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RESET - MOLBIO PRELIMS > 4. LEC > Flashcards

4. LEC Flashcards

1
Q
  • It is a process of establishing noncovalent and sequence specific interaction between two or more complementary strands of nucleic acid into a single hybrid
A

HYBRIDIZATION

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2
Q

are synthetic single stranded DNA molecule that can recognize and specifically bind to a target DNA by complementary base pairing.

A

DNA PROBE / GENE PROBE

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3
Q

Hybridization technology that is used for Gene structure

A

Southern blot

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4
Q

Hybridization technology that is used for Transcript structure, processing, gene expression

A

Northern blot

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5
Q

Hybridization technology that is used for Protein processing, gene expression

A

Western blot

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6
Q

Hybridization technology that is used for DNA-binding proteins, gene regulation

A

Southwestern blot

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7
Q

Hybridization technology that is used for Modification of western blot using enzymatic detection (PathHunter); also, detection of specific agriculturally important proteins

A

Eastern blot

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8
Q

Hybridization technology that is used for Transfer of high-performance liquid chromatography (HPLC)-
separated lipids to polyvinyl difluoride (PVDF) membranes for analysis by mass spectrometry

A

Far-eastern blot 29,30

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9
Q

Southern blot target

A

DNA

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10
Q

Northern blot target

A

RNA

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11
Q

Western blot target

A

Protein

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12
Q

Southwestern blot target

A

Protein

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13
Q

Eastern blot target

A

Protein

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14
Q

Far-eastern blot 29,30 target

A

Lipids

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15
Q

Southern blot probe

A

Nucleic acid

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16
Q

Northern blot probe

A

Nucleic acid

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17
Q

Western blot probe

A

Protein

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18
Q

Southwestern blot probe

A

DNA

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19
Q

Eastern blot probe

A

Protein

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20
Q

Far-eastern blot 29,30 probe

A

None

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21
Q

Who is the Southern blot named after?

A

Edwin Southern

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22
Q

What is the first step in the Southern blot procedure?

A

Isolate and cut genomic DNA with restriction enzymes

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23
Q

How are DNA fragments separated in Southern blotting?

A

Gel electrophoresis

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24
Q

What is done to the DNA fragments after separation? [southern blot]

A

They are denatured and transferred to a solid support

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25
Q

Example of solid support used for southern blot after DNA fragments are denatured

A

nitrocellulose

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26
Q

What probe is used to detect DNA fragments?

A

Labeled probe (complementary DNA or RNA)

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27
Q

How is the presence of a DNA sequence determined? [southern blot]

A

By detecting the signal from the probe

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28
Q

What was the original method of detection in Southern blotting?

A

Radioactively labeled probe

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29
Q

What types of DNA regions are analyzed using Southern blot?

A

Large regions (10 kb to over 100 kb)

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30
Q

What has replaced Southern blot for many applications?

A

PCR (for many, but not all, applications)

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31
Q

What does Southern blot analyze at the molecular level?

A

Any gene or gene region in prokaryotes or eukaryotes

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32
Q

What is Southern blot still commonly used for?

A

Characterization of large DNA regions

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33
Q

What is the first step in the Southern blot procedure?

A

Digestion of test DNA with restriction enzymes.

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34
Q

What determines the choice of restriction enzymes in Southern blotting?

A

Genetic locus and application.

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35
Q

What is the result of incomplete restriction enzyme cutting in Southern blotting?

A

anomalous patterns

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36
Q

How are DNA fragments resolved after restriction enzyme digestion in Southern blotting?

A

Gel electrophoresis.

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37
Q

What determines the percentage and type of gel used in Southern blotting?

A

DNA fragment size.

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38
Q

What should be run alongside test samples during electrophoresis in Southern blotting?

A

Molecular-weight standard

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39
Q

How are large DNA fragments (10,000-20,000 bp) best resolved in Southern blotting?

A

0.7% agarose gel, low voltage, long run.

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40
Q

What does a smear on an ethidium bromide-stained gel illuminated with UV light indicate during Southern blotting?

A

Complete digestion.

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41
Q

What does a large aggregate of DNA near the top of the gel lane indicate in Southern blotting?

A

Incomplete digestion.

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42
Q

What does a smear primarily located in the lower region of the gel lane indicate in Southern blotting?

A

DNA degradation.

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43
Q

What should be done if uncut or degraded DNA is present after electrophoresis in Southern blotting?

A

Repeat digestion or purify DNA.

44
Q

How much genomic DNA is typically required for each restriction enzyme digestion in Southern blotting?

A

10-50 µg.

45
Q

What should be done if multiple restriction enzymes are used for Southern blotting?

A

Digest samples separately.

46
Q

How long is the typical digestion time for complete DNA cutting in Southern blotting?

A

3+ hours.

47
Q

What is the goal of the Southern blot procedure?

A

Analyze specific DNA region.

48
Q

How are target DNA fragments detected in Southern blotting?

A

Hybridization with complementary probe.

49
Q

What must happen to double-stranded DNA fragments before hybridization in Southern blotting?

A

Denaturation.

50
Q

is used to prepare larger DNA fragments (over 500 bp) by removing purine bases, making them easier to denature.

A

Depurination

51
Q

What is used to depurinate DNA fragments in Southern blotting?

A

Dilute HCl

52
Q

How is DNA denatured in Southern blotting?

A

Exposure to sodium hydroxide (NaOH).

53
Q

What is the role of sodium hydroxide in denaturation?

A

Breaks hydrogen bonds.

54
Q

Common membranes used in Southern blotting?

A

Nitrocellulose,
nylon,

cellulose modified with:
diethyl aminoethyl,
carboxymethyl (CM).

55
Q

are used for immobilizing proteins for probing with antibodies
(western blots).

A

polyvinyl difluoride (PVDF)

56
Q

Nitrocellulose-based membranes bind ____________ of nucleic acid per square
centimeter

A

70 to 150 μ g

57
Q

Membrane pore sizes that are
suitable for DNA fragments from a few hundred bases up to those greater than 20,000 bp in length.

A

0.05 to 0.45 μ m

58
Q

has a high binding capacity for proteins as well as nucleic acids. It is the most versatile medium for molecular transfer applications. It is also
compatible with different transfer buffers and detection systems

A

Pure nitrocellulose

59
Q

Membrane used in southern blot that is more appropriate for applications where multiple probings may be necessary

A

Reinforced nitrocellulose

60
Q

These membranes can be formulated with a net neutral charge to decrease nonspecific binding when performing southern blot .

A

Mechanically stable membranes

61
Q

A covalent attachment of nucleic acid to these membranes [southern blot] is achieved by exposure of the DNA on the membrane with _____________

A

UV-light cross-linking

62
Q

Are more effective when binding small fragments of DNA.

These membranes, however, are more likely to retain protein or other contaminants that will contribute to background noise after the membrane is probed.

A

Membranes with a positive charge

63
Q

What transfer method did southern use in order to transfer the DNA sample from the gel to the nitrocellulose membrane

A

Capillary transfer

64
Q

What types of buffers can be used during capillary transfer to facilitate DNA movement?

A

10 × saline sodium citrate (10X SSC) or

commercially available transfer buffers.

65
Q

What should be avoided during the transfer to ensure effective binding of DNA to the membrane?

A

Folding or creasing of the membrane.

66
Q

What type of cellulose effectively binds nucleic acids and negatively charged proteins?

A

Diethylaminoethyl (DEAE)-conjugated cellulose.

67
Q

What types of membranes are used specifically for protein (western) blotting?

A

PVDF and charged carboxymethyl cellulose membranes.

68
Q

How do PVDF and charged carboxymethyl cellulose membranes bind nucleic acids and proteins?

A

Hydrophobic and ionic interactions.

69
Q

What is the binding capacity range for PVDF membranes?

A

20 to 150 μg/cm².

70
Q

Types of transfer methods

A

Capillary transfer
electrophoretic transfer
Vacuum transfer

71
Q

electrophoretic transfer two types

A

Tank
Semidry

72
Q

A simple and inexpensive method that requires no instruments, but can be less than optimal, especially with large gels. Bubbles and salt crystals between the membrane and gel can lead to information loss or staining artifacts. The procedure can take from a few hours to overnight for large fragments.

A

Capillary transfer

73
Q

This method uses electrodes placed above and below the gel, transferring electric current through the gel and membrane using electrophoresis buffer, allowing the DNA to move to the membrane.

A

Tank method.

74
Q

In this approach, electrodes contact the gel-membrane sandwich directly, only requiring enough buffer to soak the gel and membrane. This method is often used for smaller proteins.

A

Semidry method.

75
Q

This method is preferred for transferring large proteins resolved on acrylamide gels, utilizing a tank system for effective transfer.

A

Tank electrophoretic transfer.

76
Q

This blotting technique employs suction to facilitate the movement of DNA from the gel to the membrane, allowing for a quicker transfer time of 2 to 3 hours and preventing air trapping issues.

A

Vacuum transfer.

77
Q

What methods can be used to permanently immobilize cut, denatured DNA to the membrane after transfer?

A

Baking in a vacuum oven (80°C, 30 to 60 minutes)
UV cross-linking

78
Q

Process where we incubate the membrane in a buffer solution without the probe to block nonspecific binding.

A

prehybridization

79
Q

What are the key components of prehybridization buffer?

A

Denhardt solution
salmon sperm DNA,
and sodium dodecyl sulfate (SDS).

80
Q

Denhardt solution components

A

Ficoll,
polyvinyl
pyrrolidine,
bovine serum albumin)

81
Q

Blocking agents specially used for RNA probes

A

formamide

82
Q

The membrane is exposed to the prehybridization buffer at the optimal hybridization temperature for

A

30 minutes
to several hours

83
Q

a modification of the Southern blot technique, was designed to investigate
RNA structure and quantity

A

northern blot

84
Q

Analysis of RNA structure and quantity indirectly reveals mutations in the

A

regulatory or splicing
signals in DNA.

85
Q

Agarose concentrations of ____________ are usually employed for northern blots

A

0.8% to 1.5%

86
Q

These gels may also be used, especially for smaller transcripts—for instance, for analysis of viral gene expression in northern blot

A

Polyacrylamide gels

87
Q

In northern blot procedures After electrophoresis, representative lanes can be cut from the gel, soaked in ________ to remove the denaturant

A

ammonium acetate

88
Q

Northern blot makes use of the stains ______________
to assess quality and equivalent sample loading

A

acridine orange or ethidium bromide

89
Q

Denaturant such as formaldehyde must be removed from the gel before transfer because it inhibits binding
of the RNA to nitrocellulose. This is accomplished by rinsing the gel in

A

deionized water

90
Q

During northern blot procedures, If the RNA has been denatured in glyoxal, the
membrane must be soaked in warm ______________
to remove any residual denaturant immediately before prehybridization

A

Tris buffer (65°C)

91
Q

There are many variations on western blots.

Generally, serum, cell lysate, or extract is separated on which type of gels

A

SDS-polyacrylamide gels (SDS-PAGE) or isoelectric focusing gels (IEF)

92
Q

What does SDS-PAGE resolve proteins by?

A

Molecular weight

93
Q

What does IEF resolve proteins by?

A

Charge

94
Q

Which chemicals may be used to separate proteins into subunits in a Western blot?

A

Dithiothreitol or 2-mercaptoethanol.

95
Q

What are the typical concentrations of polyacrylamide used in Western blot gels?

A

5% to 20%

96
Q

How much protein is typically loaded per well for a Western blot?

A

1 to 50 μg

97
Q

What treatment is applied to samples before loading in a Western blot?

A

Samples are treated with denaturants, such as 0.04 M Tris HCl, pH 6.8, 0.1% SDS.

98
Q

What can denaturing gels affect in Western blotting?

A

They can affect epitopes, preventing binding with antibodies.

99
Q

How can proteins be renatured before transfer in Western blotting?

A

By pretreating gels with 20% glycerol in 50 mM Tris-HCl, pH 7.4.

100
Q

What transfer methods are used for proteins in Western blotting?

A

Capillary or electrophoretic transfer.

101
Q

What blocking agent is used to prevent antibody binding to the membrane when performing western blot

A

0.1% Tween 20 in 0.05 M Tris, 0.15 M NaCl, and 5% dry milk.

102
Q

How are proteins likely bound to nitrocellulose membranes?

A

Hydrophobically

103
Q

What other membrane types are used for protein blotting?

A

PVDF, DEAE-cellulose (anion exchange), and CM-cellulose (cation exchange).

104
Q

How long is the membrane incubated with the primary antibody in Western blotting?

A

12 to 16 hours.

105
Q

How is the signal developed in Western blotting?

A

With the addition of a chemiluminescent or colorimetric substrate.

RESET - MOLBIO PRELIMS (16 decks)

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