Southern Analysis:Southern Analysis:

Hybridization, Washing, and Hybridization, Washing, and DetectionDetection

MYB61MYB61Single or Multicopy gene in Single or Multicopy gene in

Arabidopsis ThalianaArabidopsis Thaliana??

Research PlanResearch PlanIsolate Genomic DNA

Southern Blot Analysis

Digest Genomic DNA w/ Various Restriction Enzymes

Agarose Gel Electrophoresis and Southern Transfer

Make Non-Radioactive Myb61 Probe

Hyribidize Probe to Southern Blot

Washes and Chemiluminescent Detection

Data Analysis

Theoretical Basis of SouthernTheoretical Basis of SouthernHybridization and WashingHybridization and Washing

Prehybridization: block portions of membrane where there is no bound DNA to prevent probe from binding to membrane.

Hybridization: to allow probe to bind to complementary sequences on membrane (heat denatured probe added to prehybridization solution and incubated overnight at 60°C).

Today’s Laboratory ObjectivesToday’s Laboratory Objectives

1.1. To become familiar with a Southern Hybridization, To become familiar with a Southern Hybridization, Washing and Detection MethodsWashing and Detection Methodsa. mechanics and trouble spotsa. mechanics and trouble spotsb. What variables can be manipulated to enhance signal b. What variables can be manipulated to enhance signal

2.2. Data Analysis and InterpretationData Analysis and Interpretation Positive control- efficacy of probe and hybridization conditionsPositive control- efficacy of probe and hybridization conditions Negative control- stringency of hybridizationNegative control- stringency of hybridization Experimental signal- identify restriction fragments harboring the Experimental signal- identify restriction fragments harboring the

myb61 gene myb61 gene

Theoretical Basis of SouthernTheoretical Basis of SouthernHybridization and WashingHybridization and Washing

Washing: to removes non-specifically bound probe molecules.

Variables that affect stringency of washes include: salt concentration, temperature, and SDS concentration

DigoxigeninDigoxigenin

Foxglove Plant

a hapten derived from the steroid DIGOXIN

DIGOXIN occurs exclusively in Digitalis purpurea and Digitalis lanata

Advantages of DIG LabelingAdvantages of DIG Labeling

1.1. System for labeling nucleic acids and proteinsSystem for labeling nucleic acids and proteins

2.2. Detection options include color, fluorescence, Detection options include color, fluorescence,

chemiluminescencechemiluminescence

3.3. Faster, safer, and extremely sensitive alternative Faster, safer, and extremely sensitive alternative to to radioactivityradioactivity

DIG-dUTPDIG-dUTP

DIG Detection PrincipleDIG Detection Principle

DIG labeled probes that hybridized to a target sequence detected with DIG labeled probes that hybridized to a target sequence detected with alkaline phosphatase labeled anti-DIG antibody. alkaline phosphatase labeled anti-DIG antibody.

Blot incubated with suitable reagents like NBT and BCIP, phosphatase Blot incubated with suitable reagents like NBT and BCIP, phosphatase

activity is detected by a color reaction.activity is detected by a color reaction.

Theoretical Basis of Theoretical Basis of Colorimetric DetectionColorimetric Detection

Blocking: performed with BSA to prevent non-specific binding of Blocking: performed with BSA to prevent non-specific binding of antibodyantibody

Antibody Wash: antibody binds to DIG portion of DIG-dUTP Antibody Wash: antibody binds to DIG portion of DIG-dUTP incorporated during amplification of metacaspase gene probeincorporated during amplification of metacaspase gene probe

Colorimetric Detection: phosphatase enzyme conjugated to anti-Colorimetric Detection: phosphatase enzyme conjugated to anti-DIG antibody reacts with substrate; when phosphate is removed DIG antibody reacts with substrate; when phosphate is removed blue/purple precipitate is formedblue/purple precipitate is formed

Colorimetric Detection with Colorimetric Detection with Alkaline PhosphataseAlkaline Phosphatase

BCIP/NBT together they yield an intense, insoluble black-purple precipitate when BCIP/NBT together they yield an intense, insoluble black-purple precipitate when reacted with Alkaline Phosphatase.reacted with Alkaline Phosphatase.

NBT/BCIP reaction scheme.BCIP is hydrolyzed by alkaline phosphatase to form an intermediate that undergoes dimerization to produce an indigo dye. The NBT is reduced to the NBT-formazan by the two reducing equivalents generated by the dimerization

Colorimetric Detection with Colorimetric Detection with Alkaline PhosphataseAlkaline Phosphatase

ReactionReaction SolutionSolution TimeTimeWashingWashing 2X SSC, 0.1% SDS2X SSC, 0.1% SDS 10 min10 minWashingWashing 0.5X SSC, 0.1% SDS0.5X SSC, 0.1% SDS 30 min30 minBlockingBlocking 100 mM Tris, pH 7.5, 150 mM NaCl100 mM Tris, pH 7.5, 150 mM NaCl

Blocking ReagentBlocking Reagent 30 min30 minAntibodyAntibody Wash Buffer w/ 150 mU/ml Wash Buffer w/ 150 mU/ml

Anti-Dig AbAnti-Dig Ab 30 min30 minWashingWashing Wash BufferWash Buffer 30 min30 minDetectionDetection NBT/BCIPNBT/BCIP until pptuntil ppt

DetectionDetection

Blot incubated with DIG probeBlot incubated with DIG probe Wash to eliminate non-specifically bound probe moleculesWash to eliminate non-specifically bound probe molecules Probe detected via DIG specific antibody conjugated to alkaline Probe detected via DIG specific antibody conjugated to alkaline

phosphatase enzymephosphatase enzyme Phosphatase reacts with substrate causing blue/purple precipitate to Phosphatase reacts with substrate causing blue/purple precipitate to

form when phosphate is removedform when phosphate is removed

Substrate BCIP and NBT form a redox system

BCIP is oxidized by the alkaline phosphatase to indigo by release of a phosphate group

NBT is reduced to diformazan

Reaction products form a water insoluble dark blue to brownish precipitate, depending on the type of membrane.

Color Detection

Data AnalysisData Analysis What information do you positive and negative controls provide?What information do you positive and negative controls provide? How many hybridizing fragments for each restriction enzyme?How many hybridizing fragments for each restriction enzyme? How homologous is Myb61 to other gene sequences? (BLASTn)How homologous is Myb61 to other gene sequences? (BLASTn) What size are hybridizing fragments in relation to expected Myb61 What size are hybridizing fragments in relation to expected Myb61

mRNA size? mRNA size?

Evidence for a single copy gene

Data AnalysisData Analysis

Agarose Gel of Digested Genomic DNA Southern Blot Analysis

TroubleshootingTroubleshooting

Poor signalPoor signal Probe specific activity too lowProbe specific activity too low Inadequate depurinationInadequate depurination Inadequate transfer bufferInadequate transfer buffer Not enough target DNANot enough target DNA Transfer time too shortTransfer time too short Inefficient transfer systemInefficient transfer system Probe concentration too lowProbe concentration too low Incomplete denaturation of probe and/or target DNAIncomplete denaturation of probe and/or target DNA Final wash too stringentFinal wash too stringent Hybridization time too shortHybridization time too short Inappropriate membraneInappropriate membrane

TroubleshootingTroubleshooting

Spotty BackgroundSpotty Background Unincorporated nucleotides not removed from labeled probeUnincorporated nucleotides not removed from labeled probe Particles in hybridization bufferParticles in hybridization buffer Agarose dried on membraneAgarose dried on membrane Baking or UV crosslinking when membrane contains high saltBaking or UV crosslinking when membrane contains high salt

TroubleshootingTroubleshooting

High BackgroundHigh Background Insufficient BlockingInsufficient Blocking Membrane allowing to dry out during hybridization or washingMembrane allowing to dry out during hybridization or washing Membranes adhered during hybridization or washingMembranes adhered during hybridization or washing Bubbles in hybridization bagBubbles in hybridization bag Walls of hybridization bag collapsed on to membraneWalls of hybridization bag collapsed on to membrane Not enough wash solutionNot enough wash solution Hybridization temperature too lowHybridization temperature too low Labeled probe molecules are too shortLabeled probe molecules are too short Probe Concentration too highProbe Concentration too high Inadequate prehybridizationInadequate prehybridization Probe not denaturedProbe not denatured Not enough SDS in wash solutionNot enough SDS in wash solution