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