Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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The material on this page is part of Chapter 10, which is shown in full as a preview on this site.

Chapter 10: Nucleic Acid Platform Technologies

Rando Oliver, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

INTRODUCTION

Cyanine-dCTP Labeling of DNA Using Klenow

(Protocol summary only for purposes of this preview site)

Similar to the direct RNA labeling protocol (Protocol 5), direct DNA labeling with Cy-dCTP is the simplest and fastest method for labeling DNA. This is a standard Klenow labeling protocol in which Cy-dCTP is incorporated during the labeling reaction. After stopping the reaction, labeled nucleotide is separated from unreacted Cy-dCTP, and Cy3- and Cy5-labeled materials are combined for hybridization (Protocol 10). This protocol is suitable for many applications, including detection of copy number variation, nucleosome mapping, and other location analysis (e.g., ChIP-chip).


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 Protocol 7: Cyanine-dCTP Labeling of DNA Using Klenow

Similar to the direct RNA labeling protocol (Protocol 5), direct DNA labeling with Cy-dCTP is the simplest and fastest method for labeling DNA. This is a standard Klenow labeling protocol in which Cy-dCTP is incorporated during the labeling reaction. After stopping the reaction, labeled nucleotide is separated from unreacted Cy-dCTP, and Cy3- and Cy5-labeled materials are combined for hybridization (Protocol 10). This protocol is suitable for many applications, including detection of copy number variation, nucleosome mapping, and other location analysis (e.g., ChIP-chip).


MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Recipes for reagents specific to this protocol, marked <R>, are provided at the end of the protocol. See Appendix 1 for recipes for commonly used stock solutions, buffers, and reagents, marked <A>. Dilute stock solutions to the appropriate concentrations.

Reagents

  • Cy3-dCTP and Cy5-dCTP (GE Life Sciences)
  • dNTP mixture (10) <R>
  • EDTA (0.5 M, pH 8.0)
  • Genomic DNA (or from Protocol 2)
  • Human Cot-1 DNA (1 g/L) (GIBCO)
  • Klenow fragment of E. coli DNA polymerase I (4050 units/L)
  • Poly(dA-dT) (Sigma-Aldrich)
    • Make a 5 g/L stock in nuclease-free water. Store the stock at 20C.
  • Random primer/buffer solution <R>
    • This can be obtained from Life Technologies BioPrime labeling kit or can be made in the laboratory.
  • TE (pH 7.4)
  • Yeast tRNA (GIBCO)
    • Make a 5 g/L stock in nuclease-free water.

Equipment

  • Heat block set at 37C and 95C100C
  • Microcon 30 spin column (Millipore)


METHOD

  • 1. Add 23 g of DNA to a microcentrifuge tube.
    • For high-complexity DNA, such as mammalian genomic DNA, reducing the fragment size by sonication or restriction digestion improves labeling efficiency. Shearing by sonication to 1000-bp average fragment size is sufficient for this application.
  • 2. Add H2O to the DNA to a final volume of 21 L. Add 20 L of 2.5 random primer/buffer mixture. Boil the tube for 5 min in a heat block set to 95C100C.
  • 3. Place the tubes on ice for 5 min.
  • 4. Add reagents in the following order:
    • Cy-UTP also works well, but if using UTP, adjust the 10 dNTP mix accordingly.
  • 5. Incubate the reaction for 12 h at 37C.
    • For improved labeling efficiency, add 1 L of Klenow after the reaction has been going for 1 h. Incubate the reaction for another hour.
  • 6. Stop the reaction by adding 5 L of 0.5 M EDTA (pH 8.0).

Cleanup of Labeled DNA

  • 7. Combine the Cy3- and Cy5-labeled samples, and add 450 L of TE (pH 7.4).
  • 8. Add the combined samples to a Microcon 30 spin column. Centrifuge the spin column for 1011 min at 10,000g in a microcentrifuge.
  • 9. Discard the flowthrough.
  • 10. If hybridizing to homemade microarrays, add blocking nucleotides at this step:
    • If using a commercial microarray, refer to the manufacturer's protocols for specific hybridization buffers and blocking nucleotides.
  • 11. Add another 450 L of TE, and centrifuge as in Step 8.
  • 12. Measure the volume of the liquid remaining in the column. If necessary, centrifuge for 1 min. The desired probe volume is 20 L. The exact volume will depend on the size of the coverslip that contains the microarray. For small microarrays this volume may be as low as 12 L (see Protocol 10, Step 3).
  • 13. Discard the flowthrough. Recover the labeled DNA from the filter by inverting the column onto a fresh collection tube. Centrifuge for 1 min at 10,000g.
  • 14. Proceed to hybridization with the microarray (Protocols 9 and 10).


DISCUSSION

The products of this protocol will be Cy5-labeled and Cy3-labeled cDNA, which are ready to hybridize to microarrays. This material can be stored in foil, to prevent bleaching, at 4C for up to 1 wk. A useful check for labeling success is the color of the labeled cDNA after unincorporated nucleotides have been removed: Good labeling will result in visible blue (Cy5) or red (Cy3) color in the cleaned up material.


RECIPES

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

dNTP Mixture (10)
Random Primer/Buffer Solution

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