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Molecular Biology Lecture > 4: Hybridization (PRELIMS) > Flashcards

4: Hybridization (PRELIMS) Flashcards

1
Q

Vitro technique whereby ssDNA or RNA sequence called the probe is allowed to complementary base pair with DNA or RNA

A

Hybridization

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2
Q

What is the molecule that is allowed to complementary base pair with DNA or RNA?

A

Probe

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3
Q

Prior hybridization what do you need to do to the DNA?

A

Cut the DNA in short fragments

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4
Q

These are ssDNA/RNA molecule that can recognize and specifically bind to target DNA by complementary base pairing in mixture of biomolecule

A

Probe

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5
Q

Probes contain what molecule which can help to hybridize complementary DNA or RNA and resist nuclease degradation

A

Normal nitrogen bases

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6
Q

Enzyme that degrades DNA

A

Nuclease

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7
Q

Attaches to the Gene of Interest

A

Probe

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8
Q

T or F

Width of probe determine its specificity

A

F (LENGTH of probe determine its specificity)

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9
Q

Length of probe determines its what?

A

Specificity

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10
Q

T or F

Longer probe means more specific

A

T

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11
Q

This is for visualization of bound probe to target fragments in membrane

A

Probe labeling

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12
Q

What are the types of probe labeling?

A
  1. Enzyme
  2. Fluorescence
  3. Radiolabeled
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13
Q

In this type of probe labeling, a substrate is needed in order to catalyze reaction

A

Enzyme

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14
Q

In this type of probe labeling, light of a specific wavelength is emitted when excited by an external light source

A

Fluorescence

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15
Q

What is the positive result for Fluorescence type of probe labeling?

A

Fluorescent/Light formation

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16
Q

In this type of probe labeling, probe is labeled with radioactive isotope and is visualized using X-ray or CCD camera

A

Radiolabeled

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17
Q

T or F

UV light is used in visualizing radiolabeled type of probe

A

F (X-ray and CCD camera)

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18
Q

This process immobilizes the molecule of interest in support

A

Blotting

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19
Q

Gene of Interest + Membrane

A

Blotting

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20
Q

Uses hybridization technique for identification of specific nucleic acids

A

Blotting

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21
Q

What is the Target, Probe, and Purpose of Southern Blot hybridization technique?

A

Target: DNA
Probe: NA
Purpose: Gene structure

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22
Q

What is the Target, Probe, and Purpose of Northern Blot hybridization technique?

A

Target: RNA
Probe: NA
Purpose: Transcript structure, Gene expression

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23
Q

What is the Target, Probe, and Purpose of Western Blot hybridization technique?

A

Target: Protein
Probe: Protein (globulin)
Purpose: Protein processing gene

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24
Q

What is the Target, Probe, and Purpose of Eastern Blot hybridization technique?

A

Target: Protein
Probe: Protein
Purpose: Modification of WB using Enzymatic digestion

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25
Q

What is the Target, Probe, and Purpose of South-Western Blot hybridization technique?

A

Target: Protein
Probe: DNA
Purpose: DNA binding proteins, Gene

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26
Q

What is the Target, Probe, and Purpose of Far-Eastern Blot hybridization technique?

A

Target: Lipids
Probe: None
Purpose: Transfer of HPLC, Separated lipids to PDM for analysis by mass spectrophotometry

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27
Q

Enumerate all hybridization technique

A
  1. Southern Blot
  2. Northern Blot
  3. Western Blot
  4. Eastern Blot
  5. South-Western Blot
  6. Far-eastern Blot
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28
Q

This is a hybridization technique that allows the detection of given DNA sequence

A

Southern Blot

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29
Q

T or F

Southern blot can be used to identify heterogenous sequences of genomic DNA or facilitate gene mapping

A

F (can be used to identify HOMOGENOUS sequences of genomic DNA)

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30
Q

Use this card to familiarize the process of southern blotting

A
  1. Purify DNA
  2. Restriction Digestion
  3. Gel Electrophoresis
  4. Depurination
  5. Denaturation
  6. Blotting
  7. Saturation
  8. Fixation
  9. Hybridization
  10. Detection
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31
Q

1st and crucial step in southern blotting

A

Purify genomic DNA from eukaryotic cells or bacteria

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32
Q

2nd step in southern blotting wherein DNA is cut into smaller fragments by restriction enzymes

A

Restriction digestion/fragmentation

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33
Q

3rd step in southern blotting where there is separation of fragments according to their molecular weight

A

Gel Electrophoresis

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34
Q

How many percent of gel is used in Gel electrophoresis?

A

0.7 - 2%

meansyung size ng DNA is large; .8 to 2

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35
Q

What are the two types of gel used in Gel electrophoresis?

A

Agarose or Acrylamide

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36
Q

In the Gel electrophoresis part of southern blotting, sample is stained with what substance and subjected to what device?

A

Stained with Ethidium Bromide and Subjected to fluorescence under UV Light

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37
Q

After Gel electrophoresis in southern blotting, sample undergoes what?

A

Depurination

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38
Q

4th step of souther blotting wherein the DNA is apurinated or removing purines with DNA using diluted HCL

A

Depurination

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39
Q

What substance is used in depurination of DNA?

A

diluted HCl

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40
Q

T or F

In depurination part of southern blotting, you will be left with pyrimidines and some remnants of purines

A

F (left with PYRIMIDINES only since purines were removed during this step)

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41
Q

After depurination of sample in southern blotting, what is the next step?

A

Denaturation

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42
Q

What substance is used in denaturation part of southern blotting?

A

NaOH (Alkaline Solution)

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43
Q

What is the purpose of denaturation in southern blotting?

A

Break H-bonds to turn double stranded DNA to single stranded

44
Q

After depurinating and denaturation, what step is done wherein the fragmented DNA is transferred into membrane

45
Q

In blotting, what are the 4 membrane types used?

A
  1. Nitrocellulose
  2. Nylon
  3. Modified Cellulose
  4. PVDF (polvinyl difluoride)
46
Q

Membrane Types:

Usually used in southern blotting

A

Nitrocellulose

47
Q

Membrane Type:

Used in northern blot; most sensitive

48
Q

Membrane Type:

What is used in modified cellulose?

A

Diethylaminoethyl or cARBOXYMETHYL

49
Q

Membrane Type:

Used in western blot

50
Q

In blotting, what are the methods of transferring?

A
  1. Capillary
  2. Electroblotting
  3. Vacuum
51
Q

Method of transferring:

Uses capillary action

52
Q

T or F

In capillary method of transferring, the movement of the fluid is downward

A

F (UPWARD)

53
Q

Method of transferring:

Uses electric currents based on electrodes

A

Electroblotting

54
Q

T or F

Nucleic acid are normally negatively charged

55
Q

Method of transferring:

Nitrocellulose is placed under the gel through suction

56
Q

In vacuum method of transferring, what solution is used to saturate?

57
Q

T or F

The recommended method of transferring is vacuum since it removes air bubbles

58
Q

What is the process that prevents probe from binding to non-specific sites on the membrane surface?

A

Prehybridization

59
Q

Prehybridization

What is the buffer solution used in southern blot?

A

Denhardt solution; Ficoll, Polyvinyl, Pyrolidine, Bovine serum

60
Q

After blotting in southern blotting, what is the next step?

A

Saturation

NaCl

61
Q

What are the 2 techniques of fixation in southern blotting?

A
  1. Drying at 80 degrees celsius
  2. UV radiation
62
Q

After the fixation part in souther blotting, what is the next step wherein the probes is attached to the gene of interest?

A

Hybridization

63
Q

T or F

Hybridization in southern blotting last 2-10 hours

A

F (last 1-16 HOURS)

64
Q

After the process of hybridization in souther blotting, what is the next step?

A

Washing and autoradiography

65
Q

In the washing part of southern blot, what substances are used?

A

Buffer, NaCl, Detergent

65
Q

If the DNA is not washed in southern blotting, what result can this lead to?

A

False-positive result

66
Q

Last step of southern blot is what?

67
Q

In detection process of southern blot, what enables autoradiographic detection?

A

Radioactive probes

68
Q

This hybridization technique allows detection of RNA molecules in a mixture of heterogenous NRA

A

Northern Blot

69
Q

Use this card to familiarize process of Northern blot

A
  1. Extract and purify mRNA from cells
  2. Separate by gel electrophoresis
  3. Depurination
  4. Blotting
  5. Fixation
  6. Addition of probe/Hybridization
  7. Washing
  8. Detection
70
Q

The northern blot almost has the same process as southern blot except for what step?

A

Denaturation (since RNA is already single stranded, no need to break H-bond)

71
Q

In the blotting process of northern blot, what filter paper is used?

A

Aminobenyloxymethyl filter paper (ABOM filter paper–hirap na hirap ako memorize)

72
Q

What fixation technique is used in northern blot?

A

Drying at 80 degrees c

73
Q

What buffer solution is used in prehybridization of northern blot?

A

Salmon Sperm DNA EWWWWWWW

74
Q

Hybridization technique where target proteins are transferred to hydrophobic membrane after the SDS-PAGE and detected using antibodies

A

Western Blot

75
Q

Use this card to familiarize the process of western blot

A
  1. Load and Separate protein sample on SDS-PAGE
  2. PAGE –> Nitrocellulose membrane of PVDF
  3. Block the membrane w/ neutral protein
  4. Probing with antibody
  5. Detection of band
76
Q

In western blot, where do you load and separate protein samples?

77
Q

After loading and separating in SDS-PAGE, what is the next step

Western blot

A

PAGE / fractionated proteins is electrophoretically transferred onto
nitrocellulose membrane of PVDF

OR

PAGE –> nitrocellulose membrane of PVDF

78
Q

What are the 3 transfer buffers used in western blot?

A
  1. 25 mM. TRIS HCl
  2. 192 mM Lycine
  3. 20% methanol
79
Q

After transfer into membrane in western blot, what is the next step?

A

Block membrane w/ neutral protein

80
Q

T or F

You block membrane with an acidic protein

A

F (NEUTRAL protein)

81
Q

This step in western blot, prevents non-specific binding of antibodies to membrane

A

Blocking w/ neutral protein

82
Q

What are the 3 neutral proteins used in blocking process of western blot?

A
  1. 1-3% BSA
  2. 1-5% Skim milk
  3. PBS-Tween 20
83
Q

After blocking process in western blocking, what is the next step?

A

Probing/Hybridization

84
Q

In western blot, probing is done with an?

85
Q

T or F

In western blot, probing with antibody is only a one step process

A

F (two-step since you incubate with primary and secondary antibody)

86
Q

In western blot, specifically in probing process, you first incubate membrane with what?

A

Primary antibody specific to target protein

87
Q

Low amount of primary antibody indicates what result?

A

False-negative result

88
Q

High amount of primary antibody indicates what result in western blot?

A

High non-specific reaction

89
Q

In western blot, specifically in probing process, after incubating w/ primary what is the next antibody you use?

A

HRP - Labeled Secondary antibody specific to primary antibody

90
Q

These 2 are example of secondary antibodies used to probe in western blot

A
  1. HRP
  2. Alkaline phosphatase
91
Q

What is the last step in western blot?

A

Detection of band

92
Q

In the last step of western blot which is detection of band, what do you use to incubate blot?

A

Chemiluminescent HRP Subtrate; Diaaminobenzidine, TMB, BCIP, NBT

93
Q

How is the band detected in western blot?(

A

XRAY, CCD camera

94
Q

T or F

In western blot, specifically in last step which is detection of band, an enzyme attached to antibody catalyzes reaction

95
Q

Hybridization technique where target DNA, protein, or RNA is deposited directly on membrane

96
Q

T or F

In DOT Blot, there is gel resolution of target fragments

A

F (NO gel resolution of target fragments)

97
Q

Use this card to familiarize yourself with the process of DOT Blot

A
  1. Purification of RNA/DNA
  2. Apply directly to the small dots
  3. Denature DNA (70-80 c)
  4. Add labeled probe
  5. Incubation for complementary binding
  6. Wash excess probe
  7. Visualization of result
98
Q

T or F

Dot blot is denatured at 80-90 degrees c

A

F (70-80 degrees c)

99
Q

What is the membrane used in DOT Blot?

A

Nitrocellulose or Nylon

100
Q

What is the positive result of a DOT Blot?

101
Q

What is the advantage of DOT Blot from other techniques?

A

Simple; easy to use

102
Q

This is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes

A

Fluorescent in Situ Hybridization

FISH tilapia, talaping talapong talap-talap

103
Q

–> Method for visualizing specific location on chromosome (by fluorescent complementary probes)

–> Fluorescent probes that bind to only parts of chromosomes

A

Fluorescent in Situ Hybridization

104
Q

T or F

There is cell lysis in Fluorescent in Situ Hybridization

A

F (NO cell lysis in Fluorescent in Situ Hybridization)

Molecular Biology Lecture (17 decks)

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