Sequencing of Differentially Expressed cDNA Fragments
Once proved to be differentially expressed, a cDNA fragment is sequenced to search for homology with other cloned genes and/ or putative gene functions. The sequence of a deoxyribonucleic acid molecule can be determined by chemical (Maxam and Gilbert 1977, 1980) or enzymatic (Sanger et al. 1977) methods. The enzymatic sequencing method is based on the ability of DNA polymerase to extend a primer hybridized to the template to be sequenced until a chain-terminating nucleotide (2’,3’-di-deoxyribonucleoside 5’-triphosphate, ddNTP) is incorporated. Because the ddNTP lacks the necessary 3’-OH group required for chain elongation, the growing oligonucleotide is terminated selectively at G, A, T, or C when a set of four separate reactions, each with a different ddNTP, are performed. When a radionucleotide is included in the reaction, the resulting labeled fragments, with the same origin but ending at a different nucleotide, are visualized by autoradiography after separation by denaturing gel electrophoresis.
KeywordsVortex Phenol EDTA Agarose Electrophoresis
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