Slide 1: DNA SEQUENCING Adane Mihret Februray 2011 WHAT IS DNA SEQUENCING ? : WHAT IS DNA SEQUENCING ? “Sequencing” means finding the order of nucleotides on a piece of DNA . Nucleotide order determines Amino acid order, and by extension, protein structure and function (proteomics) An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect in a plant or animal DNA sequencing usually involves enzymatic DNA synthesis in the presence of base-specific dideoxynucleotide chain terminators. Determining the DNA sequence is therefore useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or forensic research. FOUNDERS OF SEQUENCING TECHNOLOGY : FOUNDERS OF SEQUENCING TECHNOLOGY Sanger Wally Gilbert Slide 4: MAXAM & GILBERT DNA SEQUENCING (CHEMICAL DEGRADATION) In the late 1970s, A. M. Maxam and W.Gilbert devised the first method for sequencing DNA fragments containing up to ≈500 nucleotides. The sequence of a double-stranded DNA molecule is determined by treatment with chemicals that cut the molecule at specific nucleotide positions. It is the early method involving base-specific chemical modification and subsequent cleavage of DNA. most of the chemicals used in chemical degradation method are toxic and hazardous to the health of the researchers doing the DNA sequencing. Slide 5: CHEMICAL DEGRADATION METHOD PROCEDURE four samples of an end-labeled DNA restriction fragment are chemically cleaved at different specific nucleotides. the resulting sub-fragments are separated by agarose gel electrophoresis and the labeled fragments are detected by autoradiograph. the sequence of the original end-labeled restriction fragment can be determined directly from parallel electrophoretograms of the four samples. Slide 6: CHEMICALS INVOLVED Dimethyl sulphate methylates guanine. Acid removes any purines . Hydrazine modifies any pyrimidine . Hydrazine with NACL specifically modifies cytosines . Piperidine is used to remove the modified bases. Slide 7: SANGER METHOD F. Sanger and his colleagues developed a second method of DNA sequencing, which now is used much more frequently than the Maxam -Gilbert method. The sequence of a single-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains, these chains terminating at specific nucleotide positions. Sanger method is the most suitable method for automation in large scale sequencing projects, and most general sequencing is now carried out in this way. Slide 8: CHAIN TERMINATION METHOD PRINCIPLE The single-stranded DNA to be sequenced serves as the template strand for in vitro DNA synthesis; a synthetic 5′-end-labeled oligodeoxynucleotide is used as the primer. When a small amount of a specific dideoxy NTPs ( ddNTPs ) is included along with the four deoxy NTPs normally required in the reaction mixture for DNA polymerase. The products are the series of chains that are specifically terminated at the dideoxy residue. Thus four separate reactions, each containing a different dideoxy NTP,can be run, and their products displayed on a high-resolution Acryl amide gel. Slide 9: CHAIN TERMINATION METHOD PROCEDURE Prepared the starting material for a chain termination sequencing experiment is a.identical single-stranded DNA molecules. To anneal a short oligonucleotide to the same position on each molecule, this oligonucleotide subsequently acting as the primer for synthesis of a new DNA strand that is complementary to the template catalyzed by DNA polymerase requires requires the four deoxyribonucleotide triphosphates ( dNTPs - dATP , dCTP , dGTP and dTTP ) as substrates, would normally continue until several thousand nuceotides had been polymerised . The polymerase does not discriminate between dNTPs and ddNTPs , so the dideoxynucleotide can be incorporated into the growing chain, but it then blocks further elongation because it lacks the 3′-hydroxyl group needed to form a connection with the nucleotide. Slide 10: This process continue until several hundred nucleotides have been polymerized before a ddATP is eventually incorporated. The result is therefore a set of new chains, all of different lengths, but each ending in ddATP. Now the polyacrylamide gel comes on to play. The family of the presence of ddNTPAs(ddATP,ddCTP,ddGTP,ddTTP) loaded into four adjacent wells of the gel. After electrophoresis, the DNA sequence can be read directly from the positions of the bands in the gel. (Continue) Slide 11: CHAIN TERMAINTAION METHOD Slide 13: CHAIN TERMINATION SEQUENCING Thermal cycle sequencing Automated DNA sequencing Pyrosequencing Sequencing by hybridization Slide 14: THERMAL CYCLE SEQUENCING The discovery of thermo stable DNA polymerases, which led to the development of PCR has also resulted in new methodologies for chain termination sequencing. Thermal cycle sequencing has two advantages over traditional chain termination sequencing 1) uses double-stranded rather than single-stranded DNA as the starting material. 2) very little template DNA is needed, so the DNA does not have to be cloned before being sequenced. Slide 15: Thermal cycle sequencing is carried out in a similar way to PCR but just one primer is used and each reaction mixture includes one of the ddNTP . Because there is only one primer, only one of the strands of the starting molecule is copied, and the product accumulates in a linear fashion, not exponentially as is the case in a real PCR. The presence of the ddNTP in the reaction mixture causes chain termination, as in the standard methodology, and the family of resulting strands can be analyzed and the sequence read in the normal manner by polyacrylamide gel electrophoresis Slide 16: Thermal cycle sequencing . PCR is carried out with just one primer and with a dideoxynucleotide present in the reactionmixture. The result is a family of chain-terminated strands - the ‘A' family in the reaction shown. These strands, along with the products of the C, G and T reactions, are electrophoresed as in the standard methodology Slide 17: AUTOMATED DNA SEQUENCING The most dramatic advance in sequencing and the one that carried DNA sequencing into a high throughput environment was the introduction of automated sequencing using fluorescence- labeled dideoxy -terminators. In 1986, Leroy Hood and colleagues reported on a DNA sequencing method in which the radioactive labels, autoradiography, and manual base calling were all replaced by fluorescent labels, laser induced fluorescence detection, and computerized base calling. In their method, the primer was labeled with one of four different fluorescent dyes and each was placed in a separate sequencing reaction with one of the four dideoxynucleotides plus all four deoxynucleotides . Slide 18: Fluorolabeling has been equally important in the development of sequencing methodology, in particular because the detection system for fluorolabels has opened the way to automated sequence reading. (A) The chain termination reactions are carried out in a single tube, with each dideoxynucleotide labeled with a different fluorophore.In the automated sequencer, the bands in the electrophoresis gel move past a fluorescence detector, which identifies which dideoxynucleotide is present in each band. The information is passed to the imaging system. (B) The printout from an automated sequencer. The sequence is represented by a series of peaks, one for each nucleotide position. In this example, a green peak is an ‘A', blue is ‘C', black is ‘G', and red is ‘T'.