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DNA SEQUENCING Principles & methods Topic Outline Principles of DNA Sequencing Methods of DNA Sequencing Principles of DNA Sequencing Denaturation Cycle sequencing (one cycle) Extension/ termination Annealing Partial copies of DNA fragments made with DNA polymerase Collection of DNA fragments that terminate with A,C,G or T using ddNTP or chemical. Separate by gel electrophoresis Read DNA sequence Radioactive label Denaturation, Gel electrophoresis Four aliquots – treated with chemicals ( modification) Cleaved by piperidine Separated by Electrophoresis Synthesis termination Dead-end product Add in DNA polymerase I, dNTPs, ddNTPs Extension /synthesis of complementary strands Run gel divide by size Running the reaction of all the dideoxy nucleotides using different dyes generates this type of diagram in same lane. Sometimes the spacing between the bands is hard to measure. Thus use machine to run and read the electrophoresis. Capillary electrophoresis: the fragments are piped through a tiny glass-fiber capillary during the electrophoresis step, and they come out the far end in size-order. Chemical cleave method Sequence small fragments of DNA The radioactive labelling is done on the dsDNA. Division of aliquots is done by methylation or removal of base. Requires DNA Breaks DNA at different nucleotides Enzymatic cleave method Sequencing small fragments are problematic. The radioactive labelling is done on the ssDNA. Allow high throughput automated sequencing techniques. Allow Real Time detection. Requires DNA synthesis Termination of chain elongation SOUTHERN, NORTHERN AND WESTERN BLOTTING Comparison of Southern, Northern, and Western analyses of Gene X SOUTHERN BLOTTING The technique was developed by E.M. Southern in 1975. The Southern blot is used to detect the presence of a particular piece of DNA in a sample. The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome. Southern hybridization Transfer buffer Detection of an RFLP by Southern blotting Flow chart of Southern Preparing the samples and running the gel hybridization Southern transfer Probe preparation Isotope Non-isotope Prehybridization Hybridization Post-hybridization washing Signal detection SOUTHERN BLOTTING The key to this method is hybridization. Hybridization-process of forming a double- stranded DNA molecule between a singlestranded DNA probe and a single-stranded target patient DNA. SOUTHERN BLOTTING There are 2 important features of hybridization: The reactions are specific-the probes will only bind to targets with a complementary sequence. The probe can find one molecule of target in a mixture of millions of related but noncomplementary molecules. Comparison of Southern, Northern, and Western blotting techniques Molecule detected Gel electrophoresis Gel pretreatment Southern blotting DNA (ds) Northern blotting mRNA (ss) Western blotting Protein Agarose gel Formaldehyde agarose gel - Polyacrylamide gel Capillary transfer cDNA, cRNA Radioactive or nonradioactive Autoradiography Chemiluminescent Colorimetric Electric transfer primary antibody Depurination, denaturation, and neutralization Blotting method Capillary transfer Probes DNA Radioactive or nonradioactive Detection Autoradiography system Chemiluminescent Colorimetric - Chemiluminescent Colorimetric MB206 MUTAGENESIS What Is a Mutation? Genetic information is encoded by the sequence of the nucleotide bases in DNA of the gene. The four nucleotides are: adenine (A), thymine (T), guanine (G), and cytosine (C), a mutation is a change in the order of these nucleotides. A change in the order can cause the gene to encode for wrong proteins and inhibit the function of the gene or cause the gene to be virtually inactive. Mutagenesis Mutagenesis (the creation or formation of a mutation) can be used as a powerful genetic tool. By inducing mutations in specific ways and then observing the phenotype of the organism the function of genes and even individual nucleotides can be determined. Some types of Mutagenesis Directed Mutagenesis Site-directed/Site-specific Mutagenesis Mismatched Mutagenesis