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Molecular Biology > Detection and Analysis of Nucleic Acid (Gel Electrophoresis and Gel Documentation) > Flashcards

Detection and Analysis of Nucleic Acid (Gel Electrophoresis and Gel Documentation) Flashcards

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1
Q

Father of Electrophoresis; did pioneer work on moving boundary electrophoresis (1930) and later developed a zone method for the purification of biomolecules.

A

Arne Tiselius

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2
Q
  • Routinely used for detection and size analysis of proteins and nucleic acids.
  • Uses an electrical current
A

Electrophoresis

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3
Q

_______ : Vertical Gel Electrophoresis (Polyacrylamide Gel)

A

Proteins

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4
Q

Proteins : _________

A

Vertical Gel Electrophoresis (Polyacrylamide Gel)

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5
Q

____________ : Horizontal Gel Electrophoresis (Agarose Gel)

A

DNA and RNA

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6
Q

DNA and RNA : __________________

A

Horizontal Gel Electrophoresis (Agarose Gel)

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7
Q

DNA and RNA : ____-charged because of phosphor-sugar backbone

A

Negatively

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8
Q

Migration rate of a molecule depends on two factors:

A

SHAPE and charge-to-mass ratio

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9
Q

The penetration of the DNA molecules through pores of different sizes provides a _________

A

Mechanism for separation by molecular mass

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10
Q

DNA electrophoretic mobility is determined by the interplay of three factors

A
  • The relative size of the DNA molecules with respect to the pore size of the
    matrix
  • The effect of the electric field on the matrix
  • Specific interactions of the matrix with the DNA molecules during
    electrophoresis
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11
Q

3 Parts of Electrophoresis

A

Voltage, Supporting Medium, and Buffer System

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12
Q

A part of electrophoresis that is responsible for the power supply

A

Voltage

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13
Q

A part of electrophoresis that consists of a matrix in which the biomolecule separation takes place

A

Supporting Medium

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14
Q

A part of electrophoresis that conduct electricity by running buffer

A

Buffer system

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15
Q

Purified linear galactan hydrocolloid isolated from agar or agar-bearing marine algae

A

Agarose

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16
Q

Alternating copolymer of D-galactose and 3,6-anhydro-L-galactose

A

Agarose

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17
Q

Agarose is dissolved by ____________

A

Heating the fibrous powder in an aqueous

solution

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18
Q

Occurs when the fiber bundles become linked together by the formation of additional hydrogen bonds

A

Gelation

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19
Q

Charge of the Gel Matrix

A

Negatively-charged

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20
Q

_________ groups in an agarose gel are affixed to the matrix and cannot move

A

Anionic

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21
Q

Negatively charged residues are surrounded by ____________

A

Positively charged counterions from the buffer.

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22
Q

The net result is a flow of the solvent toward the cathode, called the ________

A

Electroosmotic flow

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23
Q

DNA is negatively charged. It migrates in the _____

A

Opposite direction, toward the anode.

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24
Q

Held together by covalent cross-linking of

Acrylamide and N,N’-methylene bisacrylamide

A

Polyacrylamide gel structure

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25
Q

Tougher than agarose gels

A

Polyacrylamide Gel (PAG)

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26
Q

Tougher than agarose gels

A

Polyacrylamide Gel (PAG)

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27
Q

Used for separating smaller nucleic

acids and proteins

A

Polyacrylamide Gel (PAG)

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28
Q

2 Layers of Polyacrylamide Gel (PAG) and their pH value

A

Stacking gel: Top most layer (6.8 pH)

Resolving gel: Lower layer (8.8 pH)

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29
Q

• For denaturing the proteins
• An anionic detergent that breaks
disulfide bonds

A

Sodium Dodecyl Sulfate-PAGE

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30
Q 
• Gives a negative charge to each protein
in proportion to its mass
• Composed of a hydrophilic group with a
net negative charge and a long
hydrophobic chain with neutral charge
A

SDS-PAGE

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31
Q

Factors that Determine the Rate of Migration of a DNA Molecule Through a Gel:

A 
Size of the Molecule
Agarose Concentration
DNA Conformation
Voltage Applied
Electrophoresis Buffer
Presence of DNA Stains in the Gel Electrophoresis Buffer
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32
Q

Molecular size of nucleic acid is expressed in ___________

A

Molecular weight equivalent to the number of

bases/ base pairs in the molecule

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33
Q

A linear DNA fragment of a given size migrates at __________ through gels containing different concentrations of agarose

A

Different Rates

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34
Q

Supercoiled circular DNA, relaxed circular DNA and linear DNA of the same molecular
weight will migrate at _____ through the gel (DNA Conformation)

A

Different Rates

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35
Q

Rate of migration is ______ to the voltage applied

A

Proportional

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36
Q

Composition and ionic strength affects DNA mobility; High ionic strength (10X Buffer) –> ______

A

Electrical Conductance is Efficient

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37
Q

Dyes used to stain DNA in gels are usually intercalating agents; Addition to the gel may _________

A

retard the rate of migration of the DNA

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38
Q

Materials Required for Agarose Gel Electrophoresis

A 
Electrophoresis Chamber
Gel Casting Tray
Loading Dye
Comb
Buffer
Agarose
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39
Q

The gel is placed in an ____ which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster.

A

Electropheresis Chamber

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40
Q

Available in variety of sizes and composed of
UV transparent plastic

The open ends are closed with
tape while the gel is being cast, then
removed prior to electrophoresis

A

Gel Casting Trays

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41
Q

Placed in the liquid agarose after it has been
poured

Removing it from the hardened gel produces a series of wells used to load the DNA

A

Comb

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42
Q

Placed in the liquid agarose after it has been
poured

Removing it from the hardened gel produces a series of wells used to load the DNA

A

Comb

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43
Q

A solution that contains the right ions to

conduct electricity

A

Running Buffer

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44
Q

Examples of Buffer (4):

A

TAE (Tris Acetate EDTA)
TBE (Tris Borate EDTA)
Tris phosphate
Tris Citrate Buffer

45
Q

A mixture of glycerol and tracking dyes

A

Loading Dye

46
Q

weighs down DNA sample

by increasing its density

A

Glycerol

47
Q

allows visual gauge of

the progress of electrophoresis.

A 
Tracking dye (bromphenol blue;
xylene cyanol)
48
Q

consists of known DNA sizes
used to determine the size of an
unknown DNA sample

A

DNA ladder

49
Q

usually contains regularly spaced sized samples which when run on an agarose gel looks like a “ladder”

A

DNA ladder

50
Q

Enables estimate DNA size (bp) in the

sample

A

DNA ladder

51
Q

Method for Electrophoresis (first 4 parts)

A

Add 1% of Agarose powder to 25 mL
of 10x TBE

Heat the mixture in a microwave

Add 0.75 uL of loading dye to
the mixture

Pour into casting tray with comb
and allow to solidify

52
Q

Method for Electrophoresis (second 5 parts)

A

Remove the Comb

Add Running Buffer and transfer the
agarose gel to the Casting Chamber

Dispense the DNA ladder and the
samples to the loading wells

Run gel at constant voltage until
band separation occurs

View DNA on UV light box and show
results

53
Q

Detection of DNA: ______________ to view the fluorescent DNA in the gel

A

UV transilluminator

54
Q

Quantification of DNA: _______ to DNA being measured

A

Dye and size must be the same

55
Q

Evaluation of the Quality of DNA: Bond appear as compact, no _________

A

double bond or faint band

56
Q

Characteristics of 1x TAE

A

contains 40 mM Tris-acetate, and 1 mM EDTA at pH 8.3

provide good resolution of DNA fragments (improved separation of larger size)

recommended for resolution of RNA and DNA fragments larger than 1500 bp, for genomic DNA and for large supercoiled DNA

contain a weak acid, acetic or boric acid

57
Q

Characteristics of 1x TBE

A

contains 890 mM Tris base, 890 mM boric acid, 20 mM EDTA at pH 8.3

provide good resolution of DNA fragments (improved separation of smaller size)

contain a weak acid, acetic or boric acid

58
Q

EDTA is __________, but is added as a preventative because it chelates Mg2+ ions

A

not absolutely essential

59
Q

Usually performed at constant voltage

A

Gel Buffer

60
Q

The current (measured in mA) increases and _______________

A

warming of the running buffer occurs.

61
Q

The voltage is kept low at _______

A

10V/cm

62
Q

small- or medium-sized horizontal gel (10–15 cm length)

A

power supplies are set to between 100 V and 150 V, and gels are run for 50–90 min.

63
Q

used to monitor the progress of the electrophoresis run

A

Tracking dyes, also known as loading dyes; or DNA Size Tracking Dye and Density Agents

64
Q

Ficoll, sucrose, or glycerol, can

A

increase the density of the sample

65
Q

The dyes migrate at specific speeds in a given gel concentration and

A

usually run ahead of the smallest fragments of DNA

66
Q

Tracking dyes that are commonly used

A

bromophenol blue and xylene cyanol

67
Q

Gel loading dye is typically made at _____

A

6 concentration (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol).

67
Q

Gel loading dye is typically made at _____

A

6 concentration (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol).

68
Q
  • routinely used to stain DNA in agarose and polyacrylamide gels
  • binds to DNA molecules by intercalating between adjacent base pairs
  • powerful mutagen
  • limited sensitivity
A

Ethidium bromide (EtBr)

69
Q
  • used either as a poststain after electrophoresis

* included in the buffer solution

A

nonmutagenic dyes that stain DNA green, red, or blue are now used in
many laboratories

70
Q

Some of these dyes require UV irradiation or illumination

The most sensitive dyes are able to detect bands that contain

A

less than 1ng DNA.

71
Q

Pulsed electric fields must be applied to the gel to

A

separate very large DNA molecules

72
Q

pulsed fields may be applied _______

A

intermittently in one direction, in opposite directions, or at obtuse angles

73
Q

The rate at which relaxation occurs depends on ______

A

DNA size

74
Q

are trapped in the gel matrix and scarcely move

A

large DNA molecules

75
Q

Optimal separation window is determined by ________

A

the ratio of pulse length to DNA reorientation time

76
Q

Basic Principle of Pulsed Field

A
  • DNA molecules in pulse field agarose gels are highly dependent on the electric field applied to the gel.
  • PFGE can separate DNA molecules up to 10 Mb.
77
Q

Types of PFGE

A

Contour-clamped homogeneous electric field (CHEF)

Field-inversion gel electrophoresis (FIGE):

Orthogonal-field alternation gel electrophoresis (OFAGE)

Rotating gel electrophoresis (RGE)

Programmable autonomously controlled electrodes (PACE)

78
Q
  • 4 passive electrodes are arranged hexagonally
  • can regulate the voltage on all hexagonally arranged electrodes in a unit electric field.
  • the size, location, coordination, stability, and continuity of the electric field are precisely controlled
  • DNA molecules as large as 2 Mb can be well separated with a CHEF system alternating between two orientations 120 degrees apart.
  • DNA smaller than 50 kb can be separated without distortion.
A

Contour-clamped homogeneous electric field (CHEF)

79
Q
  • Two fields are arranged in separated straight angle (180 degree)
  • can overcome the problem caused by the comigration of nucleic acids and protein-detergent complexes
  • provides good resolution, over 800kb
A

Field-inversion gel electrophoresis (FIGE)

80
Q
  • vertical electrophoresis system

* DNA molecules between 1000 kb and 2000 kb can be separated

A

Orthogonal-field alternation gel electrophoresis (OFAGE)

81
Q
  • the agarose gel is rotated between two angles periodically
  • power supply is turned off after switching the angle during electrophoresis
  • easy to change the angle of rotation
  • this method is convenient and suitable for the separation of DNA with 50kb to 6000kb.
A

Rotating gel electrophoresis (RGE)

82
Q
  • 24 electrodes are arranged in a closed contour
  • controls all the parameters of the electric field
  • flexible, and it is preferable to the other alternating electrophoresis methods
  • DNA fragments from 100 bp to more than 6 Mb are separable.
A

Programmable autonomously controlled electrodes (PACE)

83
Q

• for molecular typing
• Identification of pathogens in the prevalence of certain diseases (gold
standard method to identification of some bacteria)
• used in epidemiological studies
• used for viral DNA fingerprinting of viruses isolated from the
environment.

A

Application of PFGE

84
Q
  • carried out in buffers containing entangled hydrophilic polymers that act as the sieving medium.
  • offers significant advantages over slab gel DNA separations
A

CAPILLARY ELECTROPHORESIS

85
Q

Disadvantage of Capillary Electrophoresis

A

fragility of the capillaries and capillary coatings and the necessity of running one sample at a time in each capillary

86
Q

Setup of the Capillary Electrophoresis

A

• carried out in fused-silica capillaries with a high surface area to volume ratio
• sample is introduced into a buffer-filled capillary either electrokinetically (with
low voltage) or hydrodynamically (with pressure or suction).
• Both ends of the capillary and electrodes are then placed into a buffer solution
that also contains the electrodes, and a high voltage is applied to the system
• The applied voltage causes the analytes to migrate through the capillary and pass
a detector window

87
Q

Two electrically driven phenomena contribute to the mobility of the analyte:

A
  • electrophoretic mobility of the analyte itself

* electroosmotic flow of the bulk solution.

88
Q

The net movement of buffer toward the cathode is concurrent with

A

analyte mobility

89
Q

Separation Principle: Charge-to-mass ratio

Analytes: Small Ions, small molecules, peptides and proteins, limited DNA

A

Capillary Zone Electrophoresis

90
Q

Separation Principle: Isoelectric Point

Analytes: Peptides and Proteins

A

Capillary Isoelectric Focusing

91
Q

Separation Principle: Mobility with Buffer

Analytes: Small molecules, Peptides and Proteins

A

Capillary Isotachophoresis

92
Q

Separation Principle:

  • Charged species - charge-to-mass ratio and micelles, according to hydrophobicity
  • Neutral Species - detergent micelle organization based on hydrophobicity

Analytes: Small molecules, peptides, DNA

A

Micellular Electrokinetic Capillary Chromatography

93
Q

Separation Principle: Charge-to-mass ratio sieving

Analytes: Peptides and Proteins, DNA

A

Capillary Gel Electrophoresis Nondenaturing

94
Q

Separation Principle: Mass Sieving

Analytes: Peptides & Proteins, DNA

A

Capillary Gel Electrophoresis: Denaturing (SDS, Urea)

95
Q
  • Also known as Gel Doc. System, Gel Image, or Gel Imager
  • For Imaging and documentation of nucleic acid
  • It is composed of ultraviolet (UV) light transilluminator, a hood and CCTV camera for image capturing
A

Gel Documentation

96
Q

Principle of Gel Documentation

A
  • Principle of fluorescence with fluorescent staining of nucleic acids, a fluorescent substance that has bound to nucleic acids is excited by ultraviolet irradiation and emits
    fluorescent light.

*The fluorescent substance Ethidium Bromide binds specifically to nucleic acid and the amount of bonding depends on the molecular weight and concentration of the nucleic acid.

97
Q

Purpose of Gel Documentation

A
  • Photography of stained gel
  • Print out of photographic data
  • Image data is displayed in real time
  • Image still displayed can be printed out with a video printer or saved to a compact flash media for future use.
98
Q

Different parts in Gel-Doc System

A

Camera, lenses, filters, overhead illuminator, visible light, and adjustable stage

99
Q

Camera in Gel Documentation

A

Ultraviolet camera ranging from 1.4m

upto 8.3m

100
Q

Computer controlled and motor driven in Gel Documentation

A

Lenses

101
Q

Filters:

A

There is an extensive range of emission

filters use for an array of application

102
Q

Over head illuminator:

A

White light, Ultraviolet light, and LED lighting option

103
Q

For extending the transmitted light application

A

Visible Light

104
Q

For extending the transmitted light application

A

Visible Light

105
Q

Enables sample to be moved closer to camera

A

Adjustable Stage

106
Q

Process for Imaging

A

Remove - Remove the gel from the plastic gel tray (it slides out).

Place - Place the hood back over the glass panel.

FLIP - flip the switch to turn on the UV light.

TAKE A PHOTO - Click the “snap” button to capture the image.

107
Q

Process of Cleaning the UV Light Source

A
  1. Make sure the UV light is turned off.
  2. Remove the camera hood.
  3. Wipe the glass panel with ethanol and a KimWipe.
108
Q

Important Warnings

A
  1. UV light is extremely dangerous. NEVER turn on the light unless the hood covers the ENTIRE glass panel.
  2. ALWAYS wear gloves when working with gels… ethidium bromide is a carcinogen!
  3. ALWAYS wear CLEAN gloves when using the keyboard and mouse.
  4. NEVER dispose of gels in the trash or sink. Use the gel waste bucket

Molecular Biology (2 decks)

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