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MCM II > Lec 4 molecular diagnsostics > Flashcards

Lec 4 molecular diagnsostics Flashcards

1
Q

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

A

Hybridization

polymerase chain reaction (PCR)

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2
Q

What must be known for hybridization and PCR?

A

know the sequence of the pathogen or gene

(5000 pathogens sequenced human genome sequenced)

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3
Q

What is hybridization

A

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

or DNA-RNA hybrid

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4
Q

how does hybridization happen?

A

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

single-stranded oligonucleotides (probes)

added and attach to complimentary region

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5
Q

Southen Blotting

A

probe and target nucleic acid are DNA

determine which restriction frags are associated with a gene

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6
Q

Northern blotting

A

probe is single strand DNA and target is mRNA

measure size and quantities of mRNA molecules

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7
Q

western blotting

A

target is a protein

measure amount of protein

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8
Q

Eastern blotting

A

PTM (post translational modifications is target

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9
Q

PCR

A

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)
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10
Q

advantages and disadvantages of PCR

A

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

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11
Q

qPCR

A

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

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12
Q

Techniques used to detect variation in DNA sequences

A

Restriction Fragment Length Polymorphism (RFLP)

Variable number of tandem repeats (VNTR)

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13
Q

RFLP

A

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

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14
Q

What is RFLP used in?

A

forensic analysis

paternity testing

disease detection

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15
Q

VNTR

A

variable number of tandem repeats

short tandem repeats in genome but varies by individual

VNTR repeat regions isolated by flanking restriction sites though PCR

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16
Q

What is VNTR used in?

A

identification and severity of inherited diseases

Huntington disease!! fragile x syndrome frederich ataxia

17
Q

recombinant proteins examples

A
  • insulin
  • growth hormone
  • erythropoietin
  • clotting factors
  • vaccines (flu/viral)
18
Q

What makes a recombinant protein

A

cDNA of protein inserted into expression vectors

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

19
Q

monoclonal antibody targets and uses

A
  • inhibit platelet agg
  • prevents rejection transp kidney
  • treat metastatic colorectal cancer
  • autoimmune diseases treat
  • treat lymphomas, leukemias
  • (many uses)
20
Q

ELISA

A

enzyme-linked immunosorbent assay

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

21
Q

Indirect ELISA

A
22
Q

Sandwich ELISA

A
23
Q

ELISA applications

A
  1. Diagnose HIV
  2. MI detection
  3. pregnancy test
24
Q

ELISA and HIV

A

(indirect ELISA)

antibodies to HIV produced 4-6 weeks

efficient, sensitive, cost efficient

NEED CONF with western blotting

25
Q

ELISA and MI detection

A

cardiac form of troponin increase (antigen)

(sandwich ELISA)

which binds to the antibody

26
Q

ELISA and pregnancy test

A

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

27
Q

Western Blotting

A

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
28
Q

application of western blot?

A

confirmation of HIV

circulating HIV p24 surface antigen (proteins)

and other antigens (proteins)

can use controls to compare samples detect antigens

MCM II (11 decks)

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