Making Nonradioactive Probes:

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Making Nonradioactive Probes

• PCR DIG Labeling

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Broad and Long Term Objective
To determine the copy number of Myb transcription
factor genes in the genome of the model plant
Arabidopsis thaliana
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Research Plan
Isolate Genomic DNA
Digest Genomic DNA with Various Restriction
Enzymes
Agarose Gel Electrophoresis and Southern Transfer
Southern Blot
Make Non-Radioactive Myb Probe
Hyribidize Probe to Southern Blot
Washes and Colorimetric Detection
Data Analysis
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Todays Laboratory Objectives

• To make a homologous gene probe to the
• Myb61 gene of Arabidopsis
• To learn how to set up and run a polymerase chain
  reaction (PCR)
• Evaluate PCR products and the success of the
  reaction

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Nucleic Acid Probes

• Definition Short (lt 2000 bp), single stranded
  DNA or RNA molecule that is used to identify a
  particular nucleic acid sequence through
  hybridization
• Probe labeling
• Primary label- radioactive or fluorescent
  nucleotides
• Secondary label- hapten-bound nucleotides
• Probe Classification
• Heterologous- from a different organism
• Homologous- from the same organism

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Probes and DNA blots

• Size-separated genomic DNA fragments (single
  stranded) are covalently bound to the surface of
  a nylon membrane
• Membrane is mixed with a solution of single
  stranded Myb61 probe (labeled with digoxigenin),
  allowing hybridization of the probe to
  complementary DNA sequences
• Membrane is washed to remove unbound probe
  molecules and and colorimetric detection is
  performed to visualize the Myb-homolgous DNA
  fragments

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Why label nucleic acids with DIG?

• DIGoxigenin is a steroid found exclusively in
  Digitalis lanata and D. purpurea
• DIG can be coupled to nucleotide triphosphates
  (e.g. DIG-dUTP)
• DIG-dUTP can be incorporated into DNA or RNA
  by DNA/RNA polymerase
• Antibodies can be raised against DIG in sheep
  or rabbits. These antibodies will bind to
  DIG-labeled nucleic acid probes

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How is our probe synthesized?
Polymerase Chain Reaction (PCR)
http//pathology2.jhu.edu/molec/techniques_main.cf
m
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DIG DNA Labeling by PCR

• Reaction Components
• - Template DNA
• - Primers
• - DIG dNTP mix 2mM dATP/dGTP/dCTP, 1.3 mM dTTP,
  0.7 mM DIG-dUTP)
• - Buffer
• - dH2O
• - Taq DNA Polymerase

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PCR Template DNAMyb61 in pENTR221
Myb61 cDNA Template 1.5 Kb M13 Reverse
GGAAACAGCTATGACCATG M13 Forward
GTAAAACGACGGCCAGTG
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How does one judge the success of a PCR reaction?

• Agarose gel electrophoresis is used to size
  PCR products.
• Is a product band visible?
• Are there multiple bands?
• Is the band of the expected size?

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Detection of bound probe

• Blot incubated with DIG probe
• Wash to eliminate unbound probe molecules
• Blot incubated with anti-DIG antibody coupled to
  an alkaline phosphatase enzyme
• At sites on the blot where the DIG probe has
  hybridized, the antibody binds to the DIG. The
  phosphatase reacts with uncolored substrates
  (NBT/BCIP) to produce a localized blue-colored
  precipitate

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Detection of bound probe, ctd.

• Uncolored substrates BCIP and NBT
• BCIP and NBT are dephosphorylated by alkaline
  phosphatase, forming insoluble blue-colored
  products
• Blot incubated at 37 C for color development
• Faster and safer than radioactive labeling,
  almost as sensitive

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Next Week

• Hybridization
• Washes and Color Detection
• Analysis