Southern Analysis: Hybridization, Washing, and Detection

MYB61 Single or Multicopy gene in Arabidopsis Thaliana?

Research Plan Isolate 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 Southern Hybridization 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 Objectives 1. To become familiar with a Southern Hybridization, Washing and Detection Methods a. mechanics and trouble spots b. What variables can be manipulated to enhance signal 2. Data Analysis and Interpretation  Positive control- efficacy of probe and hybridization conditions  Negative control- stringency of hybridization  Experimental signal- identify restriction fragments harboring the myb61 gene

Theoretical Basis of Southern Hybridization and Washing Washing: to removes non-specifically bound probe molecules. Variables that affect stringency of washes include: salt concentration, temperature, and SDS concentration

Digoxigenin Foxglove Plant a hapten derived from the steroid DIGOXIN DIGOXIN occurs exclusively in Digitalis purpurea and Digitalis lanata

Advantages of DIG Labeling 1.System for labeling nucleic acids and proteins 2.Detection options include color, fluorescence, chemiluminescence 3.Faster, safer, and extremely sensitive alternative to radioactivity

DIG-dUTP

DIG Detection Principle DIG labeled probes that hybridized to a target sequence detected with alkaline phosphatase labeled anti-DIG antibody. Blot incubated with suitable reagents like NBT and BCIP, phosphatase activity is detected by a color reaction.

Theoretical Basis of Colorimetric Detection Blocking: performed with BSA to prevent non-specific binding of antibody Blocking: performed with BSA to prevent non-specific binding of antibody Antibody Wash: antibody binds to DIG portion of DIG-dUTP incorporated during amplification of metacaspase gene probe Antibody Wash: antibody binds to DIG portion of DIG-dUTP incorporated during amplification of metacaspase gene probe Colorimetric Detection: phosphatase enzyme conjugated to anti- DIG antibody reacts with substrate; when phosphate is removed blue/purple precipitate is formed Colorimetric Detection: phosphatase enzyme conjugated to anti- DIG antibody reacts with substrate; when phosphate is removed blue/purple precipitate is formed

Colorimetric Detection with Alkaline Phosphatase BCIP/NBT together they yield an intense, insoluble black-purple precipitate when 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 Alkaline Phosphatase ReactionSolutionTime Washing2X SSC, 0.1% SDS10 min Washing0.5X SSC, 0.1% SDS30 min Blocking100 mM Tris, pH 7.5, 150 mM NaCl Blocking Reagent30 min AntibodyWash Buffer w/ 150 mU/ml Anti-Dig Ab30 min WashingWash Buffer30 min DetectionNBT/BCIP until ppt

Detection  Blot incubated with DIG probe  Wash to eliminate non-specifically bound probe molecules  Probe detected via DIG specific antibody conjugated to alkaline phosphatase enzyme  Phosphatase reacts with substrate causing blue/purple precipitate to form 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 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 mRNA size? What size are hybridizing fragments in relation to expected Myb61 mRNA size? Evidence for a single copy gene

Data Analysis Agarose Gel of Digested Genomic DNASouthern Blot Analysis

Troubleshooting Poor signal  Probe specific activity too low  Inadequate depurination  Inadequate transfer buffer  Not enough target DNA  Transfer time too short  Inefficient transfer system  Probe concentration too low  Incomplete denaturation of probe and/or target DNA  Final wash too stringent  Hybridization time too short  Inappropriate membrane

Troubleshooting Spotty Background  Unincorporated nucleotides not removed from labeled probe  Particles in hybridization buffer  Agarose dried on membrane  Baking or UV crosslinking when membrane contains high salt

Troubleshooting High Background  Insufficient Blocking  Membrane allowing to dry out during hybridization or washing  Membranes adhered during hybridization or washing  Bubbles in hybridization bag  Walls of hybridization bag collapsed on to membrane  Not enough wash solution  Hybridization temperature too low  Labeled probe molecules are too short  Probe Concentration too high  Inadequate prehybridization  Probe not denatured  Not enough SDS in wash solution