Making Nonradioactive
Probes:
PCR DIG Labeling
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
Southern Blot
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
Today’s 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
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Definition: Short (< 2000 bp), single stranded DNA
or RNA molecule that is used to identify a particular
nucleic acid sequence through hybridization

Probe labeling:
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Primary label- radioactive or fluorescent nucleotides
Secondary label- hapten-bound nucleotides
Probe Classification:
Heterologous- from a different organism
Homologous- from the same organism
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
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
How is our probe synthesized?
Polymerase
Chain
Reaction
(PCR)
http://pathology2.jhu.edu/molec/techniques_main.cfm##
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
PCR Template DNA:
Myb61 in pENTR221
Myb61 cDNA Template = 1.5 Kb
M13 Reverse
GGAAACAGCTATGACCATG
M13 Forward
GTAAAACGACGGCCAGTG
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?
Detection of bound probe
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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
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
Next Week
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Hybridization
Washes and Color Detection
Analysis