the clinical use of cetuximab

Answer 11

treats metastatic colorectal cancer

Question 12

What is the clinical use of influximab

Answer 12

treats autoimmune diseases

Question 13

What is the clinical use of retuximab

Answer 13

treats lymphomas, leukemias

Question 14

Describe how ELISA is used in HIV detection

Answer 14

Indirect ELISA is used:

1. biological sample is added to the Immobilized HIV antigens in a plate
2. Plate is washed to removed unbound Abs and nonspecific Ag-Ab associations
3. enzyme linked IgG is added and washed
4. color stuff added

If there is color present, that means that there were anti-HIV antibodies present in the blood which means that the patient has been exposed and infected to the HIV virus, and has it

Question 15

Describe how ELISA can be used in the detection of MI’s

Answer 15

ELISA can be used to measure the increase in the cardiac forms of troponin T and troponin I , which are elevated following an MI

Question 16

Describe how ELISA can be used to detect pregnancy

Answer 16

The reaction site of the stick is where you pee and it has the free hCG abs that are able to bind to the hCG that is present in the urine. This then moves to the test site

At the test site, the hCG/ab complex binds to the immobilized hCG antibody and the sandwich is complete and the dye gives a color

The control site has a non-specific AB, and the de will give color regardless of the test result just to prove that the test itself is working

Question 17

Describe the process of a western blot

Answer 17

used to detect the levels of proteins

SDS-page separates out the proteins on a gel with an electric field and the proteins migrate based on size

This is then transferred to a membrane.

Primary and secondary antibodies are added and the secondary ab has an enzyme tag

substrate is added which gives a color

can be used to VERIFY HIV

Question 18

How can the western blot be used to confirm that a patient has HIV?

Answer 18

Circulating levels of HIV p24 appear before the Ab is formed

other antigens are also present (gp160, gp120, p66, p55, etc)