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MCM Exam 2 > (5) Molecular Diagnostics > Flashcards

(5) Molecular Diagnostics Flashcards

1
Q

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

A
  • Hybridization

- PCR

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2
Q

Hybridization:

Describe

A

Single stranded DNA binds to another strand of DNA or RNA with complementary sequence to form:

DNA-DNA hybrid or DNA-RNA hybrid

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3
Q

Hybridization:

useful for?

A

Detection and quantification of target DNA or RNA

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4
Q

Hybridization:

What is a probe?

A

Short, single-stranded oligonucleotide

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5
Q

Hybridization:

What do you do with Target DNA?

A

Converted to single stranded DNA then IMMOBILIZED on a solid support; called blotting

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6
Q

Hybridization:

What is southern blotting?

A

Probe=DNA

Target=DNA

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7
Q

Hybridization:

What is northern blotting?

A

Probe=Single stranded DNA

Target=mRNA

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8
Q

Hybridization:

What do probes have on them?

A

Labeled with radioactive or fluorescent tag

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9
Q

Blotting Techniques:

Purpose?

A

Detection and visualization of specific biomolecules

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10
Q

Blotting Techniques:

Southern: Target?

A

DNA

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11
Q

Blotting Techniques:

Southern: Purpose?

A

Determine which restriction fragments are associated with a gene

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12
Q

Blotting Techniques:

Northern: Target?

A

RNA

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13
Q

Blotting Techniques:

Northern: Purpose?

A

Measure size and quantities of mRNA molecule

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14
Q

Blotting Techniques:

Western: Target?

A

Protein

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15
Q

Blotting Techniques:

Western: Purpose?

A

Measures amount of protein or antibody

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16
Q

Blotting Techniques:

Eastern: Target

A

PTM (lipid, carb, phosphorylation)

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17
Q

Blotting Techniques:

Eastern: Purpose?

A

Detects post-translational modifications (PTMs) on proteins

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18
Q

Polymerase Chain Reaction (PCR)

Describe

A

DNA subjected to high temp to denature

Primers complement sequences to flank each end of DNA

Allowed to anneal

Add 4 dNTPs

Taq Polymerase; synthesizes copy of DNA by extending the primers on both ends

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19
Q

Polymerase Chain Reaction (PCR)

Advantage?

A

Very small amount of template DNA needed!

10^9 fold amplification from trace amount of DNA

20
Q

Polymerase Chain Reaction (PCR)

Disadvantage?

A

Need to know the sequence of the flanking DNA for primer design, error prone, amplification of contaminating DNA

21
Q

Polymerase Chain Reaction (PCR)

What are the 3 major steps?1

A
  1. Heat to separate
  2. Cool to anneal
  3. DNA synthesis
22
Q

Quantitative PCR (qPCR)

Used for?

A

Quantifying copy number of a specific gene in two or more samples in real time

  • Detect levels of infectious agent
  • Determine levels of gene expression
23
Q

Quantitative PCR (qPCR)

How does it differ from traditional PCR?

A

In addition to primers, includes…

[a probe that fluoresces only in presence of the PCR product]

24
Q

Detections of Variations in DNA sequences:

2 methods?

A
  1. Restriction Fragment Length Polymorphism (RFLP)

2. Variable number of tandem repeats (VNTR)

25
Q

How can determining variations in DNA sequences be useful?

A
  • Forensics

- Diagnostic (amniocentesis, newborn screening, genetic carriers)

26
Q

RFLP:

Thought process behind?

A
  • Genomes differ by 1 in every 1000 base pairs

* Some of these differences occur in the RECOGNITION SEQUENCES for restriction enzymes

27
Q

RFLP:

How are RFLP used?

A
  • DNA fingerprinting
  • Forensic analysis
  • Paternity testing
  • Disease detection
28
Q

RFLP:

Example of variation in restriction sites?

A

-Normal Beta-globulin allele has 3 restriction sites

vs

-Sickle cell only have 2 restriction sites

29
Q

What does VNTR stand for?

A

Variable number of tandem repeats

30
Q

VNTR:

Thought process behind?

A

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

31
Q

VNTR:

Useful for?

A

Identification and severity of inherited diseases

ex: Huntington disease

32
Q

What process do you use to diagnose Huntington’s disease?

A

VNTR

33
Q

What are examples RECOMBINANT PROTEINS that are produced on a large scale?

A
  • Insulin
  • Growth hormone
  • Erythropoietin
  • Clotting factors
  • Vaccines against diseases such as flu and malaria
34
Q

How are recombinant proteins formed?

A

cDNA of the protein inserted into expression vectors

35
Q

How does human synthesized insulin differ from artificial?

A

Human produced insulin: Pro at position 28, Lys at pos 29 at C terminus of B chain

Lispro: reverse position of these 2 aa

Insulin aspart: proline 28 replaced by aspartic acid

36
Q

What are the two artificial insulins?

A

Lispro, Insulin aspart

37
Q

What are the benefits of Lispro and Insulin aspart?

A

Faster acting, more readily absorbed

38
Q

What is ELISA?

A

Enzyme-linked immunosorbent assay (ELISA)

-Immunological technique which tests for levels of specific antigen or antibody concentrations

39
Q

What does:

INDIRECT ELISA measure?

A

Amount of an ANTIBODY in a sample

40
Q

What does:

SANDWICH ELISA measure?

A

Amount of an ANTIGEN in a sample

41
Q

What technique is used to diagnose HIV?

A

**Indirect ELISA

must confirm w/ Western Blotting

42
Q

What technique is used to detect an MI?

A

ELISA;

Cardiac forms of T and I increase in acute myocardial infarction

***Sandwich ELISA

43
Q

What technique is used for pregnancy tests?

A

*** Sandwich ELISA

44
Q

Application of Western Blot?

A

Confirmation of HIV

45
Q

Describe Western Blotting (aka Immunoblotting)

A
  • SDS page separates out the proteins on a gel by applying electrical field
  • Transfer proteins from gel to nitrocellulose
  • Add primary antibody
  • Add secondary antibody (*has enzyme tag)
  • Gives color

MCM Exam 2 (13 decks)

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