1. Chapter 5: Resolution and Detection of Nucleic Acids

2. Agarose Gel Electrophoresis

3. What does gel electrophoresis do?
employs electromotive force to move molecules through a porous gel
separates molecules from each other on the basis of
size and/or
charge and/or
shape
basis of separation depends on how the sample and gel are prepared

4. Why perform electrophoresis on ds DNA?
To separate fragments from each other
To determine the sizes of fragments
To determine the presence or amount of DNA
To analyze restriction digestion products

5. Where does the current come from?
A direct current power supply
Ions supplied by the buffer
The charge on the macromolecules being separated
Electrolysis of water

6. Basics of Gel Electric Circuits
V (volts) = I (milliamps) X R (resistance)
For a segment of a gel/buffer system
 cross-sectional area of buffer or gel,  resistance
 strength of buffer = [ion],  resistance
most resistance is in the agarose gel itself

7. What factors affect mobility of linear ds DNA?
Pore size of the gel
 [agarose]   pore size
 pore size   friction   mobility
Voltage across the gel
 voltage   mobility
Length of the DNA molecule
smaller molecules generate less friction and so move faster
Ethidium bromide (stain) intercalated into DNA
decreases charge to mass ratio and so decreases mobility

8. Visualization Monitoring the progress of the electrophoresis
tracking dyes visible to naked eye during run
xylene cyanol (migrates with ~5.0 kb fragments)
bromphenol blue (migrates with fragments of a few hundred base pairs)
Orange G (migrates with fragments of ~50 bp)

9. Visualization Locating the DNA fragments in the gel
ethidium bromide staining
mutagen, wear gloves!
visible under UV light
wear UV opaque face or eye shield to observe!

10. Factors affecting resolution
Resolution = separation of fragments
The “higher” the resolution, the more space between fragments of similar, but different, lengths
Resolution is affected by
agarose type
agarose concentration
salt concentration of buffer or sample
amount of DNA loaded in the sample
voltage

11. What is agarose?
linear carbohydrate polymer extracted from seaweed , agarbiose
forms a porous matrix as it gels

12. What is agarose? (cont’d)
multiple types of agarose
Standard agarose - LE
Gels at 35-38oC; Melts at 90-95oC
Becomes opaque at high concentrations
Makes a fairly sturdy gel
Low melting agarose (NuSieve)
Gels at 35oC; Melts at 65oC
Often used to isolate DNA fragments from gel
Is relatively translucent at high concentrations
Makes a fragile gel
Intermediate forms or combinations of LE and NuSieve can provide sturdy, translucent gels at high agarose concentrations
Good for resolving smaller fragments

13. Resolution of ds linear DNA fragments in agarose gels
% Agarose (w/v) Size Range (kb)
for Optimal Separation
3.0 (Nu-Sieve)
4.0 (N-S)
5.0 (N-S)
6.0 (N-S)

14. 0.7%
2.5%
Effect of agarose concentration on linear DNA fragment resolution.
The two lanes contain identical DNA samples.

15. 4M 1M 0M
Effect of salt concentration on resolution of fragments.
Samples in all three lanes are identical except for [salt].

16. Effect of sample DNA concentration on resolution.
 DNA Hind III fragments (g)
Effect of sample DNA concentration on resolution.

17. Voltage  voltage,  rate of migration to increase the voltage
increase the setting on the power supply
increase the resistance
decrease the gel thickness
decrease the ion concentration
if voltage is too high, gel melts
as voltage is increased, large molecules migrate at a rate proportionally faster than small molecules, so
lower voltages are better for resolving large fragments
but the larger ds DNA fragments are always slower than the smaller ones

18. Buffer Systems Purposes of buffer
Keep solution at pH compatible with molecules being separated
Generate ions consistently to
maintain current
keep resistance low
Both gel and the solution in the gel box are buffered.

19. Buffer Systems (cont’d)
Two commonly used buffers for routine agarose gel electrophoresis
TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA
TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA
Tris (T) is a weak base.
Acetic (A) acid and boric (B) acid are weak acids.
Acetic acid is more completely ionized at pH 8.0 than is boric acid, so TBE has a high buffer capacity than TAE.

20. Buffer Systems (cont’d)
TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA
loses buffer capacity during long or high voltage gel runs;
gel may melt from the increased resistance that results from ion depletion
resolves high MW fragments better than TBE
TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA
higher buffer capacity
resolves low MW fragments better than TAE

21. Non-denaturing agarose gel loading solutions
Composition
tracking dyes
are used to follow progress of electrophoresis
sometimes interfere with later visualization of DNA
a solute to increase density
so that sample falls to bottom of loading well with minimal dilution
solute examples: glycerol, Ficoll

22. Ethidium bromide staining
Binds to DNA by intercalation between stacked bases
+ charge,
so migrates toward negative pole
it alters DNA mobility, especially of circular covalently closed DNA

23. Ethidium bromide staining
Used to visualize DNA with UV light
uv  254 nm absorbed by DNA
>/= 10ng/band required for visualization
Bound dye fluoresces 20-25X more than dye in solution
UV light damages eyes and skin! Wear goggles and/or face shield.

24. Trouble shooting Smearing torn sample wells
voltage too high for large fragments
too much DNA
Use </= 0.5 ug / fragment / 0.25cm2 migration area
Gel melts
voltage too high
ionic strength too low
Poor resolution
wrong [agarose]
small bands are fuzzy – the gel run may have been too long at too low a voltage, allowing diffusion of the DNA and broadening of the band

25. Gel Electrophoresis Matrices
Agarose
Acrylamide

26. Comparison of Agarose Concentrations
500 bp
200 bp
50 bp
% agarose: 2% % %

27. Fragment Resolution: Agarose Gel Electrophoresis

28. Agarose Electrophoresis of Restriction Enzyme Digested Genomic DNAB

29. Gel Electrophoresis: Apparatus and Types of Gels
Horizontal Gel Units (“Submarine Gels”)
Most DNA and RNA gels
Agarose
Vertical Gel Units
Polyacrylamide gels
Typically sequencing gels
Pulse Field Gel Units
Any electrophoresis process that uses more than one alternating electric field
Large genomic DNA (Chromosomal)

30. Electrophoresis Equipment: Horizontal or Submarine Gel
DNA/RNA is negatively charged: RUN TO RED

31. Agarose Gel Electrophoresis Horizontal Gel Format
Reservoir/Tank
Power Supply
Casting Tray and Combs

32. Agarose Gel Apparatus

33. Pouring a horizontal agarose gel

34. Electrophoresis Equipment: Vertical Gel

35. Vertical Gel Format: Polyacrylamide Gel Electrophoresis
Reservoir/Tank
Power Supply
Glass Plates, Spacers, and Combs

36. Polyacrylamide Gel Electrophoresis (PAGE)

37. Electrophoresis Equipment
Combs are used to put wells in the cast gel for sample loading.
Regular comb: wells separated by an “ear” of gel
Houndstooth comb: wells immediately adjacent

38. PULSE FIELD GEL ELECTROPHORESIS APPARATUS

39. Types Of Pulse Field Gel Electrophoresis
Field inversion gel
Transverse alternating field
Crossed field (Reverse)
Contour-clamped homogeneous electric field

40. Pulse Field Gel Electrophoresis
Used to resolve DNA molecules larger than 25 kbp
Periodically change the direction of the electric field
Several types of pulsed field gel protocols
FIGE: Field inversion gel electrophoresis
TAFE: Transverse alternating field electrophoresis
RGE: Crossed field electrophoresis
CHEF: Contour-clamped homogeneous electric field

41. Critical Parameters: Pulse Field Gel Electrophoresis
Depend on time it takes molecules of various sizes to change directions in a gel
Small DNA molecules are sieved (pass through the pores in the agarose gel)
Large DNA molecules are not “sieved” but “squeezed” through the gel at about the same rate, called the limiting mobility

42. PFGE of Bacterial DNA

43. Using PFGE In The Molecular Investigation Of An Outbreak Of S
Using PFGE In The Molecular Investigation Of An Outbreak Of S. marcescens Infection In An ICU
An outbreak due to S. marcescens infection was detected in the ICU
A total of 25 isolates were included in this study:
12 isolates from infected patients
nine isolates from insulin solution
one isolate from sedative solution
one isolate from frusemide solution
two isolates from other wards which were epidemiologically-unrelated
Singapore Med J 2004 Vol 45(5) : 214

44. Using PFGE in the Molecular Investigation Of An Outbreak of S
Using PFGE in the Molecular Investigation Of An Outbreak of S. marcescens Infection in an ICU
Singapore Med J 2004 Vol 45(5) : 214

45. Using PFGE in the molecular investigation of an outbreak of S
Using PFGE in the molecular investigation of an outbreak of S. marcescens infection in an ICU
The S. marcescens from patients, insulin solution and sedative solution showed an identical PFGE fingerprint pattern.
The isolate from the frusemide solution had a closely-related PFGE pattern to the outbreak strain with one band difference.
Found that the insulin and sedative solutions used by the patients were contaminated with S. marcescens and the source of the outbreak.
Singapore Med J 2004 Vol 45(5) : 214

49. Comparison Of Agarose Gel And PFGE
Panel B: Agarose gel electrophoresis
Panel C: PFG electrophoresis
Pulsed Field Gel Electrophoresis was applied to the study of Duchenne Muscular Dystrophy. Since the DMD gene is 2.3Mbp, it was necessary to use PFGE in order to uncover the genetic defect. The use of PFGE analysis on patients with the disease soon revealed that in 50% of the cases large deletions or duplications were a responsible for the disease (Mathew, 1991).

50. Electrophoresis of Nucleic Acids Polyacrylamide Gel Electrophoresis (PAGE)
Advantages
High degree of resolving power.
Can effectively and reproducibly separate molecules displaying 1 bp differences in molecular size.
Optimal separation is achieved with nucleic acids that are 5–500 bp in size.

51. Electrophoresis of Nucleic Acids Polyacrylamide Gel Electrophoresis (PAGE)
Typical Conditions
Vertical gel setup, TBE buffer (Tris-borate/EDTA) and constant power.
Disadvantages
Acrylamide monomer is a neurotoxin.
Polyacrylamide gels can be difficult to handle.

52. PAGE: DNA
High resolution of low molecular weight nucleic acids (500bp)

53. Polyacrylamide Gel Electrophoresis of Restriction Digested PCR Products
FV SNP
FII SNP

54. Denaturation of DNA: Urea and Formamide
Both urea and formamide effectively lower the melting point of the DNA molecules, allowing the structures to fall apart at lower temperatures.
Both formamide and urea effectively lower the melting point of the DNA molecules, allowing the structures to fall apart at lower temperatures. Generally, concentrations of urea or formamide are chosen to give melting temperatures around 50° C, and gels are run at that temperature. RNA is often denatured with harsher agents, because RNA tends to form stronger structures.

55. Chapter 6: Analysis and Characterization of Nucleic Acids and Proteins

56. Restriction Enzymes Type I Type II Type III
Methylation/cleavage (3 subunits)
>1000 bp from binding site
e.g., Eco AI GAGNNNNNNNGTCA
Type II
Cleavage at specific recognition sites
Type III
Methylation/cleavage (2 subunits)
24–26 bp from binding site
e.g., Hinf III CGAAT

57. Restriction Endonucleases: Type II
Enzyme
Isolated from
Recognition sequence
Eco RI
E. coli, strain R,
1st enzyme
Gν AATTC
Eco RV
5th enzyme
Gv ATATC
Hind III
H. influenzae, strain d,
3rd enzyme
Av AGCTT

58. There are hundreds of
restriction enzymes

59. Restriction Enzymes BamH1 HaeIII KpnI Cohesive Ends Blunt Ends GGATCC
CCTAGG
HaeIII
GGCC
CCGG
Cohesive Ends
(5´ Overhang)
(3´ Overhang)
KpnI
GGTACC
CCATGG
Blunt Ends
(No Overhang)

60. Restriction Enzymes GATC CTAG GGCC CCGG CCCGGG GGGCCC GGATCC CCTAGG
DpnI
(Requires methylation)
Methylation-sensitive Enzymes
GGCC
CCGG
HaeIII
(Inhibited by methylation)
CCCGGG
GGGCCC
XmaI
(5’ Overhang)
SmaI
(Blunt Ends)
Isoschizomers
Enzymes Generating
Compatible Cohesive Ends
GGATCC
CCTAGG
BamHI
AGATCT
TCTAGA
BglII
CTCGTG
GAGCAG
BssSI
NNCAGTGNN
NNGTCACNN
TspRI
(3’ Overhang)
Enzymes Recognizing
Non palindromic Sequences

61. Ligation of Restriction Enzyme Digested DNA
Sticky ends must match (be complementary)
for optimal re-ligation.
Sticky ends can be converted to blunt ends with nuclease or polymerase. Blunt ends can be converted to sticky ends by ligating to synthetic adaptors.
Blunt ends can be re-ligated with less efficiency than sticky ends.

62. Cloning into Plasmid Vectors

63. Restriction Enzyme Mapping
Digest DNA with a restriction enzyme.
Resolve the fragments by gel electrophoresis.
The number of bands indicates the number of restriction sites.
The size of the bands indicates the distance between restriction sites.

64. Restriction Enzyme Mapping
4.3 kb
3.7 kb
2.3 kb
1.9 kb
1.4 kb
1.3 kb
0.7 kb
BamH1
XhoI
4.0 kb
2.8 kb
1.2 kb
1.7 kb
1.1 kb
BamH1
XhoI
1.1 kb
1.7 kb
1.2 kb
2.8 kb

65. Southern Blot Developed by Edwin Southern.
The Southern blot procedure allows analysis of any specific gene or region without having to clone it from a complex background.

66. Denaturation of DNA: Breaking the Hydrogen Bonds

67. Denaturation and Annealing (Re-forming the Hydrogen Bonds)
If we heat up a tube of DNA dissolved in water, the energy of the heat can pull the two strands of DNA apart (there's a critical temperature called the Tm at which this happens). This process is called 'denaturation'; when we've 'denatured' the DNA, we have heated it to separate the strands. The two strands still have the same nucleotide sequences, however, so they are still complementary. If we cool the tube again, then in the course of the normal, random molecular motion they'll eventually bump into each other ... and stick tightly, reforming double-stranded DNA. This process is called 'annealing' or 'hybridization', and it is very specific; only complementary strands will come together if it is done right. This process is used in many crime labs to identify specific strands of DNA in a mixture.

68. HYBRIDIZATION: Denaturation and Annealing of DNA

69. Southern Blot Hybridization
Transfer DNA from a gel matrix to a filter (nitrocellulose, nylon)
Fix DNA to filter (Heat under a vacuum, UV cross-link
Hybridize with single stranded radiolabeled probe

70. Southern Blot Extract DNA from cells, etc Cut with RE
Run on gel (usually agarose)
Denature DNA with alkali
Transfer to nylon (usually capillary action)
Autoradiograph

71. Blotting a Gel
Separate restriction enzyme-digested DNA by gel electrophoresis
Soak gel in strongly alkali solution (0.5 N NaOH) to melt double stranded DNA into single stranded form
Neutralize pH in a high salt concentration (3 M NaCl) to prevent re-hybridization

72. Blot to Solid Support
Originally used nitrocellulose paper, now use chemically modified nylon paper
Binds ssDNA strongly
Transferred out of gel by passive diffusion during fluid flow to dry paper toweling
Block excess binding sites with foreign DNA (salmon sperm DNA)

73. Transfer of DNA to Membrane

74. Capillary Transfer Dry paper Nitrocellulose membrane Gel Soaked
Reservoir

75. Electrophoretic Transfer- +
Buffer
Glass plates
Whatman
paper
Nitrocellulose filter
Gel

76. Vacuum Transfer Gel Recirculating buffer Nitrocellulose filter Vacuum
Porous plate
Gel
Recirculating
buffer
Vacuum

77. Southern Blot Block with excess DNA (unrelated)
Hybridize with labeled DNA probe
Wash unbound probe (controls stringency)

78. The Probe Determines What Region Is Seen
DNA, RNA, or protein
Covalently attached signal molecule
radioactive (32P, 33P, 35S)
nonradioactive (digoxigenin, biotin, fluorescent)
Specific (complementary) to target gene

79. Complementary Sequences
Complementary sequences are not identical.
Complementary strands are antiparallel.
P5′ - GTAGCTCGCTGAT - 3′OH
OH3′ - CATCGAGCGACTA - 5′P

80. Southern Blot Hybridization: Overview

81. Types Of Nucleic Acid Probes
dsDNA probes
Must be denatured prior to use (boiling, 10 min)
Two competing reactions: hybridization to target, reassociation of probe to itself
ssDNA probes
RNA probe
Rarely used due to RNAses, small quantities
PCR generated probes
ss or ds, usually use asymmetric PCR

82. Detection Methods Isotopic labels (3H, 32P, 35S, 125I)
Photographic exposure (X-ray film)
Quantification (scintillation counting, densitometry)
Non-isotopic labels (enzymes, lumiphores)
Enzymatic reactions (peroxidase, alkaline phosphatase)
Luminescence (Adamantyl Phosphate derivatives, “Lumi-Phos”)

83. Radioactive Labels 32P: t1/2 = 14.3 days 33P: t1/2 = 25.4 days
High energy beta emitter
With good probe (106 cpm/ml), overnight signal
33P: t1/2 = 25.4 days
Lower energy
3-7 days for signal
35S: t1/2 = 87.4 days
More diffuse signal
3H: t1/2 = 12.4 years
Very weak
Got grand kids?

84. Radiolabeling Probes Nick translation Random primer 5’ End label
DNase to create single strand gaps
DNA pol to repair gaps in presence of  32P ATP
Random primer
Denature probe to single stranded form
Add random 6 mers,  32P ATP, and DNA pol
5’ End label
Remove 5’ Phosphate with Alkaline phosphatase
Transfer 32P from  32P ATP with T4 polynucleotide kinase

85. Melting Temperature (Tm)
The temperature at which 50% of a nucleic acid is hybridized to its complementary strand. DS
DS = SS SS Tm
Increasing temperature

86. Melting Temperature and Hybridization
Your hybridization results are directly related to the number of degrees below the melting temperature (Tm) of DNA at which the experiment is performed.
For a aqueous solution of DNA (no salt) the formula for Tm is:
Tm = 69.3oC (% G + C)oC

87. Tm in Solution is a Function of:
Length of DNA
GC content (%GC)
Salt concentration (M)
Formamide concentration
Tm = 81.5°C logM (%G + C) (%formamide) - 600/n
(DNA:DNA)

88. Denaturation: Melting Temperatures

89. G + C Content (as a %) GC content has a direct effect on Tm.
The following examples, demonstrate the point.
Tm = 69.3oC (45)oC = 87.5oC (for wheat germ)
Tm = 69.3oC (40)oC = 85.7oC
Tm = 69.3oC (60)oC = 93.9oC

90. Tm
For short (14–20 bp) oligomers:
Tm = 4° (GC) + 2° (AT)

91. General Hybridization Times/ Temperatures
Optimal Hybridization Times
Optimal Hybridization Temperatures
ON=overnight

92. Hybridization Conditions
Three steps of hybridization reaction
Prehybridization to block non-specific binding
Hybridization under appropriate conditions
Post-hybridization to remove unbound probe
High Stringency for well matched hybrids
High temp (65o-68oC) or 42oC in presence of 50% formamide
Washing with low salt (0.1X SSC), high temp (25oC)
Low Stringency
Low temp, low formamide
Washing with high salt

93. Stringency
Stringency describes the conditions under which hybridization takes place.
Formamide concentration increases stringency.
Low salt increases stringency.
Heat increases stringency.

94. Hybridization Stringency
Closely related genes are not identical in sequence, but are similar
Conserved sequence relationship is indicator of functional importance
Use lower temperature hybridization to identify DNAs with limited sequence homology: reduced stringency

96. Determination Of Tm Values Of Probes
DNA-DNA Hybrids
Tm= X log[Na]-0.65(%formamide)+41(%G+C)
RNA-DNA Hybrids
Tm= X log [Na]-0.35(%formamide)+58.4(%G+C)+11.8(%G+C)
Oligonucleotide probes (16-30 nt)
Tm=2(No. A+T) + 4(No. G + C)-5oC

97. Overview of Southern Blot Hybridization

99. Southern Blot Results Radioactive or Chromogenic detection
chemiluminescent detection
(autoradiography film)
Chromogenic detection
(nitrocellulose membrane)

100. Rate Of Reassociation: Factors Affecting Kinetics Of Hybridization
Temperature
Usually Tm-25o C
Salt concentration
Rate increases with increasing salt
Base mismatches
more mismatches, reduce rate
Fragment lengths
Probe fragments shorter than target, increase rate
Complexity of nucleic acids
Inversely proportional
Base composition
Increases with increasing G+C
Formamide
20% reduces rate, 30-50% has no effect
Dextran sulfate
increases rate
Ionic strength
increasing ionic strength, increasing rate
pH-between
Viscosity
increasing viscosity, decreasing rate of reassociation

101. Factors Affecting Hybrid Stability
Tm of DNA-DNA hybrids
Tm= (logM)+0.41(%G+C)-0.72(%formamide)
Tm of RNA-DNA hybrids
80% formamide improves stability of RNA-DNA hybrids
Formamide-lowers hybridization temperature
Ionic Strength-higher ionic strength, higher stability
Mismatched hybrids-Tm decreases 1oC for each 1% mismatched pairs

102. Factors Affecting the Hybridization Signal
Amount of genomic DNA
Proportion of the genome that is complementary to the probe
Size of the probe (short probe = low signal)
Labeling efficiency of the probe
Amount of DNA transferred to membrane