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

Round A/Round B Amplification of DNA

(Protocol summary only for purposes of this preview site)

The goal of this procedure is to randomly amplify a sample of DNA to achieve the best possible sequence representation. This protocol has been used successfully to amplify genomic representations starting with <10 ng of DNA. The protocol consists of three sets of enzymatic reactions. In Round A, Sequenase is used to extend randomly annealed primers to generate templates for subsequent PCR. During Round B, a specific primer is used to amplify the previously generated templates. Finally, amplified material can be labeled as in Protocol 7 or 8. Alternatively, Round C in this protocol can be used to incorporate either aminoallyl-dUTP or Cy-dye-coupled nucleotides during additional PCR cycles. This protocol may be unsuitable for amplifying material smaller than 250 bp because such material will not be amplified uniformly. In those cases, Protocol 3 is recommended. This protocol was adapted from Bohlander et al. (1992).


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 Protocol 2: Round A/Round B Amplification of DNA

The goal of this procedure is to randomly amplify a sample of DNA to achieve the best possible sequence representation. This protocol has been used successfully to amplify genomic representations starting with <10 ng of DNA. The protocol consists of three sets of enzymatic reactions. In Round A, Sequenase is used to extend randomly annealed primers to generate templates for subsequent PCR. During Round B, a specific primer is used to amplify the previously generated templates. Finally, amplified material can be labeled as in Protocol 7 or 8. Alternatively, Round C in this protocol can be used to incorporate either aminoallyl-dUTP or Cy-dye-coupled nucleotides during additional PCR cycles. This protocol may be unsuitable for amplifying material smaller than 250 bp because such material will not be amplified uniformly. In those cases, Protocol 3 is recommended. This protocol was adapted from Bohlander et al. (1992).


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

  • aa-dNTP/Cy-dNTP mixture (100) <R>
  • BSA (500 g/mL)
  • DNA, isolated from the sample under study (10100 ng)
    • For example, for CGH analysis, genomic DNA is isolated as described in Chapter 1; for protein localization studies, DNA is isolated by chromatin immunoprecipitation (ChIP) as in Chapter 20.
  • dNTP mixture (3 mM)
  • dNTPs (100; 20 mM each nucleotide)
  • DTT (0.1 M)
  • MgCl2 (25 mM)
  • PCR buffer (10) (500 mM KCl, 100 mM Tris at pH 8.3)
  • Primer A: GTTTCCCAGTCACGATCNNNNNNNNN (40 pmol/L)
  • Primer B: GTTTCCCAGTCACGATC (100 pmol/L)
  • Sequenase (13 units/L) (US Biochemical, catalog no. 70775)
  • Sequenase buffer (5)
  • Sequenase dilution buffer
  • Taq polymerase (5 units/L)

Equipment

  • Agarose gel (1)
  • Microcon 30 spin column (Millipore)
  • Thermal cycler


METHOD
Round A: Extending Randomly Annealed Primers

  • 1. Prepare Round A reactions as follows:
    • As little as 10 ng of DNA can be effectively amplified by this protocol. As a control, set up a reaction in which the DNA is replaced with H2O.
  • 2. Denature the template DNA and anneal the primer by heating for 2 min at 94C and then rapidly cooling to 10C. Keep the reaction for 5 min at 10C.
  • 3. Assemble the reaction mixture:
  • 4. Combine the reaction mixture and the templateprimer mix. Place the tube(s) into a thermal cycler, and extend the primers as follows.
    • i. Ramp from 10C to 37C over 8 min.
    • ii. Hold at 37C for 8 min.
    • iii. Rapidly ramp to 94C and hold for 2 min.
    • iv. Rapidly ramp to 10C, add 1.2 L of diluted Sequenase (1:4 dilution), and hold for 5 min at 10C.
    • v. Ramp from 10C to 37C over 8 min.
    • vi. Hold at 37C for 8 min.
  • 5. Dilute samples with water to a final volume of 60 L.

Round B: PCR Amplification

  • 6. Prepare Round B reactions as follows:
  • 7. Place the tube(s) into a thermal cycler, and amplify the templates as follows.
    Run 1535 cycles, depending on the amount of starting material.
    • To optimize the number of cycles, it may be necessary to remove an aliquot every two cycles to monitor the progress of the amplification. It is best to use the minimal number of cycles that generates a visible smear (see Step 8).
      Make sure that there is no DNA in the negative control lane!
  • 8. Run 5 L of the reaction on a 1 agarose gel. A smear of DNA should be present between 500 and 1000 bp.
  • 9. Label the DNA using the Round C procedure, Protocol 7, or Protocol 8.

Round C: Cyanine Labeling or Aminoallyl Activation of DNA

  • 10. Prepare the Round C reaction:
  • 11. Place the tube(s) into a thermal cycler, and label the templates as follows:
    Run 1025 cycles.
    • The number of cycles should be determined empirically. The objective is to minimize the number of cycles required to yield 23 g of material for hybridization.
  • 12. If aa-dNTPs were used in Round C, desalt the sample to remove Tris buffer, which interferes with the dye coupling. Add 400 L of water to the sample in a Microcon 30, and centrifuge for 8 min at 12,000 rpm. Repeat again with 500 L of water.
  • 13. Proceed to Cy-dye coupling as described in Protocol 8, Step 11.


DISCUSSION

The output of this amplification protocol will be double-stranded DNA. Quantify the amount of DNA by absorbance (see Chapter 2), and calculate the fold amplification based on the amount of input DNA used. First-time users of this protocol may want to run some amplified DNA on an agarose gel to observe the size distribution.


RECIPE

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.

aa-dNTP/Cy-dNTP Mixture (100)
REFERENCE
Bohlander SK, Espinosa R III, Le Beau MM, Rowley JD, Diaz MO. . Year: 1992. A method for the rapid sequence-independent amplification of microdissected chromosomal material. Genomics13: 13221324.

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