Homology Cloning of cDNA or Genomic DNA

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Abstract
Table of Contents
Materials
Literature Cited

Abstract

The principle of cloning utilizes hybridization of single?stranded DNA probes to denatured DNA fixed on membranes to detect the identical DNA molecules present in a large population of diverse DNAs. In homology or low?stringency cloning, hybridization of oligonucleotide, cDNA, or genomic DNA probes to denatured DNA from plasmid or bacteriophage libraries is carried out under low?stringency conditions that promote hybridization in the presence of sequence mismatch (i.e., mispaired nucleotides). The procedures used are identical to those for screening libraries at high stringency, with the exception that hybridization and washing conditions are altered to permit hybridization with mismatched sequence; salt concentration is increased and the hybridization and wash temperatures are decreased. This unit describes conditions that have been used to clone and identify novel genes and cDNA clones using low?stringency hybridization of known probes to membranes that contain libraries of bacterial or bacteriophage DNA.

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Reagents and Solutions
Commentary
Literature Cited

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Materials

Basic Protocol 1:

Materials

Bacteriophage, cosmid, or plasmid library
Nitrocellulose or Nytran/Hybond N+ membrane
Low‐stringency hybridization solution (see recipe )
2200 Ci/mmol radiolabeled probe, boiled
Low‐stringency wash solution I: 2× SSC ( appendix 2A )/1.0% (w/v) SDS, room temperature and 50° to 55°C
Low‐stringency wash solution II: 1× SSC/1.0% (w/v) SDS
Membrane stripping solution: 0.02× SSC/0.1% (w/v) SDS

37°C incubator or hybridization chamber
Minimonitor (series 900, model E, Mini‐Instruments, Dosimeter)
Autoradiography film (e.g., X‐Omat AR‐5, Kodak)
Autoradiography cassette and intensifying screen

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Literature Cited

Literature Cited
	Albert, P.R., Zhou, Q.‐Y., VanTol, H.H.M., Bunzow, J., and Civelli, O. 1990. Cloning, functional expression, and mRNA tissue distribution of the rat 5‐HT1A receptor gene. J. Biol. Chem. 265:5825‐5832
	Albert, P.R. 1992. Molecular biology of the 5‐HT1A receptor: Low‐stringency cloning and eukaryotic expression. J. Chem. Neuroanat. 5:283‐288.
	Bunzow, J.R., VanTol, H.H.M., Grandy, D., Albert, P., Salon, J., Christie, M., Machida, C., Neve, K.A., and Civelli, O. 1988. Cloning and expression of a rat dopamine‐D2 receptor cDNA. Nature 336:783‐787.
	Davis, L.G., Dibner, M.D., and Battey, J.F. 1986. Basic Methods in Molecular Biology. Elsevier, New York., N.Y.
	Kobilka, B.K., Frielle, T., Collins, S., Yang‐Feng, T., Kobilka, T.S., Francke, U., Lefkowitz, R.J., and Caron, M.G. 1987. An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature 329:75‐79.
	Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Van Sande, J., Maenhaut, C., Simons, M.‐J., Dumont, J.E., and Vassart, G. 1989. Selective amplification and cloning of four new members of the G protein–coupled receptor family. Science 570:569‐572.
	Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
	Shigemoto, R., Yokota, Y., Tsuchida, K., and Nakanishi, S. 1990. Cloning and expression of a neuromedin K receptor cDNA J.Biol. Chem. 265:623‐628.
	Yokota, Y., Sasai, Y., Tanaka, K., Fujiwara, T., Tsuchida, K., Shigemoto, R., Kakizuka, A., Ohkubo, H., and Nakanishi, S. 1989. Molecular characterization of a functional cDNA for rat substance P receptor. J. Biol. Chem. 264:17649‐17652.