molecular diagnostics

Question 1

What is hybridization? what does it entail?

Answer 1

Used to detect and quantify target DNA or RNA

Must know sequence of pathogen

Single stranded DNA binds to another DNA or RNA to form hybrid

Target DNA first converted to SSDNA then immobilized

Question 2

Southern blot used for what?

Answer 2

DNA & nucleic acids

Question 3

Northern blot: used for what?

Answer 3

RNA size and quantities

Question 4

western blot: used for what?

Answer 4

proteins

Question 5

Eastern blot used for what?

Answer 5

PTM

Question 6

PCR, what does it entail?

Answer 6

Double stranded DNA obtained from source, make many copies of dna

Denatured at high temp
Primers designed to complement source DNA
Allowed to anneal (binding to DNA)
Addition of dNTPs (dAtp,dTtp,dCtp,dGtp)

Uses Taq Polymerase
DNA doubles after each cycle

Question 7

positives and negatives of pcr?

Answer 7

Advantages: very small amount of template DNA needed, quick amplification of DNA

Disadvantages: need to know sequence of the flanking DNA or primer design, error prone, amplification of contaminating DNA

Question 8

RT-qPCR

Answer 8

used to quantify copy number of a specific gene in two or more samples in real time

Detects levels of an infectious agent
Determine levels of gene expression

In addition to primers, this techniques uses a fluorescent probe only in presence of PCR product
-Probe usually a compliment with a fluorescent tag

Question 9

what are variations in DNA used for?

Answer 9

Prenatal diagnosis (amniocentesis)
Newborn screening (IEM)
Genetic carriers

forensics

Question 10

Restriction fragment length polymorphism (RFLP)

, what is it used for?

Answer 10

DNA fingerprinting

Used in forensic analysis, paternity testing and disease detection

Question 11

Restriction fragment length polymorphism (RFLP), how does it work?

Answer 11

RFLPs are recognition sequences for restriction enzymes
By running DNA through a blot, similarities are observed
Can also be used to detect mutations

Question 12

how often do genomes differ?

Answer 12

Genomes differ 1 in every 1000 base pairs

Question 13

Variable number of tandem repeats (VNTR), what are they?

Answer 13

Pattern of short tandem repeats (STR) occurs in genome but varies in individuals

Useful in Identifying and severity of inherited disease
Huntington disease, fragile X syndrome, Frederick ataxia

Question 14

Use of RLFP and VNTR in DNA typing ?

Answer 14

Prenatal diagnosis
Newborn screening
Carriers of disease

Question 15

Production of recombinant proteins, what is it and how is it done?

Answer 15

Large scale production of recombinant proteins

cDNA of protein inserted into expression vectors and engineered to allow high levels of replication, transcription and translation to occur in a host
Large scale production and purification

Question 16

what are recombinant proteins used to make?

Answer 16

Insulin
Growth hormone
Erythropoietin
Clotting factors
Vaccines

Question 17

how has insulin been improved?

Answer 17

Normal human insulin has proline @position 28 and Lysine @position 29 on C terminus of B chain

Insulin Aspart (Novo Nordisk) -Proline 28 replaced by Asp

Both faster acting and more readily absorbed

Mix with normal insulin provides longer range of glycemic control

Question 18

why are antibodies produced?

Answer 18

• Used as pharmacological drugs
• Used for research purposes

Monoclonal antibody (ab)- specific for a single epitope on antigen

Single clones of B lymphocytes fused with a tumor cell to make it immortalized

Humanization minimizes immunogenicity to prolong half life in patient

Question 19

steps in antibody production?

Answer 19

• Immunization of mouse with immunogen peptide/protein
• Antibody secreting cells harvested from spleen (B cells, plasma cells)
• Fusion using eg PEG detergent
• Tissue culture -> immortalized tumor cells + abs= hybridoma secreting antibody
• Hybridomas screened for antibody production
• Ab producing hybridoma cloned
• Most efficient hybridoma clones producing most effective abs are chosen, harvested, and stored in liquid nitrogen and culture for production of abs when required

Question 20

What does Elisa do?

Answer 20

Technique that tests for levels of specific antigen or antibody concentrations
Uses a corresponding antibody or antigen

Probe patient serum
Antigen or antibody is conjugated to a reactive enzyme (color-changing)
Antigen/antibody present -> color change observed

Question 21

what is indirect elisa?

Answer 21

measures antibodies

Question 22

what is sandwich elisa?

Answer 22

measures antigen

Question 23

Measurement of cardiac-specific troponin isoforms, why is this relevant to elisa?

Answer 23

Troponin is the antigen in MI detection
Use sandwich ELIZA
Cardiac forms of T and I increase in MI

Question 24

.Diagnosis of HIV infection, how is it done?

Answer 24

Specific antibody to HIV are produced
Indirect ELIZA is used
Can produce false-positives and false-negatives
Need confirmation with western blot

Question 25

Hormone immunoassays, what do they do?

Answer 25

Measures levels of HCG

HCG is the antigen

Free HCG antibodies bind to HCG in urine complex moves to test site

Test site: immobilized HCG ab
Sandwich ELIZA

Question 26

how is westernblot done?

Answer 26

• detects level of target protein
• SDS-PAge separates out proteins on a gel by using electric field
• transfer proteins to nitrocellulose membrane
• add primary antibody
• add secondary antibody-add substrate
• color shows

Question 27

Early detection/confirmation of HIV infection, how?

Answer 27

Confirmation of HIV

Circulating levels of HIV p2 surface antigen appear even before antibodies are formed

Question 28

Pregnacy test:, how is it done?

Answer 28

elisa