Molecular Diagnostics

Question 1

What are the techniques used to detect infectious agents and diagnose inherited disorders

Answer 1

1. Hybridization

2. Polymerase Chain Reaction (PCR)

Question 2

What must be known for hybridization and PCR to move forward

Answer 2

You must have to know the sequence of the pathogen or the sequence

Question 3

What is hybridization used for?

Answer 3

Used to detect target DNA (southern blot) or RNA (northern blot) in a sample

Question 4

In hybridization, what kind of sequence do you need for a single-stranded DNA to bind to another DNA or RNA strand?

Answer 4

You need a complementary sequence to for the DNA-DNA (southern) hybrid or the DNA-RNA (northern) hybrid

Question 5

What is meant by “blotting”

Answer 5

When the target DNA is converted to a single strand DNA, it is then IMMOBILIZED onto a membrane

Question 6

What’s the difference between Southern and Northern blotting

Answer 6

Southern: both probe and target sequence is DNA
Northern: probe is single-stranded DNA and target is mRNA

Question 7

What is the purpose of blotting techniques

Answer 7

To detect and visualize specific biomolecules present in a mixture. Why you must immobilize onto a membrane.

Question 8

What is the purpose of a Southern blot

Answer 8

Determine which restriction fragments are associated with a gene

Question 9

What is the purpose of a Northern blot

Answer 9

Measure the size and quantity of mRNA

Question 10

What is the purpose of a Western blot

Answer 10

Measures amount of protein or antibody

Question 11

What is the purpose of Eastern blot

Answer 11

Detects post-translational modifications (PTMs) on proteins

Question 12

SNOWDROP

Answer 12

S-southern D-DNA
N-northern R-RNA
O and O nothing
W- western P-protein

Question 13

What are the ingredients for PCR?

Answer 13

Double-stranded DNA
Primers
dNTPs
Taq polymerase (heat resistant)
Thermocycler

Question 14

What are the steps of PCR?

Answer 14

FIRST CYCLE
1. Heat to separate DNA strands
2. Cool to anneal primers
3. DNA synthesis (w/ dNTPs and DNA polymerase)
produces 2 double-stranded DNA molecules

SECOND CYCLE
1. Heat the 2 double-stranded strands and cool to anneal primers
2. DNA synthesis
produces 4 double-stranded DNA molecules

THIRD CYCLE
1. Heat the 4 double-stranded strands and cool to anneal primers
2. DNA synthesis
produces 8 double-stranded DNA molecules

Question 15

What is quantitative PCR used for?

Answer 15

Lets you quantify how many copies of a specific gene are present. Used to detect levels of an infectious agent and determine levels of gene expression.

Question 16

What 2 processes detect variations in DNA sequences?

Answer 16

1. Restriction Fragment Length Polymorphism (RFLP)

2. Variable number of tandem repeats (VNTR)

Question 17

When is RFLP used?

Answer 17

DNA fingerprinting, forensic analysis, paternity testing, disease detection

Question 18

How many restriction sites does a normal-globulin allele have?

Answer 18

3 Ddel restriction sites. Will produce 2 on a membrane.

Question 19

How many restriction sites dows a sickle cell pt have on their B-globulin allele?

Answer 19

2 restriction sites. Will produce 1 on a membrane.

Question 20

What can VNTR help diagnose?

Answer 20

Useful in identification and severity of inherited diseases.

Huntington, Fragile X, Frederich Ataxia

Question 21

In order to diagnose Huntington, what must you know before you carry out VNTR?

Answer 21

Must know the repeats and make primers for the repeats

Question 22

In recombinant proteins, what is inserted into the expression factors?

Answer 22

cDNA of the protein is inserted into the expression factors

Question 23

What are some examples of recombinant proteins produced?

Answer 23

Insulin
Growth hormone
Erythropoietin
Clotting factors
Vaccines against flu, malaria, etc

Question 24

How does recombinant protein manufacturing work?

Answer 24

1. Take a plasmid from a bacterium and whatever gene from a human.
2. Put that gene into the plasmid so that they’re “mixed”
3. Put the gene+plasmid (recombinant DNA) into a bacterial cell
4. Put into a fermentation tank
5. The recombinant bacteria multiply and produce whatever protein from the gene you selected for at the beginning (i.e. insulin)

Question 25

What are some “improved” forms of insulin

Answer 25

Lispro (reverse position of proline and lysine)
Insulin aspart (proline 28 replaced by aspartate
*both of these are faster acting and better absorbed

Question 26

What position are proline and lysine at in normal insulin

Answer 26

Proline at 28
Lysine at 29
At C terminus od B chain

Question 27

Apciximab

Answer 27

Inhibits platelet aggregation

Apciximab=Aggregation

Question 28

Baciliximab

Answer 28

Prevents rejection of transplanted kidney

Kids are BAD (baciliximab)

Question 29

Infliximab

Answer 29

Treats autoimmune disease

Inflix=autoIMMUNE

Question 30

Cetuximab

Answer 30

Treats metastatic colorectal cancer

Cetux=colorectal cancer

Question 31

Retuximab

Answer 31

Treats lymphomas and leukemia

LLR like the LIRR

Question 32

What does the ELISA (enzyme-linked immunosorbent assay) test for?

Answer 32

Tests for levels of a specific antigen or antibody concentrations in a biological sample using a corresponding antigen or antibody

Question 33

What does an indirect ELISA test for?

Answer 33

Measures the amount of antibody in a sample

Question 34

What does a sandwich ELISA test for?

Answer 34

Measures the amount of antigen in a sample

Question 35

What can you diagnose w/ ELISA?

Answer 35

HIV (indirect ELISA)
MI (Sandwich ELISA)
Pregnancy (Sandwich ELISA)

Question 36

What is an SDS-PAGE?

Answer 36

A type of western blot (immunoblotting). SDS-PAGE separates out the proteins on a gel by applying electrical field. Smaller proteins move faster than larger ones

Question 37

What are the steps in an SDS-PAGE?

Answer 37

1. Load protein samples onto SDS polyacrylamide gel and apply an electrical field
2. Transfer protein to the membrane
3. Add primary antibody that binds to target protein. Wash.
4. Add tagged secondary antibody that binds to the primary antibody. Wash.
5. Use visualization agent to detect the secondary antibody signal

Question 38

What test would you use to confirm HIV?

Answer 38

Western blot. Would see HIV p24 antigen even before antibodies are formed. Other antigens include gp160, gp120, p66, p55, etc.