Molecular Diagnostics (5)

Question 1

Hybridization

Answer 1

• ssDNA binds to another strand of DNA or RNA with complementary sequence to form DNA-DNA hybrid or DNA-RNA hybrid
• used for detection and quantification of target DNA or RNA in sample containing a mixture of other DNA/RNA
• short, ss oligonucleotides (probes) are designed and synthesized
• target DNA first converted to ss DNA, then immobilized on solid support (blotting)
• hybridized to complementary sequences in sample
• southern= DNA-DNA
• northern= DNA-RNA

Question 2

Blotting Technique

Answer 2

1. material separated by gel electrophoresis by size
2. transfer to membrane and material on blot
3. add probe to reveal bands of interest (solid lines= bands reactive with probe)
4. visualize bands (autoradiography)
   - only bands reactive with probes visible (fluorescence) or can use isotope (P32 xrays)

Question 3

Southern Blot

Answer 3

A. target= DNA

B. determines which restriction fragments are associated with a gene

Question 4

Northern blot

Answer 4

A. target = RNA

B. measures size and quantities of mRNA molecules (questions about gene expression)

Question 5

Western blot

Answer 5

A. target= protein

B. measures amount of protein on antibody

Question 6

Eastern blot

Answer 6

A. target= PTM (lipid, carb, phosphorylation)

B. detects PTMs on proteins

Question 7

PCR

Answer 7

1. dsDNA obtained
2. subjected to high temp for denaturation or separation to ssDNA
3. primers designed to complement sequence that flank each end of DNA in 3’-5’ direction
4. allowed to anneal
5. add dNTPs (all 4)
6. Taq polymerase synthesizes copy of DNA by extending primers on both ends
7. reactions carried out in thermocycler

Question 8

Advantages and Disadvantages of PCR

Answer 8

advantages: very small amount of template DNA needed, 10^9 fold amplification from trace amount of DNA
disadvantages: need to know sequence of the flaning DNA for primer design, error prone, amplification of contaminating DNA

Question 9

qPCR (quanititative/real-time)

Answer 9

A. used to quantify copy number of a specific gene in two or more samples in real time
B. in addition to primers, this technique includes a probe which fluoresces only in presence of the PCR product
C. probe usually a complementary oligo with a fluorescent take
-used to detect: levels of an infectious agent; determine levels of gene expression

Question 10

Restriction fragment length polymorphism

Answer 10

• individual genome differs by 1 in every 1000 bp; some of these occur in the recognition sequences for restriction enzymes; when genomes from 2 individuals cleaved with set of restriction enzymes and fragments resolved on a gel, the pattern is different
• used in DNA finger printing
• used in forensic analysis, paternity testing and disease detection
• -> extract DNA, cleave with restriction endonuclease, resolve DNA fragments on agarose gel, transfer DNA to nylon membrane, add radioactive DNA probe and wash it off; expose x-ray film to membrane and see which suspects dna matches evidence dna

Question 11

RFLPs in detection of mutations (sickle cell)

Answer 11

1) Dde 1 restriction enzyme cuts at different places and you get two fragments
- -> BUT…with sickle cell (Glu–> Val= clumping), one site is eliminated and so you get two cuts and one fragment
2. when you line up dna and cut with restriction enzymes, run fragments in agarose gel, blot transfer to membrane, and design probe (at region where you expect change to occur)

Question 12

How to Diagnose Huntington’s Disease:

Answer 12

-VNTR (variable number of tandem repeats)

1. Huntington’s Diagnosis: normally have CAG repeated; but with Huntington’s have a very LARGE NUMBER of CAG copies
   how to determine:

a. allele 1 and allele 2 looked at; if have HD have larger number
- P32 is the internal labelineg mechanism
b. digest with RE, resolve DNA on gel
c. expose x-ray film to dried gel
d. higher number of repeats in HD seen because the more repeats the bigger they are and the slower they migrate!!!)

Question 13

Variable number of tandem repeats (VNTR)

Answer 13

a. pattern of short tandem repeats (STR) occurs in genome but varies in individuals
b. VNTR repeat regions isolated from genomic samples by flanking restriction sites or through PCR
c. useful in identification and severity of inherited diseases–> Huntington’s disease, Fragile X syndrome, Frederich Ataxis

Question 14

Improving on Insulin (correlation box)

Answer 14

-can make modified forms of insulin with better activity
-normal human insulin has proline at position 28 and lysine at position 29 at C terminus of B chain
A. Lispro (Eli Lilly) reverse position of these 2 aa
B. Insulin Apart (Novo Nordisk)- proline 28 replaced by aspartic acid
C. Both Lispro and Insulin apart are faster acting, more readily absorbed
D. if mix with normal insulin, provides longer range of glycemic control
a. if mix longer and shorter forms of Insulin–> helps patients monitor their glucose

Question 15

Antibodies

Answer 15

• monoclonal antibodies are the most used and are specific for a specific protein
• to make immortilized tumor cells: take single clones of B-lymphocytes and fuse with tumor cells; then divide cells

-spleen cells (have diff colors) - fusion with myeloma cells allows them to divide indefinitely; culture in HAT medium selective for positive cells and harvest monoclonal antibodies

Question 16

ELISA

Answer 16

1. microplane pre-coatedwith captured antibody, which is immobilized to plate’s surface
2. any analyte (ex. target protein) from standard or biological samples will be captured (bound) to the antibody when added to the well
3. unbound/ nonspecifically bound molecules washed away
4. second antibody conjugated with HRP added and binds to captured analyte
5. after unbound second antibody washed away, HRP substrate, TMB, is added to wells
   a. blue color develops proportionally to amount of analyte in sample

Question 17

ELISA application for HIV

Answer 17

-indirect ELISA

• specific antibodies to HIV are produced in humans 4-6 weeks after infection
  - ELISAs are used for detection due to their efficiency, sensitivity and cost effectiveness
  - Can produce false-positives and false- negatives
  - Need confirmation with Western blotting
  - use IgG secondary antibody

Question 18

ELISA application for HIV

Answer 18

-indirect ELISA (measures amount of antibody in sample)

• specific antibodies to HIV are produced in humans 4-6 weeks after infection
  - ELISAs are used for detection due to their efficiency, sensitivity and cost effectiveness
  - Can produce false-positives and false- negatives
  - Need confirmation with Western blotting
  - use IgG secondary antibody

Question 19

ELISA application for MI detection

Answer 19

• sandwich ELISA (measures amount of antigen in sample)
• troponin is a protein present in striated muscle such as heart; attached to tropoymysin and contains 3 subunits (I, C, and T
  - cardiac forms of T and I increase in acute myocardial infarction and this is measured by ELISA
  - serves as useful markers if ischemia(inadequate supply of blood) along with other protein markers

Question 20

ELISA application for pregnancy tests

Answer 20

sandwich ELISA (measures amount of antigen in sample)

				a. reaction site: free hCG antibodies;  bind to hCG in urine complex moves to test site
				b. Test site: has immobilized hug antibody;  hug/ab complex from reaction site binds to immobilized hug antibody; sandwich complete- dye gives color
				c. control site non-specific antibody immobilized; dye gives color regardless of +/- of hCG;  confirms test is working

Question 21

Western Blot

Answer 21

-used to detect levels of a target protein in a biological sample of proteins

• Steps:
  1. SDS-page: separate out proteins based on size (gel electrophoresis)- small move faster
  2. transfer proteins from gel to nitrocellulose membrane to make them surface exposed
  3. add primary antibody
  4. add secondary antibody (with enzyme tag) that binds primary antibody
  5. add substrate
  6. color is shown