Molecular Diagnostics

Question 1

Single stranded DNA binds to another strand of DNA or RNA with complementary sequence to form DNA-DNA hybrid or DNA-RNA hybrid.

Answer 1

Hybridization

Question 2

What is the blotting when both probe and target nucleic acid are DNA? What is it’s purpose?

Answer 2

Southern Blotting

Determine which restriction fragments are associated with a gene

Question 3

What is the blotting where the probe is single stranded DNA and target is mRNA?

Answer 3

Northern Blotting Measure size and quantities of mRNA molecules (questions about gene expression

Question 4

What do Western blots target and what is their purpose?

Answer 4

Protein, measure amount of protein or antibody.

Question 5

What do Eastern blots target and what is their purpose?

Answer 5

Detects post- translational modifications (PTMs) on proteins.

Question 6

What do you need to conduct a PCR?

Answer 6

dsDNA (patient or pathogen)

Primers that flank each end of DNA sequence of interest in 3’-5’ direction

dNTPs

Taq Polymerases

Thermocycler (denaturing and annealing temp)

Question 7

Advantage of PCR

Answer 7

Very small amount of template DNA needed, 100-fold amplification from trace amount of DNA

Question 8

Disadvantage of PCR

Answer 8

Need to know the sequence of the flanking DNA for primer design, error prone, amplification of contaminating DNA

Question 9

What is the purpose of cell-free PCR

Answer 9

Amplifies isolated DNA regions

Earlier detection of microorganisms: HIV, Lyme disease

Detection of specific genetic mutations

Question 10

What does qPCR use as an “ingredient” in addition to regular PCR.

Answer 10

A probe which fluoresces only in presence of the PCR product Probe usually a complementary oligo with a fluorescent tag

Question 11

What is the purpose of qPCR?

Answer 11

Determine levels of gene expression Detect levels of an infectious agent

Question 12

What is Restriction Fragment Length Polymorphism (RFLP) and how can it be used?

Answer 12

Individual genomes differ by 1 in every 1000 base pairs. Some of these occur at restriction enzyme sites which can be utilized for DNA finger printing in forensic analysis, paternity testing and disease detection.

Question 13

RFLP can be used for detection of mutants, what is the disease given as an example and how does it work.

Answer 13

Normal β-globulin allele has 3 DdeI restriction sites

Patients with sickle cell only have 2 restriction sites

Followed by electrophoresis and Southern blotting including a β-globulin-specific probe

Question 14

What is VNTR?

Answer 14

Variable Number of Tandem Repeats

Pattern of short tandem repeats (STR) occurs in genome but varies in individuals These can be isolated by flanking restriction sites r through PCR

Question 15

Diseases screen in VNTR.

Answer 15

Huntington disease, Fragile X syndrome, Frederich Ataxia.

Question 16

How are recombinant proteins made?

Answer 16

cDNA of the protein inserted into expression vectors which are put into cell lines.

Question 17

Examples of large protein production using cDNA

Answer 17

Insulin, growth hormone, erythropoietin clotting factors, vaccines against diseases such as flu, and malaria, viral infections.

Question 18

How was insulin improved upon?

Answer 18

Normal human insulin has proline at position 28 and lysine at position 29 at C terminus of B chain.

Lispro (Eli Lilly): reversed position of these 2 aa

Insulin aspart (Novo Nordisk): proline 28 replaced by aspartic acid.

Question 19

Uses of antibodies?

Answer 19

Used as drugs (cancer, rheumatoid arthritis, Crohn’s disease, hepatitis)

And for research purposes

Question 20

How to make antibodies?

Answer 20

1. Single clones of B lymphocytes are fused with a tumor cell to make it immortalized
2. Humanization minimizes immunogenicity to prolong half life in patient

Question 21

What do monoclonal antibodies bind to?

Answer 21

Specific for a single epitope on antigen

Question 22

Immunological technique which tests for the levels of specific antigen or antibody concentrations in biological samples using a corresponding antibody or antigen.

Answer 22

Elisa

Question 23

What is measured in direct ELISA, how is this done?

Answer 23

Antigens are measured. 1. Well is coated in antibodies, of which antigen bind to. 2. A secondary antibody is added. 3. Lastly substrate is assessed

Question 24

What is measured in indirect ELISA, how is this done?

Answer 24

Antibodies are measured 1. Well is coated in antigen, and specific antibody binds. 2.Enzyme linked antibody is added which binds to specific antibody. 3. Substrate is added and converted by enzyme to color

Question 25

What disease is tested using indirect Elisa?

Why must such diseases be confirmed by Western blotting?

Answer 25

HIV - specific antibodies are developed within 4-6 weeks.

They can produce false positives and false negatives.

Question 26

What disease is tested for in sandwich Elisa?

Answer 26

Myocardial infarction.

Question 27

How are pregnancy tests conducted utilizing Elisa?

1. Reaction Site
2. Test Site
3. Control Site

Answer 27

Utilyzes hCG Antigen

1, Reaction site- hCG Antibody binds hCG Antigen

2. hCG Antibody complex moves down and bind to immobilized hCG (sandwhich complete = color)
3. A non-specific antibody immobilized. Dye gives color regardless of +/- of hCG.

Question 28

Describe Western Blotting (How is it used? What does it do?)

Answer 28

Used to detect the levels of a target protein in a biological sample consisting of a mixture of proteins

SDS-PAGE = Seperate using charge by size

Transfer proteins from gel to a nitrocellulose membrane

Add Primary

Add Secondary (HRP, Alexa Flor)

Question 29

Seperate proteins by size through providing electrical feild.

Answer 29

SDS-PAGE