Chapter 6: Analysis and Characterization of Nucleic Acids and Proteins Donna C. Sullivan, PhD Division 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 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

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

There are hundreds of restriction enzymes

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)

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

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.

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 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

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) 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.

Denaturation/Annealing: An Equilibrium Reaction

HYBRIDIZATION: Denaturation and Annealing of DNA

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)

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: Electrostatic Nitrocellulose Nylon Reinforced nitrocellulose Electrostatic Nylon, nytran Positively charged nylon

Transfer of DNA to Membrane

Capillary Transfer Dry paper Nitrocellulose membrane Gel Soaked Reservoir

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

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

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

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

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 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?

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

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) and G + 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 Optimal Hybridization Times Optimal Hybridization Temperatures ON=overnight

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 Hybrids Tm=81.5+16.6 X log[Na]-0.65(%formamide)+41(%G+C) RNA-DNA Hybrids Tm=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 Signal Detection Filter with bound DNA Radioactive isotope Probe

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

Overview of Southern Blot Hybridization

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

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 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

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 Primary antibody (probe) Secondary label

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

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

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

Comparative 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

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

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

Sequencing Gels