13- DNA sequencing.ppt

DNA Sequencing 1 Learning objectives • DNA sequencing: - Chain termination/Sanger sequencing. - Next Generation Sequencing (NGS) 2 • In 1977, 24 years after the discovery of the structure of DNA, two separate methods for sequencing DNA were developed: the chain termination (Sanger sequencing)...

DNA Sequencing 1 Learning objectives • DNA sequencing: - Chain termination/Sanger sequencing. - Next Generation Sequencing (NGS) 2 • In 1977, 24 years after the discovery of the structure of DNA, two separate methods for sequencing DNA were developed: the chain termination (Sanger sequencing) method and the chemical degradation method. Both methods were equally popular to begin with, but the chain termination method is the method more commonly used today. Chain termination method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using agarose gel electrophoresis. • The purpose of DNA sequencing is to: 1- the base sequence of DNA fragments that have been cloned and purified can be determined in the laboratory. 2- study genetic variation associated with diseases or other biological phenomena. 3 Chain termination method “Sanger DNA sequencing” • This technique requires four sequencing reactions which include the mixing/addition of all the components necessary to synthesize and label unknown DNA template: 1- A DNA template 2- A primer tagged with a mildly radioactive molecule or a light-emitting chemical 3- DNA polymerase, an enzyme that drives the synthesis of DNA 4- Four deoxynucleotides (G, A, C, and T) + one dideoxynucleotide, either ddG, ddA, ddC, or ddT 4 Deoxynucleotides Dideoxynucleotides Normal reaction Chain termination reaction 5 1- Sanger Sequencing Method 6 * Give the sequence of the unknown DNA template using the above gel electrophoresis outcome. 7 Next generation DNA sequencing (NGS) Sanger sequencing is best for analyzing small numbers of gene targets and samples and can be accomplished in a single day. NGS enables the interrogation of hundreds to thousands of genes at one time in multiple samples. NGS technologies used are associated to an optical detection system to determine nucleotide incorporation during DNA synthesis. 1- Automated cycle sequencing (see video clip) 2- Pyrosequencing reaction (see video clip) 8 Learning outcomes • DNA sequencing is essential to determine the nucleotide sequence of the gene of interest to facilitate further investigations. • Sanger Sequencing is considered as a goldstandard sequencing technology. • The development of more advanced sequencing technologies under NGS. • A successful sequencing experiment requires the understanding of PCR and gel electrophoresis techniques. 9