Molecular Diagnostics

Question 1

Southern Blotting

Answer 1

both probe and target nucleic acid are DNA

Question 2

Northern Blotting

Answer 2

probe is single stranded DNA and target is mRNA

Question 3

Western Blotting

Answer 3

measures amount of protein or antibody

Question 4

Eastern Blotting

Answer 4

Detects post-translational modification on proteins

Question 5

PCR

Answer 5

 Subjected to high temp. to denature or separate single stranded DNA
 Primers designed to complement sequences that flank each end of DNA in 3’-5’ direction
 Allowed to anneal
 Taq polymerase synthesizes copy of DNA by extending the primers on both ends
 Advantage: Very small amount of template DNA needed
 Disadvantage: Need to know the sequence of the flanking DNA for primer design, error prone

Question 6

qPCR

Answer 6

• used to quantify copy number of specific copy number of a specific gene in two or more samples in real time
• this technique includes a probe which fluoresces only in presence of PCR product
• used to detect levels of infectious agent

Question 7

Restriction fragment length polymorphism (RFLP)

Answer 7

o Individual genomes differ by 1 in every 1000 base pairs
o Some of these occur in the recognition sequences for restriction enzymes
o DNA fingerprinting
o Used in forensic analysis, paternity testing and disease detection
Ex: detection of sickle cell disease and number of restriction sites

Question 8

b. Variable number of tandem repeats (VNTR)

Answer 8

o Varies in individuals
o Regions isolated from genomic samples by flanking restriction sites or through PCR
o Useful in identification and severity of inherited diseases
o Huntington disease, Fragile X syndrome, Frederich Ataxia detection

Question 9

i. Improving on insulin

Answer 9

o Normal human insulin has proline at position 28 and lysine at position 29 at C terminus of B chain
o Lispro reverse position of these aa (made it faster)
o Insulin aspart- proline 28 replaced by aspartic acid
o Mixed with normal insulin, provides longer range of glycemic control

Question 10

 Abciximab

Answer 10

• Inhibits platelet aggregation

Question 11

 Baciliximab

Answer 11

• Prevents rejection of transplanted kidney

Question 12

 Cetuximab

Answer 12

• Treats metastatic colorectal cancer

Question 13

 Infliximab

Answer 13

• Treats autoimmune diseases

Question 14

 Retuximab

Answer 14

• Treats lymphomas, leukemias

Question 15

Diagnosis of HIV

Answer 15

• Specific antibodies to HIV are produced in humans 4-6 weeks after infection
• Immobilized antigens in the wells with the biological samples
• Wash plate to remove unbound antibody and nonspecific antigen-antibody associations
• Add enzyme-linked antihuman IgG second antibody, wash, and add chromogenic substrates
• Development of color indicates presence of anti-HIV antibodies
- after sandwhich ELISA, then western blots to confirm