BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

cDNA Libraries

Complementary DNA (cDNA) is DNA synthesized from mRNA using the enzyme Reverse Transcriptase.

Unlike genomic DNA, cDNA lacks introns. This distinction is of practical importance. cDNA clones are used to produce human Insulin or other Proteins in E. coli Cells.

First, mRNA is isolated from human cells. A key structural feature of eukaryotic mRNA is the presence of an adenylyl nucleotide sequence (poly(A) tail). mRNA can be isolated by passing an RNA preparation through a Column containing an inert matrix with bound thymine residues. rRNA and tRNA pass through the column quickly, whereas mRNA binds to it via A-T hydrogen bonding between the tail and the Ligand. mRNA can then be eluted using low Ionic strength solutions.

The scheme of cDNA synthesis is shown in Fig. 1.1.

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Fig. 1.1. Synthesis of cDNA from mRNA using reverse transcriptase

Phage λ or Plasmids are used as Vectors for Cloning cDNA molecules. Although cDNA molecules lack sticky ends, Methods have been developed to attach sticky ends to them.

Introducing cDNA into bacterial cells enables the synthesis of A large number of its copies. Cloning is one of the methods used for the Amplification of DNA nucleotide sequences.