Chapter 5: Resolution and Detection of Nucleic Acids

Agarose Gel Electrophoresis

What does gel electrophoresis do? employs electromotive force to move molecules through a porous gel separates molecules from each other on the basis of size and/or charge and/or shape basis of separation depends on how the sample and gel are prepared

Why perform electrophoresis on ds DNA? To separate fragments from each other To determine the sizes of fragments To determine the presence or amount of DNA To analyze restriction digestion products

Where does the current come from? A direct current power supply Ions supplied by the buffer The charge on the macromolecules being separated Electrolysis of water

Basics of Gel Electric Circuits V (volts) = I (milliamps) X R (resistance) For a segment of a gel/buffer system  cross-sectional area of buffer or gel,  resistance  strength of buffer = [ion],  resistance most resistance is in the agarose gel itself

What factors affect mobility of linear ds DNA? Pore size of the gel  [agarose]   pore size  pore size   friction   mobility Voltage across the gel  voltage   mobility Length of the DNA molecule smaller molecules generate less friction and so move faster Ethidium bromide (stain) intercalated into DNA decreases charge to mass ratio and so decreases mobility

Visualization Monitoring the progress of the electrophoresis tracking dyes visible to naked eye during run xylene cyanol (migrates with ~5.0 kb fragments) bromphenol blue (migrates with fragments of a few hundred base pairs) Orange G (migrates with fragments of ~50 bp)

Visualization Locating the DNA fragments in the gel ethidium bromide staining mutagen, wear gloves! visible under UV light wear UV opaque face or eye shield to observe!

Factors affecting resolution Resolution = separation of fragments The “higher” the resolution, the more space between fragments of similar, but different, lengths Resolution is affected by agarose type agarose concentration salt concentration of buffer or sample amount of DNA loaded in the sample voltage

What is agarose? linear carbohydrate polymer extracted from seaweed , agarbiose forms a porous matrix as it gels

What is agarose? (cont’d) multiple types of agarose Standard agarose - LE Gels at 35-38oC; Melts at 90-95oC Becomes opaque at high concentrations Makes a fairly sturdy gel Low melting agarose (NuSieve) Gels at 35oC; Melts at 65oC Often used to isolate DNA fragments from gel Is relatively translucent at high concentrations Makes a fragile gel Intermediate forms or combinations of LE and NuSieve can provide sturdy, translucent gels at high agarose concentrations Good for resolving smaller fragments

Resolution of ds linear DNA fragments in agarose gels % Agarose (w/v) Size Range (kb) for Optimal Separation 0.5 2-30 0.75 0.7-20 1.0 0.5-10 1.5 0.2-3 2.0 0.1-2 3.0 (Nu-Sieve) 0.07-1.5 4.0 (N-S) 0.04-0.9 5.0 (N-S) 0.03-0.6 6.0 (N-S) 0.01-0.4

0.7% 2.5% Effect of agarose concentration on linear DNA fragment resolution. The two lanes contain identical DNA samples.

4M 1M 0M Effect of salt concentration on resolution of fragments. Samples in all three lanes are identical except for [salt].

Effect of sample DNA concentration on resolution. 0.1 0.25 0.5 1.0 5.0  DNA Hind III fragments (g) Effect of sample DNA concentration on resolution.

Voltage  voltage,  rate of migration to increase the voltage increase the setting on the power supply increase the resistance decrease the gel thickness decrease the ion concentration if voltage is too high, gel melts as voltage is increased, large molecules migrate at a rate proportionally faster than small molecules, so lower voltages are better for resolving large fragments but the larger ds DNA fragments are always slower than the smaller ones

Buffer Systems Purposes of buffer Keep solution at pH compatible with molecules being separated Generate ions consistently to maintain current keep resistance low Both gel and the solution in the gel box are buffered.

Buffer Systems (cont’d) Two commonly used buffers for routine agarose gel electrophoresis TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA Tris (T) is a weak base. Acetic (A) acid and boric (B) acid are weak acids. Acetic acid is more completely ionized at pH 8.0 than is boric acid, so TBE has a high buffer capacity than TAE.

Buffer Systems (cont’d) TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA loses buffer capacity during long or high voltage gel runs; gel may melt from the increased resistance that results from ion depletion resolves high MW fragments better than TBE TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA higher buffer capacity resolves low MW fragments better than TAE

Non-denaturing agarose gel loading solutions Composition tracking dyes are used to follow progress of electrophoresis sometimes interfere with later visualization of DNA a solute to increase density so that sample falls to bottom of loading well with minimal dilution solute examples: glycerol, Ficoll

Ethidium bromide staining Binds to DNA by intercalation between stacked bases + charge, so migrates toward negative pole it alters DNA mobility, especially of circular covalently closed DNA

Ethidium bromide staining Used to visualize DNA with UV light uv  254 nm absorbed by DNA >/= 10ng/band required for visualization Bound dye fluoresces 20-25X more than dye in solution UV light damages eyes and skin! Wear goggles and/or face shield.

Trouble shooting Smearing torn sample wells voltage too high for large fragments too much DNA Use </= 0.5 ug / fragment / 0.25cm2 migration area Gel melts voltage too high ionic strength too low Poor resolution wrong [agarose] small bands are fuzzy – the gel run may have been too long at too low a voltage, allowing diffusion of the DNA and broadening of the band

Gel Electrophoresis Matrices Agarose Acrylamide

Comparison of Agarose Concentrations 500 bp 200 bp 50 bp % agarose: 2% 4% 5%

Fragment Resolution: Agarose Gel Electrophoresis

Agarose Electrophoresis of Restriction Enzyme Digested Genomic DNA B

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 Large genomic DNA (Chromosomal)

Electrophoresis Equipment: Horizontal or Submarine Gel DNA/RNA is negatively charged: RUN TO RED

Agarose Gel Electrophoresis Horizontal Gel Format www.biorad.com Reservoir/Tank Power Supply Casting Tray and Combs

Agarose Gel Apparatus

Pouring a horizontal agarose gel

Electrophoresis Equipment: Vertical Gel

Vertical Gel Format: Polyacrylamide Gel Electrophoresis www.biorad.com Reservoir/Tank Power Supply Glass Plates, Spacers, and Combs

Polyacrylamide Gel Electrophoresis (PAGE)

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

PULSE FIELD GEL ELECTROPHORESIS APPARATUS

Types Of Pulse Field Gel Electrophoresis Field inversion gel Transverse alternating field Crossed field (Reverse) Contour-clamped homogeneous electric field

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

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

PFGE of Bacterial DNA

Using PFGE In The Molecular Investigation Of An Outbreak Of S 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

Using PFGE in the Molecular Investigation Of An Outbreak of S Using PFGE in the Molecular Investigation Of An Outbreak of S. marcescens Infection in an ICU Singapore Med J 2004 Vol 45(5) : 214

Using PFGE in the molecular investigation of an outbreak of S 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

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).

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 5–500 bp in size.

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.

PAGE: DNA High resolution of low molecular weight nucleic acids (500bp)

Polyacrylamide Gel Electrophoresis of Restriction Digested PCR Products FV SNP FII SNP

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. Both formamide and urea effectively lower the melting point of the DNA molecules, allowing the structures to fall apart at lower temperatures. Generally, concentrations of urea or formamide are chosen to give melting temperatures around 50° C, and gels are run at that temperature. RNA is often denatured with harsher agents, because RNA tends to form stronger structures.

Chapter 6: Analysis and Characterization of Nucleic Acids and Proteins

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.

HYBRIDIZATION: Denaturation and Annealing of DNA

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)

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 (digoxigenin, 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)

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

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

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