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5.2 Typical Result from the Nonradioactive Gel Mobility Shift Assay

Figure 34. DIG Gel Shift Assay with Oct2A ControlProtein and Oligonucleotide. Gel mobility shift as-says were performed with the control Oct2A protein

and varying amounts of Oct2A-binding oligonucleo-tide (included in the DIG Gel Shift Kit). The reaction

components were separated on a 12.5% native poly-acrylamide gel, then transferred to a nylon membraneby electroblot. DIG-labeled moieties were detected ina chemiluminescent assay (CSPD substrate, 30 minexposure, X-ray film). Lane 1: DIG-labeled control oli-gonucleotide (0.8 ng) containing Oct2A binding site.Lane 2: DIG-labeled control oligonucleotide (30 fmol)after incubation with 50 ng Oct2A control protein.Lanes 3– 6: Same as lane 2, except the incubations alsocontained increasing amounts of unlabeled controlnucleotide (25-, 62-, 125-, and 250-fold excess, left toright). Upper band, protein-oligonucleotide complex;lower band, unbound oligonucleotide.

Result: The Oct2A control protein specifically bound asite on the DIG-labeled control oligonucleotide (lane2). The reaction was specific because the protein

could be competed off the labeled oligonucleotideby increasing amounts of unlabeled oligonucleotide(lanes 3– 6).

6. Direct Detection of a DIG-labeled DNA

DIG labeling of PCR products and directimmunodetection of the DIG-labeledDNA transferred to nylon membrane of-fer a thousandfold increase in sensitivity

over detection by ethidium bromide stai-ning. (See Section 6.2, “ Typical Resultswith the PCR DIG Labeling Mix” below.)

Key Product for This Assay

Product Catalog Number Reagents included

PCR DIG Labeling Mix, 10x conc.

1 585 550 Mixture (10x conc.) of nucleotides in water, pH 7, including:● dATP, 2 mM● dCTP, 2 mM● dGTP, 2 mM● dTTP, 1.9 mM● DIG-11-dUTP, alkali-stable, 0.1 mM

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Direct Detection of a DIG-labeled DNAOverview of Assay

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Also Required

6.1 Overview of Assay

Direct immunodetection of DNA prepared with the PCR DIG Labeling Mix involvesthe following major stages:

Product1 Catalog Number

Anti-DIG-AP, Fab fragments 1 093 274

Nylon Membrane, positively charged 1 209 299

Ready-to-use CDP-Star 2 041 677

DIG Wash and Block Buffer Set 1 585 762

1 For more information on these products, see Chapter 2 or the Ordering Information in Appendix E.

Stage Description Time

A Amplify a target DNA by PCR with a reaction mix containing the PCR DIG Labeling Mix1.

Approx. 2 h

B Separate the samples on an agarose gel (in TBE or TAE buffer). 30 min– 2 h

C Transfer separated components to a positively charged nylon membrane by capillary transfer2.Note: You do not need to denature the DNA before transfer.

over-night

D Detect2 DIG-labeled DNA-protein complexes with alkaline phosphatase-conjugated anti-DIG antibody and CDP-Star chemiluminescent alkaline phosphatase substrate.Note: The detection procedure also uses components of the DIG Wash and Block Buffer Set.

Approx. 2.5 h

E Record the chemiluminescent signals with X-ray film or the Lumi-Imager F1 Workstation (Cat. No. 2 012 847).

5– 20 min

1 For details of the labeling procedure, see the package insert for the PCR DIG Labeling Mix, Cat. No. 1 585 550,available at our Web site (http://biochem.roche.com).

2 For details of these procedures, see Sections 3.1 and 4.1 of Chapter 2.

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6.2 Typical Results with the PCR DIG Labeling Mix

Comparison of Sensitivity, Direct Detection of DIG-labeled Products vs. Ethidium Bromide Staining

6.3 Relative Sensitivity of Hybridization Probes Prepared with the PCR DIG Probe Synthesis Kit and the PCR DIG Labeling Mix

Use the PCR DIG Labeling Mix only tolabel PCR products that will be directlydetected. Do not use it to label PCR prod-ucts that will be used as hybridizationprobes.Explanation: The ratio of DIG label tounlabeled nucleotide (DIG-dUTP:dTTP)in the PCR DIG Labeling Mix is very low(1:20). This allows low level DIG labeling

without significantly altering the molec-ular weight of the labeled product. Thislevel of DIG labeling is sufficient fordirect detection, but is inadequate (Figure35) for producing the sensitive hybridiza-tion probes needed to detect single-copygenes on Southern blots (or rare RNAs onNorthern blots).

Detection Method Could Detect This PCR Product

Produced from

Ethidium bromide staining Unlabeled DNA on a gel 1 pg template

Direct immunodetection DIG-labeled DNA on a blot 1 fg template

1 For details of method, see Section 6.1, “Overview of Assay” , above.

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Figure 35. Comparison of the Sensitivity of DIG-labeled Probes Generated with Different Concen-trations of DIG-dUTP. We compared the sensitivity oftwo probes, one prepared with the 1:3 DIG-dUTP:dTTPratio found in the PCR DIG Probe Synthesis Kit (PanelB), and the other with the 1:20 DIG-dUTP:dTTP ratiofound in the PCR DIG Labeling mix (Panel A). Eachprobe was 442 bp and recognized a human tPA se-quence. The probes were used to detect the targetsequence in different amounts of human DNA (10, 5,and 2.5 µg, left to right in each panel) on a Southernblot. A DIG-labeled DNA Molecular Weight Markerwas included at the left of each blot.

Result: The probe prepared with 1:3 DIG-dUTP:dTTPcould easily detect the three tPA bands in 2.5 µgDNA (panel B). The probe prepared with 1:20 DIG-dUTP:dTTP could only detect the tPA fragments asfaint bands in the 10 and 5 µg samples; it could notdetect the fragments in the 2.5 µg sample at all.Conclusion: The PCR Labeling Mix does not produceprobes that are sensitive enough to detect single-copygenes in complex samples. For labeling of high sen-sitivity hybridization probes, always use the PCRDIG Probe Synthesis Kit (as described in Section2.2, Chapter 2).

A B

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