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Diagnostic Molecular Biology > Ch. 6 - Proteins > Flashcards

Ch. 6 - Proteins Flashcards

1
Q

Purified DNA can be labeled with _______ in vitro.

A

radioisotopes

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2
Q

What are the two methods of labeling purified DNA? Which is stronger and why?

A
  1. DNA polymerase copies DNA in the presence of nt labeled with 32P or chemical tags
  2. Polynucleotide kinase labels one end of the DNA chain with 32P or a chemical marker

Method 1 is stronger because each nt is labeled, giving off a stronger signal. Method 2 only labels the 5’ or 3’ end of the DNA.

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3
Q

Give an example of both radioactive and chemical markers.

A

Radioactive: 32P
Chemical: digoxigenin, biotin, fluorescent)

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4
Q

What are the methods used to label DNA probe?

A
  1. Nick labeling
  2. Nick translation - not used often
  3. Random priming
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5
Q

Nick labeling

A

x

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6
Q

Random priming

A

x

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7
Q

Nucleic acid hybridization

A

Sensitive way to detect specific nt sequences

Consists of DNA denaturation and DNA renaturation

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8
Q

A _______ or an _______ probe is used for the detection/visualization of a particular gene of DNA region.

A

DNA; RNA

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9
Q

DNA probe

A

Used to detect where a gene is on a chromosome
ssDNA
15-1000s of nt long
Carry radioactive or chemical markers

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10
Q

RNA probe

A

Used to detect where a gene is on a chromosome
ssRNA
Made in vitro via transcription from synthetic DNA template
T7 RNA polymerase will turn DNA to RNA via transcription

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11
Q

DNA or RNA probes can be used to find exact matches at _______ conditions.

A

stringent

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12
Q

DNA or RNA can be used to find _______, non-identical matches at _______ conditions.

A

related; non-stringent

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13
Q

Stringent conditions

A

Exact bp match at 45C

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14
Q

Non-stringent conditions

A

Imperfect bp, related but not exact match

35C

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15
Q

Western Blot

A

Detects proteins

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16
Q

Southern Blot

A

Detects DNA
Allows analysis of any specific DNA region or gene
No single bands; looks like a smear

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17
Q

Northern Blot

A

Detects RNA
No restriction digestion required
Look for mRNA to look at protein expression

18
Q

What does it mean if all the DNA looks like a smear at the top of a Southern blot?

A

Digestion incomplete

19
Q

What does it mean if all the DNA looks like a smear at the bottom of a Southern blot?

A

Digestion too long; degradation

20
Q

Is the DNA on an agarose gel ds or ss?

A

Ds

21
Q

How do we make the dsDNA from a gel into ssDNA for blotting?

A

Treat the DNA with NaOH (denaturation) + a depurinating agent

22
Q

Depurinating agent

A

Loosens the ds to aid in denaturation

23
Q

DNA is transferred from a gel to a _______ for blotting techniques.

A

membrane

24
Q

The membrane used for blotting is usually _______ to attract the negatively charged DNA.

A

positively charged

25
Q

Two types of blotting membranes are _______ and _______.

A

nylon; nitrocellulose

26
Q

What are the three ways to transfer DNA to the membrane during blotting?

A
  1. Capillary transfer
  2. Electrical transfer
  3. Vacuum transfer
27
Q

Capillary transfer

A

Method of transferring DNA to membrane during blotting
DNA moves up to the membrane via capillary action
Very slow
Cheap and convenient

28
Q

Electrical transfer

A

Method of transferring DNA to membrane during blotting

Uses an electrical current to pull DNA from gel to membrane

29
Q

Vacuum transfer

A

Method of transferring DNA to membrane during blotting

Suction pulls DNA from gel to membrane

30
Q

Permanent immobilization

A

Bake the membrane or use cross-linking via UV light to covalently attach DNA to the membrane
Prevents loss of DNA

31
Q

Probe

A

ssDNA or ssRNA covalently attached to a radioactive or chemical marker
Longer probes are more specific
Shorter probes are more likely found in many locations and are less specific (better for mutational analysis)

32
Q

What three factors influence stringency and how?

A

Formamide concentration increases stringency
Low salt concentration increases stringency
High temperature increases stringency

33
Q

What happens if stringency is too low? Too high? At a good level?

A

Too low - all DNA binds to probe
Too high - no DNA binds to probe
Good level - only DNA of interest binds to probe

34
Q

What are the two ways of visualizing DNA?

A
  1. Radioactive or chemiluminescent probe - put membrane on a film that captures radiation autoradiography
  2. Chemical markers - detected by an antibody bound to an enzyme (HRP) that produces color or light in the presence of its substrate
35
Q

_______ is the technique used to separate proteins.

A

SDS-PAGE

36
Q

SDS

A

Detergent used to denature proteins and give them a negative charge

37
Q

β-mercaptoethanol

A

Reducing agent that gets rid of disulfide bonds

38
Q

SDS PAGE uses _______ and _______ to denature and unfold proteins for electrophoresis.

A

SDS; β-mercaptoethanol

39
Q

A protein with 2 subunits is processed via SDS-PAGE. What would you expect to see if the protein was denatured with SDS + β-mercaptoethanol? Just with SDS?

A

Two bands with both - β-mercaptoethanol breaks disulfide bonds between the two subunits, separating them.

One band with only SDS, since it only denatures them but doesn’t break the disulfide bonds.

40
Q

Western blotting is also called _______.

A

immunoblotting

41
Q

Monoclonal Ab

A 
Ab made from one clone of a B cell  
Only recognizes one epitope 
Cleaner band (than polyclonal) 
Weaker signal (than polyclonal) 
Specific 
Expensive
42
Q

Polyclonal Ab

A 
Ab made from many clones of a B cell 
Recognizes all epitopes 
Noisier band 
Stronger signal 
Less specific (than monoclonal) 
Cheap

Diagnostic Molecular Biology (22 decks)

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