PCR cloning of neural gene products.
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.
Grandy
D K
DK
Oregon Health Sciences University, Portland, Oregon, USA.
Bunzow
J R
JR
Dorit
R L
RL
D000818:Animals; D002999:Clone Cells; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis / Q000235:genetics*
D018076:DNA, Complementary; D043562:Receptors, G-Protein-Coupled