Central Dogma of Molecular Genetics Restriction-Modification Systems of Bacteria Cloning Vectors cDNA Cloning Clone Library Screening DNA Sequencing Southern and Northern Analysis Exons and Introns Polymerase Chain Reaction (or PCR) Study Questions Cloning and Molecular Analysis of Genes WWW Links Genetic Topics

DNA Sequencing These cloning techniques have been widely used to isolate many genes from nearly all species. Once these genes have been isolated what can they be used for? The nucleic acid sequence of the gene can be derived. If a partial or complete sequence of the protein that it encodes is available the gene can be confirmed in this manner. If the protein product is not known then the sequence of the gene can be compared with those of known genes to try to derive a function for that gene. The clone can then be used to study the sequences of the regulatory region of the gene. This is possible only for genomic clones because cDNA clones just contain coding sequences. The clone can be used to isolate similar genes from other organisms. Thus it can serve as a heterologous probe. If the gene is of clinical importance, the clone can be used for diagnostic purposes. The gene which encodes for one type of hemophilia has been used for this purpose. These are termed RFLP analyzes. A major goal of molecular genetics is to correlate the sequence of a gene with its function. Thus obtaining the sequence is of primary importance. DNA sequencing can be performed by the chemical procedure or the dideoxy-chain-termination procedure. Because the later procedure is far more popular we will only discuss that procedure. The dideoxy-chain-termination DNA sequencing technique is a DNA polymerase-based technique. This technique is based on the ability of a specific nucleotide (dideoxy nucleotide) to terminate the DNA polymerase reaction. These nucleotides do not have a free 3'-OH group, an absolute requirement for DNA polymerase activity. Thus, any time this nucleotide is inserted into the growing chain DNA synthesis stops. Technically, four polymerase reactions are performed, each containing the four nucleotides dATP, dTTP, dCTP and dGTP. In addition the reactions contain a limited amount of the one of the four dideoxybases so that all possible terminations can occur. After the reactions are finished, the products from the four reactions are separated side-by-side on a polyacrylamide gel. Each of the fragments within a lane ends with the base corresponding to the dideoxy nucleotide used in the reaction. Thus by reading the four lanes from the bottom of the gel to the top, the sequence of the DNA can be obtained. Diagrammatic Representation of a Sequencing Reaction Products 5' A T C T A G G-----------3' ddC-----------5' ddC--------------------5' ddT-----------------------------5' ddA-------------------------------------5' ddG--------------------------------------------5' ddA----------------------------------------------------5' ddT-------------------------------------------------------------5' Diagrammatic Representation of the Sequencing Gel G A C T ---------------------- __ Longest product (5' end of gene) __ __ __ __ __ __ Shortest product (3' end of gene) ---------------------- Copyright © 1997. Phillip McClean