Basics of gene manipulation

Restriction endonucleases and DNA ligase Cutting, rejoining DNA Reverse transcriptase Conversion of mRNA into cDNA Chemical DNA synthesis Oligonucleotides Polymerase chain reaction (PCR) 

Rapid amplification of specific DNA segments Vectors for gene isolation and expression DNA sequencing techniques Sequence verification 

Protein microsequencing, peptide synthesis Site-directed mutagenesis Creation of specific mutant proteins Protein overproduction Appropriate host/vector systems 

The DNA content of the smallest genome such as that of the DNA virus SV40 is of the order of thousands of base pairs and the analysis of even such small genomes would be impossible if they could not be reproducibly fragmented into smaller pieces. The discovery of restriction endonucleases from several bacterial species provided the tool for the specific cleavage of DNA. Once cleaved, the fragments of different sizes could be separated electrophoretically, isolated, and analyzed. 

Restriction enzymes are endonucleases which cleave specific sequences within double-stranded DNA stretches. The recognition and cleavage sequences of a routinely used group of endonucleases (called type 11 enzymes) may be four, five, six, or eight bases long and are symmetric across the two strands of DNA. Fig. 2.12 illustrates the cleavage specificity of a widely used sixbase-motif-specific endonuclease, EcoR 1. 

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