DNA Sequencing

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DNA Sequencing •determining the exact base sequence of a cloned piece of DNA. •The Sanger Chain-Termination Sequencing MethodTheoriginal method of sequencing a piece of DNA by the Sanger method •single-stranded DNA one hybridizes an oligonucleotide primer about 20 bases long. •Extending the primer using the Klenowfragment of DNA polymerase produces DNA complementary to the insert •Sanger’s method is to carry out such DNA synthesis reactions in four separate tubes.Include in each tube a different chain terminator

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DNA Sequencing R.Hari Prakash

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DNA Sequencing • determining the exact base sequence of a cloned piece of DNA. • The Sanger Chain-Termination Sequencing MethodThe original method of sequencing a piece of DNA by the Sanger method • single-stranded DNA one hybridizes an oligonucleotide primer about 20 bases long. • Extending the primer using the Klenow fragment of DNA polymerase produces DNA complementary to the insert • Sanger’s method is to carry out such DNA synthesis reactions in four separate tubes. Include in each tube a different chain terminator

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• The chain terminator is a dideoxy nucleotideDNA synthesis stops. • it cannot form a phosphodiester bond because it lacks the necessary 3′- hydroxyl group. • This random arrest of DNA growth means that some strands will terminate. • Each tube contains a different dideoxy nucleotide: • 1-ddATP • 2-ddCTP • 3-ddGTP • 4-ddTTP

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• high-resolution electrophoresis gel and electrophoresed to separate them according to size.

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automated sequencing methods • based on Sanger’s chain-termination method. • The primers used in each of the four reactions are tagged with a different fluorescent molecule. • each tube will emit a different color fluorescence when excited by light.

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• Then the color of the fluorescent light emitted from each oligonucleotide is detected electronically. • This information then passes to a computer programmed to convert the color information to a base sequence. • computer for analysis. • Large genome projects use many automated sequencers running simultaneously to obtain millions or even billions of bases of sequence

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Maxam –Gilbert Sequencing • synthesizing DNA in vitro and stopping the synthesis reactions with chain terminators • method starts with full-length end-labeled DNA and cleaves it with base-specific reagents.5′- or 3′-end labeling. • dimethyl sulfate DMS to methylate guanines. • the methylation under mild conditions that lead to an average of only one methylated guanine per DNA strand. • Then the methylated DNA is treated with piperidine. • First removes the methylated base and then breaks the DNA strand at the apurinic site. • This leaves a 3′-phosphate guanine nucleotide.

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COMPARISON SANGER METHOD MAXAM GILBERT METHOD enzymatic chemical Requires DNA synthesis DNA synthesis Termination of chain elongation Break DNA at different nucleotides automation Automation is not available Single- stranded DNA Double- stranded or Single -stranded DNA

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Application of dna sequencing Forensics: • Identify individuals • Determine the paternity of a child • Identifies endangered and protected species Agriculture: • Map the genome of microorganisms • Medicine: • Detect genes that are hereditary or cause diseases

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DNA SEQUENCING • advantages: • Improve health care • Helping plants and animals to be able to resist certain diseases • Helps in forensic science for identifying criminals • Disadvantage: • The provide the wrong DNA of that fragment

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