PCR Cloning of Neural Gene Products

David K. Grandy1, James R. Bunzow1, Robert L. Dorit2

1 Oregon Health Sciences University, Portland, Oregon, 2 Yale University, New Haven, Connecticut
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.2
DOI:  10.1002/0471142301.ns0402s08
Online Posting Date:  May, 2001
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Abstract

Of the many proteins that are known to be involved in neuronal signaling, one family of gene products, collectively referred to as the G protein‐coupled receptors (GPCRs), has received considerable attention. Within the transmembrane domains of GPCRs are clusters of amino acids that tend to be conserved among receptors that bind related ligands. Polymerase chain reaction (PCR)‐based approaches to cloning novel GPCRs typically begin with the identification of these well‐conserved amino acid motifs, which are then back‐translated into degenerate oligonucleotide primers. These pools of degenerate oligonucleotides are the most important variables in PCR cloning of GPCRs. Although GPCRs are used as the focus of this unit, the strategies and techniques described are applicable to the cloning of a wide variety of neuronal gene products. In the first procedure in this unit, either total or poly(A)+ purified RNA is reverse transcribed into first‐strand cDNA. In subsequent steps the cDNA product serves as the template for synthesis and amplification of target receptor sequences by PCR primed with degenerate oligodeoxynucleotides. The product is ready to be cloned and screened as described. Guidelines for database searching are provided to help identify the cloned gene from the known sequence. Typically, only a portion of the receptor coding region is cloned by the above approach. Rapid amplification of cDNA ends (RACE) or anchored PCR is described in this unit and is used to obtain a full‐length cDNA amenable for expression studies.

     
 
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Table of Contents

  • Basic Protocol 1: Amplification Using Degenerative Primers
  • Basic Protocol 2: Anchored Amplification of Regions Downstream (3′) of Known Sequence
  • Basic Protocol 3: Anchored Amplification of Regions Upstream (5′) of Known Sequence
  • Support Protocol 1: Cloning and Screening the PCR Product
  • Support Protocol 2: Database Searching
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Amplification Using Degenerative Primers

  Materials
  • 0.5 µg/µl oligo(dT) 12–18or 50 ng/µl random hexamers
  • 1 µg/µl total or poly(A)+ RNA ( appendix 1A)
  • Diethylpyrocarbonate (DEPC)‐treated H 2O (see appendix 2A)
  • 5× first‐strand cDNA buffer: 250 mM Tris⋅Cl (pH 8.3)/375 mM KCl/15 mM MgCl 2
  • 0.1 M dithiothreitol (DTT)
  • 10 mM 4dNTP mix: 10 mM each dATP, dCTP, dGTP, dTTP in H 2O or TE buffer, pH 7.5
  • 200 U/µl SuperScript II reverse transcriptase (RT; Life Technologies)
  • 2 U/µl E. coli RNase H
  • 10× degenerate PCR amplification buffer (usually supplied by the manufacturer; also see recipe)
  • 10 to 20 µM degenerate oligonucleotide primers in H 2O
  • 2.5 mM 4dNTP mix: 2.5 mM each dATP, dGTP, dCTP, dTTP in TE buffer, pH 7.5
  • 5 U/µl Pfu DNA polymerase (Taq Extender, Stratagene)
  • 5 U/µl Taq DNA polymerase
  • 37° or 42°, 55°, and 70°C water baths or constant‐temperature heating blocks
  • Programmable thermal cycler
  • Additional reagents and equipment for agarose or nondenaturing polyacrylamide gel electrophoresis (see CPMB UNITS & and appendix 1A in this manual)
NOTE: All reagents and solutions used for RNA procedures should be treated with DEPC to inhibit RNase activity.CAUTION: DEPC is a suspected carcinogen and should be handled carefully.

Basic Protocol 2: Anchored Amplification of Regions Downstream (3′) of Known Sequence

  Materials
  • ≥100 ng/µl poly(A)+ RNA ( appendix 1I)
  • 5× MMLV RT buffer (see recipe)
  • 5 µg/µl bovine serum albumin (BSA)
  • 10 mM 4dNTP mix: 10 mM each dNTP in recipeTE buffer, pH 7.5
  • 500 ng/µl actinomycin D
  • 200 U/µl MMLV reverse transcriptase
  • 100 ng/µl oligo(dT) 20 primer
  • TE buffer ( appendix 2A)
  • 10× downstream PCR amplification buffer (see recipe)
  • 100 pmol/µl each of sequence‐specific primers 1 and 2 (Fig. )
  • 2.5 mM 4dNTP mix: 2.5 mM each dNTP in TE buffer, pH 7.5
  • 2.5 U/µl Taq DNA polymerase
  • Mineral oil

Basic Protocol 3: Anchored Amplification of Regions Upstream (5′) of Known Sequence

  Materials
  • 100 pmol/µl sequence‐specific primers 3 and 4 (Fig. )
  • 1 M NaCl
  • 200 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
  • 25 mM EDTA
  • 100 ng/µl poly(A)+ RNA or 1 µg/µl total RNA
  • 100% and 70% ethanol, ice cold
  • 5× MMLV RT buffer (see recipe)
  • 5 µg/µl bovine serum albumin (BSA)
  • 10 mM 4dNTP mix: 2.5 mM each dNTP in TE buffer, pH 7.5
  • 500 ng/µl actinomycin D
  • 200 U/µl MMLV reverse transcriptase
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (see appendix 2A)
  • 3 M sodium acetate ( appendix 2A)
  • TE buffer ( appendix 2A)
  • 5× TdT buffer (see recipe)
  • 15 mM CoCl 2
  • 1 mM dATP
  • Terminal transferase
  • 40°C and 37°C water baths
  • Additional reagents and equipment for amplification reactions (see protocol 2)
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Figures

Videos

Literature Cited

Literature Cited
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Internet Resources
   www.ncbi.nlm.nih.gov
  National Center for Biotechnology Information site for database searching and other resources. NCBI database searches can also be conducted via e‐mail by addressing the message to blast@ncbi.nlm.nih.gov. Instructions for use of the NCBI's resources is available by typing the word help. Sequences may also be retrieved by addressing the message to retrieve@ncbi.nlm.nih.gov and again typing help for additional information.
   http://www.receptor.mgh.harvard.edu/GCRDBHOME.html. or http://www.receptor.mgh.harvard.edu/Alignments.html
  Informal collaboration of G protein–coupled receptor databases.
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