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025. My MCM Test 2 > Moleuclar Diagnostics > Flashcards

Moleuclar Diagnostics Flashcards

1
Q

What are the two reasons for molecular techniques, given you know the sequence of the pathogen (1) or gene (2)?

A
  1. To rapidly detect/identify disease causing organism

2. To dignose inherited disorders in humans

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2
Q

What is hybridization?

A

When ssDNA binds to another strand of DNA or RNA (complementary sequence) to form DNA-DNA or DNA-RNA hybrids.

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3
Q

What is hybridization useful for?

A

For detection and quantification of target DNA or RNA in a sample containg a mix of both

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4
Q

What are single-stranded oligonuclotides called?

A

probes that are labeled with radioactive or fluorescent tags

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5
Q

What is southern blotting?

A

DNA+DNA, both probe and target nucleic acid are ssDNA

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6
Q

What is northern blotting?

A

DNA +RNA, probe is ssDNA and target is mRNA

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7
Q

What is the purpose of southern blotting?

A

to determine which restriction fragments are associated with a gene

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8
Q

What is the purpose of northern blotting?

A

measure size and quantities of mRNA molecules (gene expression)

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9
Q

What is the purpose of a western blot?

A

to measure the amount of protein or antibody

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10
Q

Steps of blotting techniques?

A

DNA or RNA is electrophoresed in agarose gel to separate and then transfered to membrane so surface exposed. Probe DNA/RNA is added and hydrolyzed to ssDNA

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11
Q

What is polymerase chain reaction (PCR)?

A

A process to amplify a small amount of dsDNA to however large you would like.

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12
Q

What is the process of PCR?

A
  1. Denature DNA with high temps to break H bonds
  2. Annealing: sample cools, anneals primer to both strands (3’-5’)
  3. TAQ polymerase (heat stable) & all 4dNTPs added to synthesize DNA
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13
Q

What is the final product of PCR?

A

DNA doubles in each cycle and can quickly multiple cycle after cycle (in thermocycler)

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14
Q

Disadvantages vs advantages of PCR?

A

A:Only need small amount of template DNA
Dis: Need to know sequence for flanking, error prone, contamination

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15
Q

Difference between PCR and qPCR (quantitative)?

A
  1. Used to quantify copy number

2. Uses probe that fluoresces only in presence of PCR product, by primer

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16
Q

What is qPCR used for?

A

detect levels of an infectious agent and determine levels of gene expression

17
Q

What are restriction fragment length polymorphism (RFLP) and variable number tandem repeats (VNTR) used for?

A

Diagnostics and forensics

  • prenatal dianosis
  • newborn screening
  • genetic carriers
18
Q

RFLP use?

A

Restriction enzymes used to match suspect DNA with evidence DNA. If the two are the same DNA, endonucleases will cut the DNA at the same area. Southern blotting then used to trasnfer to membrane, add probe (radioactive)

19
Q

How many bans will you have if there are 3 restriction sites? (where endonucleases will cut)
2 sites?

A

3 sites = 2 bans
2 sites = 1 ban
So if disease has 2, normal 3, and you run RFLP+ blotting and have 1 ban, you will have the disease

20
Q

When running gel electrophoresis, what proteins will be at the top and which will be at the bottom of the gel post electrophoresis?

A

Large proteins will be at the top of the gel, small at the bottom

21
Q

What is variable number of tandem repeat (VNTR) and what is it useful for?

A

Pattern of short repeats, they can be isolated by restriction sites or pcr.
Useful in identification and severity of disease (HD)

22
Q

Process of VNTR for HD?

A

Take healthy and HD DNA, do PCR, probe with Alpha32P (phosphorus uses xray), # of repeats will be represented, small at bottom, large at top

23
Q

Generally, how are medications made?

A

Using recomibinant proteins to make insulin, growth hormone, clotting factors, vaccines

24
Q

Example of Recombinant protein steps with insulin?

A
  1. Human insulin gene cut from DNA from pancreas
  2. bacterial plasmid DNA cut with restriction enzymes and insulin added to vector to form recomb. DNA
  3. REcombinant Bact multiply in fermentation tank, producing insulin
  4. insulin extracted and purified
25
Q

Insulin has been improved by switching what to AAs in chain B? By switching what for aspartic acid?

A

Changed proline28 and lysine29 at C term of B (lispro)
Insulin aspart: switched proline 28 with aspartate
both of these drugs act faster and more readily absorbed

26
Q

How are monoclonal antibodies made?

A
  1. Antigen injected to mouse, produces antibodies (many epitopes)
  2. combined with myeloma tumor cells to form hybridomas
  3. culture in HAT medium selective for positive cells
  4. Harvest monoclonal antibodies
27
Q

What is ELISA and what are the two types?

A

It is a immunological technique which tests for levels of specific antigen or antibody concentrations in biological samples using a corresponding antigen/antibody

  1. indirect
  2. sandwich
28
Q

What are the steps for generalized ELISA principle

A
  1. Immobilized antibodies bound to plate
  2. Biological antigens added
  3. Plate washed to remove unbound/nonspecific antigens
  4. Second antibody HRP enzyme bound added to mix, binding to captured antigen-antibody
  5. Chromogenic substrates and enzyme added for color to develop
    * darker/more defined color = higher amount of bound anylate* rate of color formation = amount of specificity
29
Q

what does indirect ELISA measure?

A

Measures the amount of an antibody in a sample, starts with antigen coated well

30
Q

What does sandwich ELISA measure?

A

Measures the amount of an antigen in a sample, starts with antibody coated well

31
Q

What ELISA is used to determine HIV?

A

Use indirect with HIV antigens bound to coat, if person has HIV their biological sample antibodies will attach and fluorese post-procedure

32
Q

What ELISA is used to determine MI?

A

Sandwich, uses immobilized antibody coated well, adds sample, will fluorese if had MI

33
Q

What ELISA is used to determine preganancy?

A

Sandwich, immob antibody, if HCG (pregnant) attaches, will turn the stick a color (fluoreses)

34
Q

What are the steps for western blotting?

A

Always antigen

  1. Proteins run on gel by electrophoresis (acryl)
  2. transfered to membrane
  3. Primary antibody added, binds to target protein
  4. wash, add secondary antibody, binds to primary
  5. wash, add visualization agents
  6. results: target protein have the darkest thickest line!

025. My MCM Test 2 (15 decks)

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