Results from Probe Synthesis pn st rd sw do lk Wed, Thurs lab resultsEscobar/Read -DIG +DIG increased extension time (2 min, extension rate of Taq = nt/min, Taq + Tgo polymerase mix extension rate = ?) reagent contamination?

Southern Analysis: Hybridization, Washing, and Detection

Broad and Long Term Objective To determine the copy number of Myb transcription factor genes in the genome of the model plant Arabidopsis thaliana

Research Plan Isolate Genomic DNA Digest Genomic DNA with Various Restriction Enzymes Agarose Gel Electrophoresis and Southern Transfer Make Non-Radioactive Myb Probe Hyribidize Probe to Southern Blot Washes and Colorimetric Detection Data Analysis Southern Blot

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 myb genes

Theoretical Basis of Southern Hybridization and Washing Prehybridization: prehybridization solution contains a mix of proteins and nucleic acids that will bind to the membrane, covering regions where there is no fixed DNA (membrane blocking). This prevents the single stranded probe from binding nonspecifically to the membrane. Hybridization: Heat denatured probe is then added to the prehybridization solution and incubated overnight. On a fully blocked membrane, probe can associate with the membrane only by hybridizing with complementary ssDNA sequences fixed to the membrane. Washing: removes probe molecules that are weakly associated with the surface of the membrane or the genomic DNA.

Stringency of Hybridization The incubation conditions during the hybridization and washing steps can be varied to require greater or lesser complementarity between probe and bound DNA (stringency) Stringency is determined primarily by salt concentration, temperature, and the presence/absence of organic solvents (esp. formamide) Successful hybridization between probe and a target DNA is determined by the T m T m (ºC)= log 10 ([Na+]/{ [Na+]}) (%[G+C]) – 500/n – 1(% mismatch) n = length of duplex (bp) Applicable for sequences >15 bp

DIG Detection Principle DIG labeled probes that hybridized to a target sequence are detected with an anti-DIG antibody that is covalently attached to a phosphatase enzyme. If the blot is incubated with suitable reagents like NBT and BCIP, phosphatase activity is detected by a color reaction.

 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 Development

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 Myb61 gene probe Antibody Wash: antibody binds to DIG portion of DIG- dUTP incorporated during amplification of Myb61 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

Flow Diagram of Colorimetric Detection with NBT/BCIP ReactionSolutionTime Washing 2X SSC, 0.1% SDS 2 x 5 min Washing 0.5X SSC, 0.1% SDS 2 x 15 min Rinse 0.1 M Tris (pH 7.5), 0.15 M NaCl 1 min Blocking 0.1 M Malate, 0.15 M NaCl,0.5% 30 min Blocking Reagent Antibody Blocking Reagent, 150 mU/ml 30 min Anti-Dig Antibody Washing 0.1 M Malate, 0.15 M NaCl, 0.3% 2 x 15 min Tween 20 Detection 0.1 M Tris, 0.1 M NaCl, 1/50 vol 1-12 hr NBT/BCIP stock solution

Data Analysis* What information do your positive and negative controls provide? What information do your positive and negative controls provide? How many hybridizing fragments for each restriction enzyme- what does this indicate about Myb gene copy number? How many hybridizing fragments for each restriction enzyme- what does this indicate about Myb gene copy number? How homologous is Myb61 to other gene sequences? (BLASTn) From your blot, does it appear that these sequences hybridized with the Myb61 probe? How homologous is Myb61 to other gene sequences? (BLASTn) From your blot, does it appear that these sequences hybridized with the Myb61 probe? Evidence for a single copy gene

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 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