This chapter discusses the cloning of hormone genes from a mixture of complementary deoxyribonucleic acid (cDNA) molecules. The molecular cloning of cDNA synthesized from a purified messenger ribonucleic acid (mRNA) is a well-established method for obtaining purified DNA for sequence analysis and use as a hybridization probe. Only a limited number of purified mRNAs are currently available that include those from specialized cells producing predominantly a single protein, such as globin, immunoglubin, or ovalbumin. The chapter describes the procedures used to isolate and clone specific hormone cDNAs from an impure mRNA population. The method employs the restriction endonuclease cleavage of double-stranded cDNA transcribed from a complex mixture of mRNA. The method does not require any extensive purification of RNA but instead makes use of the transcription of RNA into cDNA, the sequence-specific fragmentation of this cDNA, with one or two restriction endonucleases, and the fractionation of the cDNA restriction fragments on the basis of their lengths. The use of restriction endonucleases eliminates size heterogeneity and produces homogeneous-length DNA fragments from any cDNA species that contains at least two restriction sites. From the initially heterogeneous population of cDNA transcripts, uniform-sized fragments of desired sequence are produced. The chapter discusses a complete scheme for cDNA cloning.
ASJC Scopus subject areas
- Molecular Biology