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
2
Q
What is the molecule that is allowed to complementary base pair with DNA or RNA?
A
Probe
3
Q
Prior hybridization what do you need to do to the DNA?
A
Cut the DNA in short fragments
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
5
Q
Probes contain what molecule which can help to hybridize complementary DNA or RNA and resist nuclease degradation
A
Normal nitrogen bases
6
Q
Enzyme that degrades DNA
A
Nuclease
7
Q
Attaches to the Gene of Interest
A
Probe
8
Q
T or F
Width of probe determine its specificity
A
F (LENGTH of probe determine its specificity)
9
Q
Length of probe determines its what?
A
Specificity
10
Q
T or F
Longer probe means more specific
A
T
11
Q
This is for visualization of bound probe to target fragments in membrane
A
Probe labeling
12
Q
What are the types of probe labeling?
A
- Enzyme
- Fluorescence
- Radiolabeled
13
Q
In this type of probe labeling, a substrate is needed in order to catalyze reaction
A
Enzyme
14
Q
In this type of probe labeling, light of a specific wavelength is emitted when excited by an external light source
A
Fluorescence
15
Q
What is the positive result for Fluorescence type of probe labeling?
A
Fluorescent/Light formation
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
17
Q
T or F
UV light is used in visualizing radiolabeled type of probe
A
F (X-ray and CCD camera)
18
Q
This process immobilizes the molecule of interest in support
A
Blotting
19
Q
Gene of Interest + Membrane
A
Blotting
20
Q
Uses hybridization technique for identification of specific nucleic acids
A
Blotting
21
Q
What is the Target, Probe, and Purpose of Southern Blot hybridization technique?
A
Target: DNA
Probe: NA
Purpose: Gene structure
22
Q
What is the Target, Probe, and Purpose of Northern Blot hybridization technique?
A
Target: RNA
Probe: NA
Purpose: Transcript structure, Gene expression
23
Q
What is the Target, Probe, and Purpose of Western Blot hybridization technique?
A
Target: Protein
Probe: Protein (globulin)
Purpose: Protein processing gene
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
25
What is the Target, Probe, and Purpose of South-Western Blot hybridization technique?
Target: Protein
Probe: DNA
Purpose: DNA binding proteins, Gene
26
What is the Target, Probe, and Purpose of Far-Eastern Blot hybridization technique?
Target: Lipids
Probe: None
Purpose: Transfer of HPLC, Separated lipids to PDM for analysis by mass spectrophotometry
27
Enumerate all hybridization technique
1. Southern Blot
2. Northern Blot
3. Western Blot
4. Eastern Blot
5. South-Western Blot
6. Far-eastern Blot
28
This is a hybridization technique that allows the detection of given DNA sequence
Southern Blot
29
T or F
Southern blot can be used to identify heterogenous sequences of genomic DNA or facilitate gene mapping
F (can be used to identify HOMOGENOUS sequences of genomic DNA)
30
Use this card to familiarize the process of southern blotting
1. Purify DNA
2. Restriction Digestion
3. Gel Electrophoresis
4. Depurination
5. Denaturation
6. Blotting
7. Saturation
8. Fixation
9. Hybridization
10. Detection
31
1st and crucial step in southern blotting
Purify genomic DNA from eukaryotic cells or bacteria
32
2nd step in southern blotting wherein DNA is cut into smaller fragments by restriction enzymes
Restriction digestion/fragmentation
33
3rd step in southern blotting where there is separation of fragments according to their molecular weight
Gel Electrophoresis
34
How many percent of gel is used in Gel electrophoresis?
0.7 - 2%
## Footnote
meansyung size ng DNA is large; .8 to 2
35
What are the two types of gel used in Gel electrophoresis?
Agarose or Acrylamide
36
In the Gel electrophoresis part of southern blotting, sample is stained with what substance and subjected to what device?
Stained with Ethidium Bromide and Subjected to fluorescence under UV Light
37
After Gel electrophoresis in southern blotting, sample undergoes what?
Depurination
38
4th step of souther blotting wherein the DNA is apurinated or removing purines with DNA using diluted HCL
Depurination
39
What substance is used in depurination of DNA?
diluted HCl
40
T or F
In depurination part of southern blotting, you will be left with pyrimidines and some remnants of purines
F (left with PYRIMIDINES only since purines were removed during this step)
41
After depurination of sample in southern blotting, what is the next step?
Denaturation
42
What substance is used in denaturation part of southern blotting?
NaOH (Alkaline Solution)
43
What is the purpose of denaturation in southern blotting?
Break H-bonds to turn double stranded DNA to single stranded
44
After depurinating and denaturation, what step is done wherein the fragmented DNA is transferred into membrane
Blotting
45
In blotting, what are the 4 membrane types used?
1. Nitrocellulose
2. Nylon
3. Modified Cellulose
4. PVDF (polvinyl difluoride)
46
Membrane Types:
Usually used in southern blotting
Nitrocellulose
47
Membrane Type:
Used in northern blot; most sensitive
Nylon
48
Membrane Type:
What is used in modified cellulose?
Diethylaminoethyl or cARBOXYMETHYL
49
Membrane Type:
Used in western blot
PVDF
50
In blotting, what are the methods of transferring?
1. Capillary
2. Electroblotting
3. Vacuum
51
Method of transferring:
Uses capillary action
Capillary
52
T or F
In capillary method of transferring, the movement of the fluid is downward
F (UPWARD)
53
Method of transferring:
Uses electric currents based on electrodes
Electroblotting
54
T or F
Nucleic acid are normally negatively charged
T
55
Method of transferring:
Nitrocellulose is placed under the gel through suction
Vacuum
56
In vacuum method of transferring, what solution is used to saturate?
NaCl
57
T or F
The recommended method of transferring is vacuum since it removes air bubbles
T
58
What is the process that prevents probe from binding to non-specific sites on the membrane surface?
Prehybridization
59
# Prehybridization
What is the buffer solution used in southern blot?
Denhardt solution; Ficoll, Polyvinyl, Pyrolidine, Bovine serum
60
After blotting in southern blotting, what is the next step?
Saturation
| NaCl
61
What are the 2 techniques of fixation in southern blotting?
1. Drying at 80 degrees celsius
2. UV radiation
62
After the fixation part in souther blotting, what is the next step wherein the probes is attached to the gene of interest?
Hybridization
63
T or F
Hybridization in southern blotting last 2-10 hours
F (last 1-16 HOURS)
64
After the process of hybridization in souther blotting, what is the next step?
Washing and autoradiography
65
In the washing part of southern blot, what substances are used?
Buffer, NaCl, Detergent
65
If the DNA is not washed in southern blotting, what result can this lead to?
False-positive result
66
Last step of southern blot is what?
Detection
67
In detection process of southern blot, what enables autoradiographic detection?
Radioactive probes
68
This hybridization technique allows detection of RNA molecules in a mixture of heterogenous NRA
Northern Blot
69
Use this card to familiarize process of Northern blot
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
The northern blot almost has the same process as southern blot except for what step?
Denaturation (since RNA is already single stranded, no need to break H-bond)
71
In the blotting process of northern blot, what filter paper is used?
Aminobenyloxymethyl filter paper (ABOM filter paper--hirap na hirap ako memorize)
72
What fixation technique is used in northern blot?
Drying at 80 degrees c
73
What buffer solution is used in prehybridization of northern blot?
Salmon Sperm DNA EWWWWWWW
74
Hybridization technique where target proteins are transferred to hydrophobic membrane after the SDS-PAGE and detected using antibodies
Western Blot
75
Use this card to familiarize the process of western blot
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
In western blot, where do you load and separate protein samples?
SDS-PAGE
77
After loading and separating in SDS-PAGE, what is the next step
| Western blot
PAGE / fractionated proteins is electrophoretically transferred onto
nitrocellulose membrane of PVDF
OR
PAGE --> nitrocellulose membrane of PVDF
78
What are the 3 transfer buffers used in western blot?
1. 25 mM. TRIS HCl
2. 192 mM Lycine
3. 20% methanol
79
After transfer into membrane in western blot, what is the next step?
Block membrane w/ neutral protein
80
T or F
You block membrane with an acidic protein
F (NEUTRAL protein)
81
This step in western blot, prevents non-specific binding of antibodies to membrane
Blocking w/ neutral protein
82
What are the 3 neutral proteins used in blocking process of western blot?
1. 1-3% BSA
2. 1-5% Skim milk
3. PBS-Tween 20
83
After blocking process in western blocking, what is the next step?
Probing/Hybridization
84
In western blot, probing is done with an?
Antibody
85
T or F
In western blot, probing with antibody is only a one step process
F (two-step since you incubate with primary and secondary antibody)
86
In western blot, specifically in probing process, you first incubate membrane with what?
Primary antibody specific to target protein
87
Low amount of primary antibody indicates what result?
False-negative result
88
High amount of primary antibody indicates what result in western blot?
High non-specific reaction
89
In western blot, specifically in probing process, after incubating w/ primary what is the next antibody you use?
HRP - Labeled Secondary antibody specific to primary antibody
90
These 2 are example of secondary antibodies used to probe in western blot
1. HRP
2. Alkaline phosphatase
91
What is the last step in western blot?
Detection of band
92
In the last step of western blot which is detection of band, what do you use to incubate blot?
Chemiluminescent HRP Subtrate; Diaaminobenzidine, TMB, BCIP, NBT
93
How is the band detected in western blot?(
XRAY, CCD camera
94
T or F
In western blot, specifically in last step which is detection of band, an enzyme attached to antibody catalyzes reaction
T
95
Hybridization technique where target DNA, protein, or RNA is deposited directly on membrane
DOT Blot
96
T or F
In DOT Blot, there is gel resolution of target fragments
F (NO gel resolution of target fragments)
97
Use this card to familiarize yourself with the process of DOT Blot
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
8. Visualization of result
98
T or F
Dot blot is denatured at 80-90 degrees c
F (70-80 degrees c)
99
What is the membrane used in DOT Blot?
Nitrocellulose or Nylon
100
What is the positive result of a DOT Blot?
Black dot
101
What is the advantage of DOT Blot from other techniques?
Simple; easy to use
102
This is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes
Fluorescent in Situ Hybridization
| FISH tilapia, talaping talapong talap-talap
103
--> Method for visualizing specific location on chromosome (by fluorescent complementary probes)
--> Fluorescent probes that bind to only parts of chromosomes
Fluorescent in Situ Hybridization
104
T or F
There is cell lysis in Fluorescent in Situ Hybridization
F (NO cell lysis in Fluorescent in Situ Hybridization)
