DNA SEQUENCING BY SANGER METHOD

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1 DNA SEQUENCING BY SANGER METHOD First method described by Sanger and Coulson,1975 for DNA sequencing was called plus and minus. This method used E.coli DNA polymerase I and DNA ploymerase from bacteriophage T4 with different limiting nucleoside triphophates. Due to inefficacy of this method, 2 year later Sanger and his coworkers descibed a breakthrough method for sequencing oligonucleotides via enzymic polymerization( Sanger et al,1977). This method was known as chain termination or dideoxynucleotide method.

2 Chain termination or Dideoxynucleotide method of Sequencing This method makes use of the mechanism of DNA synthesis by DNA polymerases DNA polymerases require both a primer (a short oligonucleotide strand), to which nucleotides are added, and a template strand to guide selection of each new nucleotide. In cells, the 3-hydroxyl group of the primer reacts with an incoming deoxynucleoside triphosphate (dntp) to form a new phosphodiester bond.

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4 The Sanger sequencing procedure uses dideoxynucleoside triphosphate (ddntp) analogs to interrupt DNA synthesis. (The Sanger method is also knownas the dideoxy method.) When a ddntp is inserted in place of a dntp, strand elongation is halted after the analog is added, because it lacks the 3-hydroxyl group needed for the next step. Four sets of chain-terminated fragments (one for each dideoxy analog) then undergo electrophoresis, and the base sequence of the new DNA is read from the autoradiogram of the four lanes.

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6 Because shorter fragments migrate faster, the fragments near the bottom of the gel represent the nucleotide positions closest to the primer (the 5ʹ end), and the sequence is read (in the 5'-3ʹ direction) from bottom to top. Note that the sequence obtained is that of the strand complementary to the strand being analyzed.

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8 Automating DNA sequencing reactions Each dideoxynucleotide used in the Sanger method can be linked to a fluorescent molecule that gives all the fragments terminating in that nucleotide a particular color. All four labeled ddntps are added to a single tube. The resulting colored DNA fragments are then separated by size in a single electrophoretic gel contained in a capillary tube (a refinement of gel electrophoresis that allows for faster separations). All fragments of a given length migrate through the capillary gel in a single peak, and the color associated with each peak is detected using a laser beam. The DNA sequence is read by determining the sequence of colors in the peaks as they pass the detector. This information is fed directly to a computer, which determines the sequence.

9 Sanger and co-workers determined the complete sequence of the 5386 bases in the DNA of the f X174 DNA virus in 1977, just a quarter century after Sanger's pioneering elucidation of the amino acid sequence of a protein. This accomplishment is a landmark in molecular biology because it revealed the total information content of a DNA genome. This tour de force was followed several years later by the determination of the sequence of human mitochondrial DNA, a double-stranded circular DNA molecule containing 16,569 base pairs. It encodes 2 ribosomal RNAs, 22 transfer RNAs, and 13 proteins. In recent years, the complete genomes of free-living organisms have been sequenced. The first such sequence to be completed was that of the bacterium Haemophilus influenzae. Its genome comprises 1,830,137 base pairs and encodes approximately 1740 proteins.

10 Many other bacterial and archaeal genomes have since been sequenced. The first eukaryotic genome to be completely sequenced was that of baker's yeast, Saccharomyces cerevisiae, which comprises approximately 12 million base pairs, distributed on 16 chromosomes, and encodes more than 6000 proteins. This achievement was followed by the first complete sequencing of the genome of a multicellular organism, the nematode Caenorhabditis elegans, which contains nearly 100 million base pairs. The human genome is considerably larger at more than 3 billion base pairs, but it has been essentially completely sequenced. The ability to determine complete genome sequences has revolutionized biochemistryand biology.

11 Maxam and Gilbert method of sequencing In this method, end-labelled DNA fragments are subjected to random cleavage at adenine, cytosine, guanine, thymine positions using specific chemical agents. The chemical attack is based on three steps: base modification, removal of the modified base from its sugar and DNA strand breaking at that sugar position. Purines will react with dimethyl sulfate and pyrimidines will react with hydrazine in such a way as to break the glycoside bond between the ribose sugar and the base displacing the base. Piperidine will then catalyze phosphodiester bond cleavage where the base has been displaced. Moreover, dimethyl sulfate and piperidine alone will selectively cleave guanine nucleotides but dimethyl sulfate and piperidine in formic acid will cleave both thymine and cytosine nucleotides whereas hydrazine and piperidine in 1.5M NaCl will only cleave cytosine nucleotides.

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14 Maxam and Gilbert method of sequencing The products of these four reactions are then separated by using polyacrylamide gel electrophoresis. The sequence can be easily read from the four parallel lanes in sequencing gel.

15 Advantages of Maxam and Gilbert sequencing method over Sanger s method A fragment can be sequenced from the original DNA fragment instead from enzymic copies No subcloning and no PCR reaction is involved. Foe location of rare bases, chemical cleavage analysis can not be replaced by the dideoxynucleotide method as the latter analyses the DNA of interest via its complementary sequence. It can give only sequence information in terms of four canonical bases. This method is less susceptible to mistakes with regard to sequencing of secondary structures or enzymic mistakes. Some of the chemical protocols are recognised by the different authors as being simple,easy to control,and the chemical distinctions between the different bases are clear.

16 Uses of Maxam and Gilbert sequencing method Chemical degradation method is used for 1. Genome sequencing,where information about DNA methylation and chromatin structure could be obtained. 2. To confirm the accuarcy of synthesised oligonucleotides or to verify the sequence of DNA regions with hairpin loops. 3. To locate rare bases such as Hoogsteen base pairs 4. To detect point mutations 5. To resolve the ambiguities that arise during dideoxy sequencing 6. To analyse DNA-protein interactions. 7. To sequence short DNA fragments in general.

17 Disadvantages of Maxam and Gilbert sequencing The chemical reactions of the most of the protocols are slow and the use of hazardous chemicals requires special handling care. The worst problem, however is the, occurrence of incomplete reactions introduce electrophoretic mobility polidispersion(caused by chemical and physical inhomogeneties among the DNA chains within a given band) which enlarges the band widths and this in turn reduces the interband resolution.