Lec 4 molecular diagnsostics

Question 1

What are some techniques to detect infectious agents and diagnose inherited disorders?

Answer 1

Hybridization

polymerase chain reaction (PCR)

Question 2

What must be known for hybridization and PCR?

Answer 2

know the sequence of the pathogen or gene

(5000 pathogens sequenced human genome sequenced)

Question 3

What is hybridization

Answer 3

single stranded DNA binds to DNA or RNA with complementary sequence DNA-DNA hybrid

or DNA-RNA hybrid

Question 4

how does hybridization happen?

Answer 4

target DNA converted to single stranded DNA and imobilized (blotting)

single-stranded oligonucleotides (probes)

added and attach to complimentary region

Question 5

Southen Blotting

Answer 5

probe and target nucleic acid are DNA

determine which restriction frags are associated with a gene

Question 6

Northern blotting

Answer 6

probe is single strand DNA and target is mRNA

measure size and quantities of mRNA molecules

Question 7

western blotting

Answer 7

target is a protein

measure amount of protein

Question 8

Eastern blotting

Answer 8

PTM (post translational modifications is target

Question 9

PCR

Answer 9

polymerase chain reaction

1. double-stranded DNA from target
2. high temp denature/separate
3. primers complement sequences in flank each end of DNA (3’-5’)
4. anneal (bind DNA)
5. add deoxy nucleotide triphosphates (dNTPS) all 4
6. Taq polymerase synthesizes copy of DNA from primer
7. thermocycler (does temp, number of cycles)

Question 10

advantages and disadvantages of PCR

Answer 10

A-small amount of template DNA needed to amplify

D-need to know sequence of flanking DNA for primer, error prone, amp of contaminating DNA

Question 11

qPCR

Answer 11

quantitative PCR

quantify copy number

primers added and a probe which flouresces in presence of PCR product

(complementary oligo with tag)

Detect levels of infectious agent/ levels gene express

Question 12

Techniques used to detect variation in DNA sequences

Answer 12

Restriction Fragment Length Polymorphism (RFLP)

Variable number of tandem repeats (VNTR)

Question 13

RFLP

Answer 13

restriction fragment length polymorphism

genomes differ 1 in 1000 base pairs

some in recognition sequences for restriction enzyme

cleaved with restriction enzyme show two differnt pattern for different genomes

DNA fingerprinting

Question 14

What is RFLP used in?

Answer 14

forensic analysis

paternity testing

disease detection

Question 15

VNTR

Answer 15

variable number of tandem repeats

short tandem repeats in genome but varies by individual

VNTR repeat regions isolated by flanking restriction sites though PCR

Question 16

What is VNTR used in?

Answer 16

identification and severity of inherited diseases

Huntington disease!! fragile x syndrome frederich ataxia

Question 17

recombinant proteins examples

Answer 17

• insulin
• growth hormone
• erythropoietin
• clotting factors
• vaccines (flu/viral)

Question 18

What makes a recombinant protein

Answer 18

cDNA of protein inserted into expression vectors

for ex. plasmid vector from bacteria which is then replicated by bacteria making proteins and end products

Question 19

monoclonal antibody targets and uses

Answer 19

• inhibit platelet agg
• prevents rejection transp kidney
• treat metastatic colorectal cancer
• autoimmune diseases treat
• treat lymphomas, leukemias
• (many uses)

Question 20

ELISA

Answer 20

enzyme-linked immunosorbent assay

tests for levels of specific antigen or antibody concentrations using anitbody or antigen

Question 21

Indirect ELISA

Answer 21

Question 22

Sandwich ELISA

Answer 22

Question 23

ELISA applications

Answer 23

1. Diagnose HIV
2. MI detection
3. pregnancy test

Question 24

ELISA and HIV

Answer 24

(indirect ELISA)

antibodies to HIV produced 4-6 weeks

efficient, sensitive, cost efficient

NEED CONF with western blotting

Question 25

ELISA and MI detection

Answer 25

cardiac form of troponin increase (antigen) (sandwich ELISA) which binds to the antibody

Question 26

ELISA and pregnancy test

Answer 26

Sandwich ELISA hCG antibodies (free) bind hCG in urine complex moves to test site that has immoblized hCG antibody sandwich complete dye gives color control site dye gives color regardless (non-specific antibody) confirm test working

Question 27

Western Blotting

Answer 27

immunoblotting * transfer protein to SDS page * add primary antibody (binds target) * wash excess * add secondary antibody (binds primary)(enzyme tag) * wash excess * use visualization to see enzyme tag

Question 28

application of western blot?

Answer 28

confirmation of HIV circulating HIV p24 surface antigen (proteins) and other antigens (proteins) can use controls to compare samples detect antigens