Lecture 16: Molecular Diagnostics

Question 1

Hybridization and Southern/Northern Blotting

Answer 1

• ssDNA binds to another DNA/RNA complementary sequence to form hybrid
• for detection/quantification of target DNA/RNA
• must know sequence of pathogen

North Blot: probe/target are DNA
South Blot: probe DNA, target RNA

Question 2

What is the purpose of blotting techniques?

Answer 2

• detection/visualization of specific biomolecules and proteins from complex mixture of molecules

Question 3

Target and purpose of Northern, Southern, Western, and Eastern Blots

Answer 3

North: target = DNA, determine restriction fragments associated with gene

Southern: target = RNA, measure size/quantity of mRNA molecules

Western: target = protein, measure protein/Ab amount

Eastern: target = PTM, detect post-translation mods

Question 4

Polymerase Chain Reaction (PCR)

Answer 4

• dsDNA obtained from patient, use primers designed to flank each end of DNA
• Taq Polymerase - synth DNA copy by extending primers on ends

Adv: small amount of template needed
Dis: need to know sequence of flanking DNA for primer

Question 5

Cell-Free Cloning (PCR)

Answer 5

• amplify isolated DNA regions

- detect microorganisms earlier and detect specific genetic mutations

Question 6

Quantitative PCR (qPCR) and purpose

Answer 6

• quantify copy number of specific gene in two samples
• includes fluorescent probe that fluoresces only in presence of PCR product (complementary oligo)
  purpose: detect infectious agent lvl, determine gene expression lvl

Question 7

Restriction Fragment Length Polymorphisms (RFLP) and Sickle Cell Anemia

Answer 7

• cleave DNA of 2+ individuals with same enzymes that generate fragments; matches will have same fragment lengths
• DNA fingerprinting (forensic analysis, paternity tests, disease detection)
  ex: individuals w/sickle cell anemia will have 2 restriction site cuts instead of 3 (1 fragment vs 2)

Question 8

Variable Number Tandem Repeats (VNTR)

Answer 8

• short tandem repeat pattern varies amongst individuals
• isolated via flanking restriction sites or PCR
• for identification and severity of inherited diseases (Huntington = get longer CAG repeats)

Question 9

Recombinant Proteins

Answer 9

• use plasmid vector to generate large amounts of recombinant proteins
• cDNA of protein inserted into expression vector
• high lvls of replications/transcription/translation
• large scale production/purification

Question 10

Improving Insulin

Answer 10

• can make modified forms with better biological activity
• Lipso reverse position of 28 Proline and 29 Lysine
• Insulin aspart replaces 28 Proline w/aspartic acid
• makes faster acting insulin that is more easily absorbed

Question 11

Antibody Production

Answer 11

• single clones of B lymphocytes fused w/tumor cell to make it immortalized (hybridomas)
• most effective hybridomas grown in culture and harvested for Abs

Question 12

Clinical use of : Abciximab, Baciliximab, Cetuximab, Infliximab, Retuximab

Answer 12

A: inhibits platelet aggregation
B: prevents rejection of kidney transplant
C: treats metastatic colorectal cancer
I: treats autoimmune disease
R: treats lymphomas, leukimias

Question 13

Enzyme-linked Immunosorbent Assay (ELISA)

Answer 13

• tests for lvls of specific antigen or antibody concentrations
• probe w/test antibody or antigen (more color = more Ab or Ag)

Question 14

Indirect ELISA vs Sandwich ELISA

Answer 14

Indirect = amount of antibody in sample
- uses probe Ag and fluorescent anti-Ab

Sandwich = amount of antigen in sample
- uses probe Ab and fluorescent anti-Ag antibody

Question 15

ELISA Application: HIV Detection

Answer 15

• HIV Abs produced 4-6 weeks after infection
• use indirect ELISA (efficient, sensitive, cost-effective)
• can produce FALSE POS/NEG –> need confirmation w/Western blotting

Question 16

ELISA Application: MI Detection

Answer 16

• cardiac forms of troponin T and I are increased in acute MI
• can measure w/ELISA
• serve as makers for ischemia along w/protein markers

Question 17

ELISA Application: Pregnancy Test sites

Answer 17

Rxn Site: free hCG Abs bind to hCG Ags in urine, then complex moves to test site

Test Site: immobilized hCG Abs binds hCG Ab:Ag complex; dye gives off color

Control Site: nonspecific Ab immobilized that catches free Abs; confirms test is working

Question 18

Western Blotting (Immunoblotting) and SDS-PAGE

Answer 18

• detects lvls of target protein in sample of mixed proteins
• SDS-PAGE: separate proteins in gel via electrical field (proteins migrate based on size)
• proteins transferred to nitrocellulose
• add primary Ab, then 2nd Ab (binds to primary; fluorescent)
• add substrate –> give color

Question 19

Applications of Western Blot

Answer 19

• HIV confirmation

- HIV p24 Ag appear before Abs are formed