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Isolation and Purification of Nucleic Acids: Sample

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Isolation and Purification of Nucleic Acids: Sample Processing Donna C. Sullivan, PhD Division of Infectious Diseases University of Mississippi Medical Center – PowerPoint PPT presentation

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Title: Isolation and Purification of Nucleic Acids: Sample


1
Isolation and Purification of Nucleic Acids
Sample Processing
  • Donna C. Sullivan, PhD
  • Division of Infectious Diseases
  • University of Mississippi Medical Center

2
Outline
  • Principles for handling clinical specimens
  • Types of specimens
  • Fundamentals of specimen handling
  • Nucleic acid preparation
  • DNA isolation methods
  • RNA isolation methods
  • Methods of analysis of nucleic acids

3
Principles for Handling of All Clinical Specimens
  • Observe universal precautions for biohazards.
  • Use protective gowns, gloves, face and eye
    shields.
  • Decontaminate all spills and work areas with 10
    bleach.
  • Dispose of all waste in appropriate biologic
    waste containers.
  • Use gloves. Your RNA depends on it!

4
Types of Specimens for the Molecular Diagnostics
Laboratory
  • Whole blood
  • Bone marrow
  • PBSC (phoresis product)
  • Serum/plasma
  • Buccal cells
  • Cultured cells
  • Blood spots
  • Body fluids
  • CSF
  • Bronchial lavage
  • Amniotic
  • Semen
  • Urine
  • Tissue samples
  • Fresh/frozen
  • Paraffin-embedded
  • Hair (shaft/root)

5
Fundamentals of Specimen Handling Specimen
Labeling
  • Patient name, date of birth, and medical record
    number
  • Ordering physician
  • Type of specimen
  • Accession number
  • Date and time of collection
  • Laboratory technician identification (initials)
  • Requested test(s)

6
Blood and Bone Marrow
  • Isolation of nucleic acids
  • Genomic DNA
  • RNA
  • Collection
  • Collect in an anticoagulant, mix well but gently
    to avoid disruption of cells

7
Anticoagulants
  • EDTA
  • Lavender-top Vacutainer
  • Preferred specimen
  • ACD
  • Yellow-top Vacutainer
  • Heparin
  • Green-top Vacutainer
  • Inhibits several enzymes used in molecular assays

8
Specimen Packaging and Shipping Blood and Bone
Marrow
  • DO NOT FREEZE!!!
  • Protect from temperature extremes
  • Overnight delivery preferred
  • Packaging must comply with shipping rules for
    bloodborne pathogens
  • Protective container
  • Absorbent material in packing
  • Sealed container in plastic bag
  • Labeled as Biohazard

9
The Effect of Tissue Fixatives on the
Purification of Nucleic Acid
10
Paraffin-embedded Tissue Sections
  • Genetic testing, infectious disease testing,
    identity testing
  • Formalin-fixed tissue is suitable.
  • Mercury or other heavy metal fixatives are not
    acceptable.
  • Tissue sections on glass slides can be used for
    in situ applications and microdissection
    techniques.

11
Specimen Storage RequirementsDNABlood, Bone
Marrow, Other Fluids
  • 2225 C Not recommended (lt24 hours)
  • 28 C Suitable condition for up to 72 hours
  • 20 C Not recommended
  • NOTE Do not freeze blood or bone marrow before
    lysing red blood cells (RBCs). Leukocyte pellet
    can be frozen for up to 1 year.
  • 70 C Not recommended
  • NOTE Do not freeze blood or bone marrow before
    lysing red blood cells (RBCs). Leukocyte pellet
    can be frozen for gt1 year.

12
Specimen Storage Requirements RNA Blood, Bone
Marrow, Other Fluids
  • 2225 C Not recommended within 2 hours
  • 28 C Not recommended within 2 hours
  • 20 C Not recommended 24 weeks
  • NOTE Do not freeze blood or bone marrow before
    lysing red blood cells (RBCs).
  • 70 C Preferred storage condition
  • NOTE Do not freeze blood or bone marrow before
    lysing red blood cells (RBCs)

13
Nucleic Acid Storage Requirements Storage of DNA
Specimens
lt4 Months
13 Years
lt7 Years
gt7 Years
225 C
28 C
20 C
70 C
Not recommended
Recommended for long-term storage in ethanol
14
Nucleic Acid Preparation Application?
  • DNA
  • Amplification methods (PCR, LCR)
  • Restriction enzyme digest
  • Hybridization methods (Southern analysis)
  • Sequencing

15
Nucleic Acid Preparation Application?
  • RNA
  • Amplification methods (RT-PCR)
  • Hybridization methods (Northern analysis)

16
Nucleic Acid Preparation Sample Source?
  • Whole blood
  • Buffy coat
  • Serum or plasma
  • Bone material
  • Buccal cells
  • Cultured cells
  • Amniocytes or amniotic fluid
  • Dried blood spots
  • Fresh or frozen tissue (biopsy material)
  • Sputum, urine, CSF, or other body fluids
  • Fixed or paraffin-embedded tissue

17
Nucleic Acid Preparation Other Considerations
  • What is the size or volume of each sample?
  • Amount of DNA or RNA required
  • Equipment and tube sizes required
  • How many samples are being processed?
  • Capacity of the centrifuge
  • Isolation method speed
  • Is a high-throughput or automated system
    available?
  • 96-well plate methods
  • Walk-away or semi-automation

18
Nucleic Acid Preparation Choosing an Isolation
Method
  • Important factors are
  • Processing speed
  • Ease of use
  • Yield of DNA or RNA
  • Quality of DNA and RNA prepared (amplification
    performance)
  • Shelf life/storage conditions
  • Quality assurance criteria
  • Cost of preparation

19
Basic Steps in Isolating DNA from Clinical
Specimens
20
DNA Isolation Methods Liquid Phase Organic
Extraction
  • Phenol (50)chloroform/isoamyl alcohol (50491)
  • Lysed samples mixed with above two layers are
    formed.
  • Proteins remain at interface.
  • DNA is removed with top aqueous layer.
  • DNA is precipitated with alcohol and rehydrated.
  • Disadvantages
  • Slow, labor-intensive, toxic (phenol, chloroform)
  • Fume hood required, disposal of hazardous
    materials required

21
DNA Isolation Methods Liquid Phase Nonorganic
Salt Precipitation
  • Cell membranes are lysed and proteins are
    denatured by detergent (such as SDS).
  • RNA is removed with RNase.
  • Proteins are precipitated with salt solution.
  • DNA is precipitated with alcohol and rehydrated.
  • Advantages
  • Fast and easy method
  • Uses nontoxic materials, no fume hood required,
    no hazardous materials disposal issues
  • Produces high-quality DNA

22
DNA Isolation Methods Solid Phase Procedures
  • Uses solid support columns, magnetic beads, or
    chelating agents
  • Solid support columns Fibrous or silica matrices
    bind DNA allowing separation from other
    contaminants.
  • Magnetic beads DNA binds to beads beads are
    separated from other contaminants with magnet.
  • Chelating resins
  • Advantages
  • Fast and easy, no precipitation required

23
DNA Purification Method Comparison
24
Basic Steps in Isolating RNA from Clinical
Specimens
25
Precautions for Working with RNA in the Clinical
Laboratory
  • RNA is not a stable molecule!
  • It is easily degraded by RNase enzymes.
  • Use sterile, disposable plastic ware (tubes,
    filter tips) marked For RNA Use Only.
  • Always wear gloves and work in a hood whenever
    possible/practical.
  • Treat liquids with DEPC, except Tris-based
    buffers.

26
RNA Isolation Methods Cesium Chloride Gradient
  • Used mainly to get clean RNA for Northern blots
  • Homogenize cells in guanidinium isothiocyanate
    and b-mercaptoethanol solution.
  • Add to CsCl gradient and centrifuge for 1220
    hours RNA will be at the bottom of tube.
  • Re-dissolve in TE/SDS buffer.
  • Precipitate RNA with salt and ethanol, then
    rehydrate.
  • Advantage high quality
  • Disadvantages extremely time-consuming,
    hazardous materials disposal issues

27
RNA Isolation Methods Guanidinium-based Organic
Isolation
  • Phenol/guanidinium solution disrupts cells,
    solubilizes cell components, but maintains
    integrity of RNA.
  • Add chloroform, mix, and centrifuge.
  • Proteins/DNA remain at interface.
  • RNA is removed with aqueous top layer.
  • RNA is precipitated with alcohol and rehydrated.
  • Advantage faster than CsCl method
  • Disadvantages fume hood required, hazardous
    waste disposal issues

28
RNA Isolation Methods Nonorganic Salt
Precipitation
  • Cell membranes are lysed and proteins are
    denatured by detergent (such as SDS) in the
    presence of EDTA or other RNase inhibitors.
  • Proteins/DNA are precipitated with a high
    concentration salt solution.
  • RNA is precipitated with alcohol and rehydrated.
  • Advantages
  • Fast and easy, nontoxic
  • Produces high quality RNA

29
Resuspending Final Nucleic Acid Samples
  • Have some idea of expected nucleic acid yield.
  • Choose diluent volume according to desired
    concentration.
  • Calculating Expected DNA Yield
  • Example
  • 1 X 107 cells X 6 pg DNA/cell X 80 yield 48 mg
    DNA
  • Resuspend DNA in TE buffer or ultra pure
    DNAse-free water.
  • Resuspend RNA in ultra pure RNase-free water.

30
Nucleic Acid Analysis
  • DNA or RNA is characterized using several
    different methods for assessing quantity,
    quality, and molecular size.
  • UV spectrophotometry
  • Agarose gel electrophoresis
  • Fluorometry
  • Colorimetric blotting

31
Quantity from UV Spectrophotometry
  • DNA and RNA absorb maximally at 260 nm.
  • Proteins absorb at 280 nm.
  • Background scatter absorbs at 320 nm.

32
Quantity from UV Spectrophotometry
  • DNA
  • (A260 A320) X dilution factor X 50 µg/mL
  • RNA
  • (A260 A320) X dilution factor X 40 µg/mL
  • Concentration µg of DNA or RNA per mL of
    hydrating solution

33
Quantity from UV Spectrophotometry Calculating
Yield
34
Quality from UV Spectrophotometry
35
Quality from Agarose Gel Electrophoresis
  • Genomic DNA
  • 0.6 to 1 gel, 0.125 µg/mL ethidium bromide in
    gel and/or in running buffer
  • Electrophorese at 7080 volts, 4590 minutes.
  • Total RNA
  • 1 to 2 gel, 0.125 µg/ml ethidium bromide in gel
    and/or in running buffer
  • Electrophorese at 80100 volts, 2040 minutes.

36
DNA Size from Agarose Gel Electrophoresis
Compares unknown DNA to known size standards
37
DNA Quality from Agarose Gel Electrophoresis
  • High molecular weight band (gt48.5 kb)
  • Smearing indicates DNA degradation (or too much
    DNA loaded).

38
DNA Quality from Agarose Gel Electrophoresis
39
RNA Size and Quality from Agarose Gel
Electrophoresis
  • Size mRNA may be smaller or larger than
    ribosomal RNA (rRNA).
  • Quality High-quality RNA has these
    characteristics
  • 28S rRNA band 18S rRNA band 21 intensity
  • Little to no genomic DNA (high MW band)
  • Note If 18S rRNA is more intense than 28S rRNA,
    or if both bands are smeared, RNA degradation is
    probable.

40
Cultured Cell RNA
41
Storage Conditions
  • Store DNA in TE buffer at 4 C for weeks or at
    20 C to 80 C for long term.
  • Store RNA in RNase-free ultra pure water at 70
    C.

42
Troubleshooting Nucleic AcidPreparation Methods
  • Problem No or low nucleic acid yield.
  • Make sure that ample time was allowed for
    resuspension or rehydration of sample.
  • Repeat isolation from any remaining original
    sample (adjust procedure for possible low cell
    number or poorly handled starting material).
  • Concentrate dilute nucleic acid using ethanol
    precipitation.

43
Troubleshooting Nucleic AcidPreparation Methods
  • Problem Poor nucleic acid quality
  • If sample is degraded, repeat isolation from
    remaining original sample, if possible.
  • If sample is contaminated with proteins or other
    substances, clean it up by re-isolating
    (improvement depends on the extraction procedure
    used).

44
Chapter 5 Resolution and Detection of Nucleic
Acids
45
Objectives
  • Explain the principle and performance of
    electrophoresis as it applies to nucleic acids.
  • Compare and contrast agarose and polyacrylamide
    gel polymers.
  • Explain the principle and performance of
    capillary electrophoresis as it is applies to
    nucleic acid separation.
  • Describe the general types of equipment used for
    electrophoresis.
  • Discuss methods and applications of pulsed field
    gel electrophoresis.
  • Compare and contrast detection systems used in
    nucleic acid applications.

46
Gel Electrophoresis
  • Electrophoresis is the movement of molecules by
    an electric current.
  • Nucleic acid moves from a negative to a positive
    pole.
  • Nucleic acid has a net negative charge, they RUN
    TO RED

47
Electrophoresis of Nucleic Acids
  • Nucleic acids are separated based on size and
    charge.
  • DNA molecules migrate in an electrical field at a
    rate that is inversely proportional to the log10
    of molecular size (number of base pairs).
  • Employs a sieve-like matrix (agarose or
    polyacrylamide) and an electrical field.
  • DNA possesses a net negative charge and migrates
    towards the positively charged anode.

48
Applications of Electrophoretic Techniques in the
Molecular Diagnostics Laboratory
  • Sizing of Nucleic Acid Molecules
  • DNA fragments for Southern transfer analysis
  • RNA molecules for Northern transfer analysis
  • Analytical separation of PCR products
  • Detection of Mutations or Sequence Variations

49
Principles of Gel Electrophoresis
  • Electrophoresis is a technique used to separate
    and sometimes purify macromolecules
  • Proteins and nucleic acids that differ in size,
    charge or conformation
  • Charged molecules placed in an electric field
    migrate toward either the positive (anode) or
    negative (cathode) pole according to their charge
  • Proteins and nucleic acids are electrophoresed
    within a matrix or "gel"

50
ELECTROPHORESIS
DNA and RNA are negatively charged they RUN TO
RED!
51
Principles of Gel Electrophoresis
  • The gel itself is composed of either agarose or
    polyacrylamide.
  • Agarose is a polysaccharide extracted from
    seaweed.
  • Polyacrylamide is a cross-linked polymer of
    acrylamide.
  • Acrylamide is a potent neurotoxin and should be
    handled with care!

52
Gel Electrophoresis Matrices
53
Types Of Nucleic Acid Electrophoresis
  • Agarose gel electrophoresis
  • DNA or RNA separation
  • TAE or TBE buffers for DNA, MOPS with
    formaldehyde for RNA
  • Polyacrylamide gel electrophoresis (PAGE)
  • Non-denaturing (Special applications in research)
  • Denaturing contain 6-7 M Urea (Most common)

54
Agarose Gel Electrophoresis
  • Separates fragments based on mass, charge
  • Agarose acts as a sieve
  • Typically resolve 200 bp-20 kbp
  • fragments lt200 bp, polyacrylamide gels
  • fragmentsgt 20 kbp, pulse field gels
  • Include DNA size standards

55
Factors That Effect Mobility Of DNA Fragments In
Agarose Gels
  • Agarose Concentration
  • Higher concentrations of agarose facilitate
    separation of small DNAs, while low agarose
    concentrations allow resolution of larger DNAs
    (Remember-inversely proportional!)
  • Voltage
  • As the voltage applied to a gel is increased,
    larger fragments migrate proportionally faster
    that small fragments
  • Charge is evenly spread (uniform) so the larger
    fragments will have more charged groups

56
Factors That Effect Mobility Of DNA Fragments In
Agarose Gels
  • Electrophoresis Buffer
  • The most commonly used for double stranded
    (duplex) DNA are TAE (Tris-acetate-EDTA) and TBE
    (Tris-borate-EDTA).
  • Effects of Ethidium Bromide
  • Staining dye that inserts (intercalates) into the
    DNA between the nitrogenous bases (rungs of the
    ladder) and glows when exposed to UV light
  • Binding of ethidium bromide to DNA alters its
    mass and rigidity, and therefore its mobility

57
Comparison of Agarose Concentrations
58
Fragment Resolution Agarose Gel Electrophoresis
59
Gel Electrophoresis The Basics
  • The movement of molecules is impeded in the gel
    so that molecules will collect or form a band
    according to their speed of migration.
  • The concentration of gel/buffer will affect the
    resolution of fragments of different size ranges.
  • Genomic DNAs usually run as a smear due to the
    large number of fragments with only small
    differences in mass

60
Agarose Electrophoresis of Restriction Enzyme
Digested Genomic DNA
61
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
  • Agarose
  • Large genomic DNA (Chromosomal)

62
Electrophoresis Equipment Horizontal or
Submarine Gel
63
Agarose Gel Electrophoresis
64
Agarose Gel ElectrophoresisHorizontal Gel Format
65
Agarose Gel Apparatus
66
Electrophoresis Equipment Vertical Gel
67
Vertical Gel Format Polyacrylamide Gel
Electrophoresis
68
Polyacrylamide Gel Electrophoresis (PAGE)
69
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

70
PULSE FIELD GEL ELECTROPHORESIS APPARATUS
71
Types Of Pulse Field Gel Electrophoresis
72
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

73
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

74
Size of Fragments and Distance Traveled Not
Linear When Large Fragments Are Analyzed
75
Movement Of DNA In Gels
76
Pulse Field Gel Electrophoresis
  • PFGE works by periodically altering the electric
    field orientation
  • The large extended coil DNA fragments are forced
    to change orientation
  • Size dependent separation is re-established
    because the time taken for the DNA to reorient is
    size dependent

77
Comparison of Migration Horizontal vs. CHEF
78
Preparation Of Intact DNA For PFGE
  • Conventional techniques for DNA purification
    (organic extraction, ethanol precipitation)
    produce shear forces
  • DNA purified is rarely greater than a few hundred
    kb in size
  • This is clearly unsuitable for PFGE which can
    resolve mb DNA
  • The problem of shear forces was solved by
    performing DNA purification from whole cells
    entirely within a low melting temperature (LMT)
    agarose matrix

79
Preparation Of Intact DNA For PFGE
  • Intact cells are mixed with molten low melting
    point (LMT) agarose and set in a mold forming
    agarose plugs
  • Enzymes and detergents diffuse into the plugs and
    lyse cells
  • Proteinase K diffuses into plugs and digests
    proteins
  • If necessary restriction digests are performed in
    plugs (extensive washing or PMSF treatment is
    required to remove proteinase K activity)
  • Plugs are loaded directly onto PFGE and run

80
FIGE GEL
81
CHEF Contour-Clamped Homogenous Gel
Electrophoresis
  • Based on hexagonal array of alternate electric
    fields at 120 degree angle
  • Generates a more uniform electric field when
    compared to other PFGE systems
  • Programmable, autonomously controlled electrodes
  • Extremely versatile system based on CHEF
    hexagonal array
  • All electrophoretic parameters can be controlled
    at each electrode
  • Can generate electric field and switching
    characteristics of any PFGE system

82
PFGE of Bacterial DNA
83
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
84
Using PFGE in the Molecular Investigation Of An
Outbreak of S. marcescens Infection in an ICU
Singapore Med J 2004 Vol 45(5) 214
85
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
86
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).
87
Polyacrylamide Gel Electrophoresis (PAGE)
  • PAGE is the preferred method for PROTEINS but can
    be used for DNA/RNA
  • Gel prepared immediately before use by
    copolymerization of acrylamide and N,N'-methylene
    bis acrylamide under UV light.
  • Porosity controlled by proportions of the two
    components.
  • Larger pore size for larger proteins.
  • Gradient gels also possible.

88
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 5500 bp in size.

89
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.

90
Electrophoresis of Nucleic AcidsAgarose Gel
Electrophoresis
  • Advantages
  • Greater range of separation of nucleic acid
    molecules.
  • Optimal separation is achieved with nucleic acids
    that are 200 bp to 30 kb in size.
  • Ease of preparation and handling.

91
PAGE Critical Parameters
  • Polymerization reaction critical
  • High grade acrylamide, bis-acrylamide
  • Break down into acrylic acid (long shelf life
    solutions incorporate inhibitors of
    polymerization)
  • Must have even heat distribution to prevent
    smiling

92
Polymerization Of Polyacrylamide
93
PAGE DNA
  • High resolution of low molecular weight nucleic
    acids (500bp)
  • Polymerization of acrylamide monomers into long
    chains
  • Cross link chains with bis-acrylamide
  • Initiated by free radicals in ammonium
    persulfate, stabilized by TEMED
  • Pore size determined by acrylamide

94
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.

95
PAGE Fragment Resolution Denaturing Conditions
(6M Urea)
96
PAGE Fragment Resolution Non Denaturing PAGE
97
Polyacrylamide Gel Electrophoresis of Restriction
Digested PCR Products
98
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.
99
Preparation of Polyacrylamide Gel
  • Pour into glass plate gel sandwich and
    polymerize.
  • Prepare DNA samples by adding loading buffer.
  • Document and verify loading order of samples and
    electrophoretic conditions (voltage).
  • Stain gel, visualize DNA, photograph/ document
    and dispose of gel properly.

100
PAGE of Restriction Digested PCR Products
101
Chapter 6 Analysis and Characterization of
Nucleic Acids and Proteins
102
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).

103
Restriction Enzymes
  • Type I
  • Methylation/cleavage (3 subunits)
  • gt1000 bp from binding site
  • e.g., Eco AI GAGNNNNNNNGTCA
  • Type II
  • Cleavage at specific recognition sites
  • Type III
  • Methylation/cleavage (2 subunits)
  • 2426 bp from binding site
  • e.g., Hinf III CGAAT

104
Restriction Endonucleases Type II
105
There are hundreds of restriction enzymes
106
Restriction Enzymes
107
Restriction Enzymes
108
Ligation of Restriction Enzyme Digested DNA
109
Cloning into Plasmid Vectors
110
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.

111
Restriction Enzyme Mapping
112
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.

113
Denaturation of DNA Breaking the Hydrogen Bonds
114
Denaturation and Annealing (Re-forming the
Hydrogen Bonds)
115
Denaturation/Annealing An Equilibrium Reaction
116
HYBRIDIZATION Denaturation and Annealing of DNA
117
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)

118
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

119
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

120
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

121
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

122
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)

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DNA Binding Media
  • Electrostatic and hydrophobic
  • Nitrocellulose
  • Nylon
  • Reinforced nitrocellulose
  • Electrostatic
  • Nylon, Nytran
  • Positively charged nylon

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Transfer of DNA to Membrane
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Capillary Transfer
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Electrophoretic Transfer
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Vacuum Transfer
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Southern Blot
  • Block with excess DNA (unrelated)
  • Hybridize with labeled DNA probe
  • Wash unbound probe (controls stringency)

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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

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

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Southern Blot Hybridization Overview
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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

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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)

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Radioactive Labels
  • 32P t1/2 14.3 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?

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Radiolabeling Probes
  • Nick translation
  • 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

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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
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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 0.41( G C)oC

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Tm in Solution is a Function of
  • Length of DNA
  • GC content (GC)
  • Salt concentration (M)
  • Formamide concentration
  • Tm 81.5C 16.6 logM 0.41 (G C) - 0.61
    (formamide) - 600/n
  • (DNADNA)

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Denaturation Melting Temperatures
140
G C Content (as a )
  • GC content has a direct effect on Tm.
  • The following examples, demonstrate the point.
  • Tm 69.3oC 0.41(45)oC 87.5oC (for wheat
    germ)
  • Tm 69.3oC 0.41(40)oC 85.7oC
  • Tm 69.3oC 0.41(60)oC 93.9oC

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Tm
  • For short (1420 bp) oligomers
  • Tm 4 (GC) 2 (AT)

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Melting Temperature (Tm) andG C Content
143
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 degree 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 81.5 16.6(log M Na) 0.41(GC) -
    0.72( formamide)

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General Hybridization Times/ Temperatures
ONovernight
145
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

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

147
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

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

150
Determination Of Tm Values Of Probes
  • DNA-DNA Hybrids
  • Tm81.516.6 X logNa-0.65(formamide)41(GC)
  • RNA-DNA Hybrids
  • Tm79.818.5 X log Na-0.35(formamide)58.4(GC
    )11.8(GC)
  • Oligonucleotide probes (16-30 nt)
  • Tm2(No. AT) 4(No. G C)-5oC

151
Hybridization On A Surface
152
Annealing On A Surface
153
Detection Of Labeled Probe
154
Radioactive Signal Detection

Filter with bound DNA
155
Non-Radioactive Signal Detection
156
Overview of Southern Blot Hybridization
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Southern Blot Results
160
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 GC
  • Formamide
  • 20 reduces rate, 30-50 has no effect
  • Dextran sulfate
  • increases rate
  • Ionic strength
  • increasing ionic strength, increasing rate
  • pH-between 6.8-7.4
  • Viscosity
  • increasing viscosity, decreasing rate of
    reassociation

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Factors Affecting Hybrid Stability
  • Tm of DNA-DNA hybrids
  • Tm81.516.6(logM)0.41(GC)-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

162
Factors Affectingthe 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

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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?

164
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?

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

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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

167
SDS PAGE Proteins
168
Function Of SDS
169
SDS PAGE Proteins
170
DISC ELECTROPHORESIS
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SDS PAGE Coomassie Blue Stain
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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 11 with 0.04 M Tris HCl, pH 6.8, 0.1
    SDS.
  • 520 polyacrylamide gels

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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.

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Western Blot Signal Detection
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Filter-based Hybridization Technologies
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Blotting Formats
  • Dot blots
  • amplification analysis
  • expression analysis (RNA)
  • mutation analysis
  • Reverse dot blots
  • Slot blots
  • amplification analysis
  • expression analysis

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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)
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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

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Comparative Genomic Hybridization
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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.

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

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

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

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DNA Sequencing
185
Dideoxy or Sanger DNA Sequencing
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Sequencing Gels
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