BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

Chemical DNA Synthesis and Gene Construction

The phosphoramidite method is the most widespread technique for the Chemical synthesis of DNA. Modified deoxynucleosides serve as the starting Materials.

The modification involves attaching a benzoyl group to the amino groups of deoxyadenosine and deoxycytidine, and an isobutyryl group to the amino group of deoxyguanosine. Thymidine, which lacks an amino group, is not modified. The modification of nitrogenous bases is necessary to protect nucleosides from undesirable Side Reactions during the elongation of the polynucleotide chain.

Specialized devices for automated Chemical DNA Synthesis—known as DNA synthesizers—have been developed, capable of synthesizing single-stranded DNA fragments up to 50 NUCLEOTIDES in length. A DNA molecule 100 - 120 Base Pairs long can be synthesized within a 24-hour period. Chemically synthesized oligonucleotides

can be used for Gene construction, Amplification of specific DNA sequences, Site-Directed Mutagenesis of isolated DNA, as probes for DNA Hybridization, and as primers.

Gene construction is carried out using chemically synthesized oligonucleotides. This process involves the Enzymes E. coli DNA polymerase and DNA ligase. Genes exceeding 300 base pairs in length are assembled from individual modules—single-stranded fragments 20 - 100 nucleotides long. Genes whose length exceeds 1000 base pairs are constructed from double-stranded fragments.