1. Chapter 6: Analysis and Characterization of Nucleic Acids and Proteins
Donna C. Sullivan, PhD
Division of Infectious Diseases
University of Mississippi Medical Center

2. Objectives Describe how restriction enzyme sites are mapped on DNA.
Construct a restriction enzyme map of a DNA plasmid or fragment.
Diagram the Southern blot procedure.
Define hybridization, stringency, and melting temperature.
Calculate the melting temperature of a given sequence of dsDNA.
Describe comparative genomic hybridization (CGH).

3. 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

4. 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

5. There are hundreds of
restriction enzymes

6. 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)

7. 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

8. 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.

9. Cloning into Plasmid Vectors

10. 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.

11. 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

12. 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.

13. Denaturation of DNA: Breaking the Hydrogen Bonds

14. 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.

15. Denaturation/Annealing: An Equilibrium Reaction

16. HYBRIDIZATION: Denaturation and Annealing of DNA

17. Basic Techniques for Analysis of Nucleic Acids
Enzymatic modification (polymerase, kinase, phosphatase, ligase)
Endonuclease digestion (DNAse, RNase, restriction enzymes)
Electrophoresis (agarose and polyacrylamide gel electrophoresis)

18. Molecular Search Tools: Blots
Southern blots
DNA immobilized on solid support
Northern blots
RNA immobilized on solid support
Western blots
Proteins immobilized on solid support

19. 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

20. 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

21. 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

22. 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)

23. DNA Binding Media Electrostatic and hydrophobic: Electrostatic
Nitrocellulose
Nylon
Reinforced nitrocellulose
Electrostatic
Nylon, nytran
Positively charged nylon

24. Transfer of DNA to Membrane

25. Capillary Transfer Dry paper Nitrocellulose membrane Gel Soaked
Reservoir

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

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

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

29. The probe determines what region is seen.
DNA, RNA, or protein
Covalently attached signal molecule
radioactive (32P, 33P, 35S)
nonradioactive (digoxygenin, biotin, fluorescent)
Specific (complementary) to target gene

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

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

32. Southern Blot Hybridization: Overview

33. 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

34. 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”)

35. 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?

36. 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

37. 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

38. 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

39. 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)

40. Denaturation: Melting Temperatures

41. 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

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

43. Melting Temperature (Tm) and G + C Content

44. Formula Which That Takes The Salt Concentration Into Account
Hybridizations though are always performed with salt.
Under salt-containing hybridization conditions, the effective Tm is what controls the degeree of homology between the probe and the filter bound DNA is required for successful hybridization.
The formula for the Effective Tm (Eff Tm).
Eff Tm = (log M [Na+]) (%G+C) (% formamide)

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

46. 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

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

48. 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

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

51. 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

52. Hybridization On A Surface

53. Annealing On A Surface

54. Detection Of Labeled Probe

55. Radioactive Signal Detection
Filter with bound DNA
Radioactive isotope
Probe

56. Non-Radioactive Signal Detection
Antidigoxygenin antibody or avidin conjugated
to alkaline phosphatase or horseradish peroxidase.
Probe covalently attached to digoxygenin or biotin.
Substrate Color or light

57. Overview of Southern Blot Hybridization

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

61. 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

62. 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

63. 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

64. Trouble Shooting Southern Blots
Was enough DNA loaded/well (10 g)?
Was DNA completely digested with restriction enzyme?
Was DNA denatured and neutralized prior to transfer?
Was DNA transfer complete?
Was DNA immobilized on membrane?

65. Trouble Shooting Southern Blots
Was the probe prepared properly?
Was hybridization time adequate?Was exposure time adequate?
Was the probe labeled sufficiently?
How many total cpm were added?
What was the specific activity (cpm/g)?
How many times has the membrane been probed and stripped?

66. Southern Blot Applications
Genetics, oncology (translocations, gene rearrangements)
Typing/classification of organisms
Cloning/verification of cloned DNA
Forensic, parentage testing (RFLP, VNTR)

67. Molecular Search Tools: Blots
Southern blots
DNA immobilized on solid support
Northern blots
RNA immobilized on solid support
Western blots
Proteins immobilized on solid support

68. SDS PAGE: Proteins

69. Function Of SDS

70. SDS PAGE: Proteins

71. DISC ELECTROPHORESIS

72. SDS PAGE: Coomassie Blue Stain

73. Western Blot
Serum, cell lysate, or protein extract is separated on SDS-polyacrylamide gels (SDS-PAGE) or isoelectric focusing gels (IEF).
Samples are treated with denaturant, such as mixing 1:1 with 0.04 M Tris HCl, pH 6.8, 0.1% SDS.
5–20% polyacrylamide gels

74. Western Blot
Proteins may be renatured before blotting to optimize antibody (probe)-epitope binding.
Proteins are blotted to membranes by capillary or electrophoretic transfer.
Probes are specific binding proteins, polyclonal antibodies, or monoclonal antibodies.

75. Western Blot Signal Detection
Target
protein
Primary
antibody
(probe)
Secondary
label

76. Filter-based Hybridization Technologies
Target
Probe
Southern blot
DNA
Nucleic acid
Northern blot
RNA
Western blot
Protein
Southwestern blot

77. Blotting Formats Dot blots Reverse dot blots Slot blots
amplification analysis
expression analysis (RNA)
mutation analysis
Reverse dot blots
Slot blots
expression analysis

78. Comparative Genomic Hybridization (CGH)
Immobilized, denatured normal chromosomes.
Test and reference DNA are labeled by incorporation of nucleotides covalently attached to fluorescent dyes.
(Test) (Reference)

79. Comparative Genomic Hybridization
The labeled DNA is hybridized to the normal chromosomes on a microscope slide.
Differences between normal and reference will be revealed
amplification: test color dominates
deletion: reference color dominates
(Amplification at this locus)
(Deletion at this locus)
Normal reference DNA
Test sample DNA

80. Comparative Genomic Hybridization
Amplification
Deletion
Deletion
Deletion

81. Summary Restriction enzymes cut DNA at specific recognition sequences.
DNA can be characterized by restriction enzyme mapping.
Specific DNA regions in a complex mixture are characterized using Southern blot.
Specific proteins in a complex mixture are characterized using Western blot.
Regions of genomic amplification or deletion are characterized using comparative genomic hybridization.

82. DNA Sequencing Methods
Technology
Chain termination
Cycle sequencing
Chemistry
Maxam and Gilbert
Sanger
Platform
Manual
Automated

83. Maxam and Gilbert DNA Sequencing
Chemical cleavage of specific bases
Piperidine cleavage of phosphate backbone
Fragment size analysis by gel electrophoresis
Not commonly used

84. Sanger (Dideoxy) DNA Sequencing
Incorporation of 2´,3´-dideoxynucleotides by DNA polymerase
Termination of elongation reaction
Fragment size analysis (manual vs. automated)
Gel
Capillary

85. DNA Sequencing Dideoxy (Sanger) Sequencing (ddNTP) 2,3-dideoxyribose H
CH2OH OH 1 5 4 3 2
2,3-dideoxyribose

86. Dideoxy or Sanger DNA Sequencing
ATTAGACGT A
ATTA
ATTAGA T AT
ATT G
ATTAG
ATTAGACG C
ATTAGAC
A T G C

87. Sequencing Gels