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MCM test 2 > Molecular diagnostics > Flashcards

Molecular diagnostics Flashcards

1
Q

Techniques to Detect Infectious Agents and Diagnose Inherited Disorders

A
  • Hybridization

- Polymerase Chain reaction (PCR)

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2
Q

hybridization steps

A

-Single stranded DNA binds to another strand of DNA or RNA with
complementary sequence to form DNA-DNA hybrid or DNA-RNA
hybrid.

-detection and quantification of target DNA or RNA

-single-stranded oligonucleotides (called probes) are
designed and synthesized.

  • target DNA converted to SS and immobolized
  • hybridized to complementary sequences in samples
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3
Q

southern blotting

A
  • both probe and nucleic acid are DNA
    -Determine which
    restriction fragments are
    associated with a gene
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4
Q

northern blotting

A 
probe is ss DNA and target is mRNA
-Measure size and
quantities of mRNA
molecules (questions
about gene expression
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5
Q

western blotting

A

-target is protein
-Measures amount of
protein or antibody

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6
Q

polymerase chain reaction (PCR) steps

A

-Double stranded DNA (ds DNA) obtained from a
patient/individual/pathogen
-denature
-primers flank end of DNA
-primers anneal
-add all 4 dNTPS
-taq polymerase : synthesizes copy of DNA

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7
Q

PCR advantage

A

-Very small amount of template DNA needed, 109-fold

amplification from trace amount of DNA

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8
Q

PCR disadvantage

A
  • need to know sequence of flanking DNA for primer design
  • error prone
  • amplification of contaminating DNA
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9
Q

quantitative PCR (qPCR) overview

A 
-Used to quantify copy number of a
specific gene in two or more
samples in real time.
- In addition to primers, this
technique includes a probe which
fluoresces only in presence of the PCR product
-Probe usually a complementary
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10
Q

what is qPCR used for?

A
  • Detect levels of an infectious agent

- Determine levels of gene expression

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11
Q

what can you use to detect variations in DNA sequences

A

1) Restriction Fragment Length Polymorphism (RFLP)
2) Variable number of tandem repeats (VNTR)
- forensic and diagnostic

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12
Q

Restriction Fragment Length

Polymorphism (RFLP)

A

-diff in genomes based on recognition for restriction enzyme
-DNA fingerprinting
-Used in forensic analysis,
paternity testing and disease
detection

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13
Q

what can you use to detect mutations in offspring passed on by parent?

A

RFLP

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14
Q

Variable number of tandem repeats (VNTR)

A

-Pattern of short tandem repeats (STR) occurs in
genome but varies in individuals
-Useful in identification and severity of inherited
diseases
-Huntington disease, Fragile X syndrome,
Frederich Ataxia

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15
Q

How could you diagnose Huntington’s disease?

A

using VNTR

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16
Q

what are recombinant proteins used for?

A 
medications such as:
Insulin
 growth hormone
 erythropoietin
 clotting factors
 vaccines against diseases such as flu
and malaria, viral infections
17
Q

how are recombinant proteins produced?

A
  • take plasmid of gene of interest and insert cDNA into expression vector
  • bacteria internalize plasmid with gene of interest
  • grow bacteria to make more plasmic
  • gene translated to protein
18
Q

what are two modifications that improve the action of insulin

A

-lispro: reversed postion of 2 a.a on normal human insulin

-Insulin aspart (Novo Nordisk) – proline 28 replaced by aspartic acid.
-Both Lispro and Insulin aspart faster acting, more readily absorbed.
-Mix with normal insulin, provides
longer range of glycemic control

19
Q

monoclonal Antibodies

A

specific for a

single epitope on antigen

20
Q

what is the production of antibodies in lab used for?

A

drugs (cancer,
rheumatoid arthritis, Crohn’s
disease, hepatitis)

21
Q

Enzyme-linked Immunosorbent Assay

ELISA

A

-tests for levels of specific antigen or antibody concentrations

22
Q

Indirect ELISA

A

measures amount of antibody in sample

23
Q

sandwich ELISA

A

measures amount of antigen in sample

24
Q

what technique could you use to diagnose HIV

A

indirect ELISA

-looks at amount of antibody formed

25
Q

what technique could you use to diagnose MI

A

sandwich ELISA

-mesures antigen (cardiac forms of T and I increase in acute myocardial infarction

26
Q

what technique do pregnancy tests use?

A

sandwich ELISA

  • looks for hCG which is an antigen
  • antibody built in stick
27
Q

what is Western Blotting (immunoblotting) used for?

A

-to detect levels of target protein

28
Q

what are steps to western blotting

A
  • SDS page to separate proteins
  • transfer proteins from gel to nitrocellulose
  • add primary antibody
  • add secondary antibody
  • secondary antibody= enzyme tag which gives color
29
Q

after diagnosing HIV how could you CONFIRM it

A

western blot

MCM test 2 (16 decks)

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