Making Nonradioactive Making Nonradioactive Probes:Probes:

PCR DIG LabelingPCR DIG Labeling

Broad and Long Term ObjectiveBroad and Long Term Objective

To determine the copy number of MybTo determine the copy number of Myb

transcription factor genes in the genome oftranscription factor genes in the genome of

the model plant the model plant Arabidopsis thalianaArabidopsis thaliana

Research PlanResearch PlanIsolate 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

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Today’s Laboratory ObjectivesToday’s Laboratory Objectives

To make a homologous gene probe to theTo make a homologous gene probe to the

Myb61 gene of Myb61 gene of ArabidopsisArabidopsis To learn how to set up and run a To learn how to set up and run a

polymerase chain reaction (PCR)polymerase chain reaction (PCR) Evaluate PCR products and the success Evaluate PCR products and the success

of the reactionof the reaction

Nucleic Acid ProbesNucleic Acid Probes Definition: Short (< 2000 bp), single stranded DNA or Definition: Short (< 2000 bp), single stranded DNA or

RNA molecule that is used to identify a particular nucleic RNA molecule that is used to identify a particular nucleic acid sequence through hybridization acid sequence through hybridization

Probe labeling: Probe labeling: Primary label- radioactive or fluorescent nucleotidesPrimary label- radioactive or fluorescent nucleotides Secondary label- hapten-bound nucleotidesSecondary label- hapten-bound nucleotides

Probe Classification: Probe Classification:

Heterologous- from a different organismHeterologous- from a different organism

Homologous- from the same organismHomologous- from the same organism

Probes and DNA blotsProbes 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

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

Why label nucleic acids with DIG?Why label nucleic acids with DIG?

How is our probe synthesized?How is our probe synthesized?

Polymerase ChainReaction(PCR)

http://pathology2.jhu.edu/molec/techniques_main.cfm##

DIG DNA Labeling by PCRDIG DNA Labeling by PCR

Reaction Components:Reaction Components:- Template DNA- Template DNA- Primers- Primers- DIG dNTP mix = - DIG dNTP mix = 2mM dATP/dGTP/dCTP,

1.3 mM dTTP, 0.7 mM DIG-dUTP)- Buffer- Buffer

- dH- dH22OO

- Taq DNA Polymerase- Taq DNA Polymerase

PCR Template DNA:PCR Template DNA:Myb61 in pENTR221Myb61 in pENTR221

Myb61 cDNA Template = 1.5 Kb

M13 Reverse GGAAACAGCTATGACCATG

M13 Forward

GTAAAACGACGGCCAGTG

How does one judge the success of How does one judge the success of a PCR reaction?a PCR reaction?

Agarose gel electrophoresis is used to Agarose gel electrophoresis is used to

size PCR productssize PCR products**..

Is a product band visible?Is a product band visible?

Are there multiple bands?Are there multiple bands?

Is the band of the expected size?Is the band of the expected size?

Detection of bound probeDetection of bound probe

Blot incubated with DIG probeBlot incubated with DIG probe Wash to eliminate unbound probe moleculesWash to eliminate unbound probe molecules Blot incubated with anti-DIG antibody coupled to an alkaline Blot incubated with anti-DIG antibody coupled to an alkaline

phosphatase enzymephosphatase enzyme At sites on the blot where the DIG probe has hybridized, the antibody At sites on the blot where the DIG probe has hybridized, the antibody

binds to the DIG. The phosphatase reacts with uncolored substrates binds to the DIG. The phosphatase reacts with uncolored substrates (NBT/BCIP) to produce a localized blue-colored precipitate(NBT/BCIP) to produce a localized blue-colored precipitate

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

Detection of bound probe, ctd.Detection of bound probe, ctd.

Next WeekNext Week

HybridizationHybridization Washes and Color DetectionWashes and Color Detection AnalysisAnalysis