Chapter 6: Analysis and Characterization of Nucleic Acids and Proteins Donna C. Sullivan, PhD...
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Transcript of Chapter 6: Analysis and Characterization of Nucleic Acids and Proteins Donna C. Sullivan, PhD...
Chapter 6: Analysis and Characterization of Nucleic Acids and Proteins
Donna C. Sullivan, PhDDivision of Infectious Diseases
University of Mississippi Medical Center
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).
Restriction Enzymes Type I
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
Restriction Endonucleases: Type II
Enzyme Isolated from
Recognition sequence
Eco RI E. coli, strain R, 1st enzyme
Gν AATTC
Eco RV E. coli, strain R, 5th enzyme
Gv ATATC
Hind III H. influenzae, strain d, 3rd enzyme
Av AGCTT
BamH1
GGATCCCCTAGG
HaeIII
GGCCCCGG
Cohesive Ends(5´ Overhang)
Cohesive Ends(3´ Overhang)
KpnI
GGTACCCCATGG
Blunt Ends(No Overhang)
Restriction Enzymes
GATCCTAG
DpnI(Requires methylation)
Methylation-sensitive Enzymes
GGCCCCGG
HaeIII(Inhibited by methylation)
CCCGGGGGGCCC
XmaI(5’ Overhang)
CCCGGGGGGCCC
SmaI(Blunt Ends)
Isoschizomers
Enzymes GeneratingCompatible Cohesive Ends
GGATCCCCTAGG
BamHI(5’ Overhang)
AGATCTTCTAGA
BglII(5’ Overhang)
CTCGTGGAGCAG
BssSI(5’ Overhang)
NNCAGTGNNNNGTCACNN
TspRI(3’ Overhang)
Enzymes RecognizingNon palindromic Sequences
Restriction Enzymes
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.
Ligation of Restriction Enzyme Digested DNA
Cloning into Plasmid Vectors
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.
Restriction Enzyme Mapping
BamH1
XhoI
XhoI
1.1 kb
1.7 kb
1.2 kb
2.8 kb4.3 kb3.7 kb
2.3 kb1.9 kb
1.4 kb1.3 kb
0.7 kb
BamH1 XhoIBamH1XhoI
4.0 kb
2.8 kb
1.2 kb
1.7 kb
1.2 kb
1.1 kb
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.
Denaturation of DNA: Breaking the Hydrogen Bonds
Denaturation and Annealing (Re-forming the Hydrogen Bonds)
Denaturation/Annealing: An Equilibrium Reaction
HYBRIDIZATION: Denaturation and Annealing of DNA
Basic Techniques for Analysisof Nucleic Acids
Enzymatic modification (polymerase, kinase, phosphatase, ligase)
Endonuclease digestion (DNAse, RNase, restriction enzymes)
Electrophoresis (agarose and polyacrylamide gel electrophoresis)
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
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
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
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
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)
DNA Binding Media Electrostatic and hydrophobic:
Nitrocellulose Nylon Reinforced nitrocellulose
Electrostatic Nylon, nytran Positively charged nylon
Transfer of DNA to Membrane
Capillary Transfer
Drypaper Nitrocellulose
membrane
Gel Soakedpaper
Reservoir
- +
Buffer Buffer
Glass plates
Whatmanpaper Nitrocellulose filter
Gel
Electrophoretic Transfer
Nitrocellulose filter
Porous plate
Gel Recirculatingbuffer
Vacuum
Vacuum Transfer
Southern Blot
Block with excess DNA (unrelated) Hybridize with labeled DNA probe Wash unbound probe (controls
stringency)
TheThe probe probe determines determines what region is seen.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
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
Complementary Sequences Complementary sequences are not
identical. Complementary strands are
antiparallel. P5′ - GTAGCTCGCTGAT - 3′OH
OH3′ - CATCGAGCGACTA - 5′P
Southern Blot Hybridization: Overview
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
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”)
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?
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
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
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
Tm in Solution is a Function of: Length of DNA GC content (%GC) Salt concentration (M) Formamide concentration
Tm = 81.5°C + 16.6 logM + 0.41 (%G + C) - 0.61 (%formamide) - 600/n
(DNA:DNA)
Denaturation: Melting Temperatures
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
Tm
For short (14–20 bp) oligomers: Tm = 4° (GC) + 2° (AT)
Melting Temperature (Tm) andG + C Content
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 = 81.5 + 16.6(log M [Na+]) +
0.41(%G+C) - 0.72(% formamide)
General Hybridization Times/ Temperatures
ON=overnight
Optimal Hybridization Times
Optimal Hybridization Temperatures
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
Stringency
Stringency describes the conditions under which hybridization takes place.
Formamide concentration increases stringency.
Low salt increases stringency. Heat increases stringency.
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
Stringency
Stringency describes the conditions under which hybridization takes place.
Formamide concentration increases stringency.
Low salt increases stringency. Heat increases stringency.
Determination Of Tm Values Of Probes
DNA-DNA HybridsTm=81.5+16.6 X log[Na]-0.65(%formamide)
+41(%G+C)
RNA-DNA HybridsTm=79.8+18.5 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
Hybridization On A Surface
Annealing On A Surface
Detection Of Labeled Probe
Radioactive isotope
Probe
Filter with bound DNA
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
Non-Radioactive Signal Detection
Overview of Southern Blot Hybridization
Radioactive orchemiluminescent detection(autoradiography film)
Chromogenic detection(nitrocellulose membrane)
Southern Blot Results
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 6.8-7.4 Viscosity
increasing viscosity, decreasing rate of reassociation
Factors Affecting Hybrid Stability Tm of DNA-DNA hybrids
Tm=81.5+16.6(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
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
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?
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?
Southern Blot Applications Genetics, oncology (translocations,
gene rearrangements) Typing/classification of organisms Cloning/verification of cloned DNA Forensic, parentage testing (RFLP,
VNTR)
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
SDS PAGE: Proteins
Function Of SDS
SDS PAGE: Proteins
DISC ELECTROPHORESIS
SDS PAGE: Coomassie Blue Stain
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
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.
Western Blot Signal Detection
Target protein
Primaryantibody(probe)
Secondaryantibody
label
Filter-based Hybridization Technologies
Target Probe
Southern blot
DNA Nucleic acid
Northern blot RNA Nucleic acid
Western blot Protein Protein
Southwestern blot
Protein DNA
Blotting Formats Dot blots
amplification analysis expression analysis (RNA) mutation analysis
Reverse dot blots Slot blots
amplification analysis expression analysis
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)
Normal reference DNA
Test sample DNA
(Amplification at this locus)
(Deletion at this locus)
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
Comparative Genomic HybridizationComparative Genomic Hybridization
Amplification
Deletion
Deletion
Deletion
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.
DNA Sequencing Methods Technology
Chain termination Cycle sequencing
Chemistry Maxam and Gilbert Sanger
Platform Manual Automated
Maxam and Gilbert DNA Sequencing
Chemical cleavage of specific bases
Piperidine cleavage of phosphate backbone
Fragment size analysis by gel electrophoresis
Not commonly used
Sanger (Dideoxy) DNA Sequencing
Incorporation of 2´,3´-dideoxynucleotides by DNA polymerase
Termination of elongation reaction Fragment size analysis (manual vs.
automated) Gel Capillary
Dideoxy (Sanger) Sequencing (ddNTP)
H
O
CC
C C
CH2OH OH
H
H
H
1
5
4
32
2,3-dideoxyribose
DNA Sequencing
ATTAGACGT
A
AATTAATTAGA
T
ATATTATTAGACGT
G
ATTAGATTAGACG
C
ATTAGAC
A T G C
Dideoxy or Sanger DNA Sequencing
Sequencing Gels