sathish fpm 39

DNA Sequencing : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 1 DNA Sequencing Sathish t Fpm 39

INTRODUCTION : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 2 INTRODUCTION “Sequencing” means finding the order of nucleotides on a piece of DNA . 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

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6/11/2009 Comp 590/Comp 790-90 Fall 2008 3 2 dimensional chromatography Understanding a particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment Environmental, agricultural and forensic applications

DNA Sequencing : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 4 DNA Sequencing Shear DNA into millions of small fragments Read 500 – 700 nucleotides at a time from the small fragments (Sanger method)

Fragment Assembly : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 5 Fragment Assembly Assembles the individual overlapping short fragments (reads) into a genomic sequence Problems: DNA read error rate of 1% to 3% Can’t separate coding and template strands DNA is full of repeats

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6/11/2009 Comp 590/Comp 790-90 Fall 2008 6 Prior to the mid-1970’s no method existed by which DNA could be directly sequenced. Maxam-Gilbert chemical cleavage method Sanger chain-termination method .

Maxam-Gilbert Sequencing : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 7 Maxam-Gilbert Sequencing Chemical cleavage In the late 1970s, A. M. Maxam and W.Gilbert devised the first method for sequencing DNA fragments containing up to ˜500 nucleotides Most of chemicals are toxic

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6/11/2009 Comp 590/Comp 790-90 Fall 2008 8 Watson (1960) Interest in genetics Brookes and Lawley (1963) Alkylation of nucleic acids Mirzabekov (1975) Methylation

REACTION : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 9 REACTION Chemical modification of the bases Modified base is removed from its sugar, pyperidine cleaves phosphodiester bonds 5’ and 3’ and base is released DISADVANTAGES Time consuming A lot of very nasty chemicals are used

CHEMICALS INVOVLED : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 10 CHEMICALS INVOVLED 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.

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Sanger Method : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 17 Sanger Method Chain termination method Single-chain Frances Crick (1962) Conversion to sequencing Brownlee and Barrell (1965) Radioactive labeling of small oligonucleotides C. Weissmann (1969) RNA copying

REQUIREMENT : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 18 REQUIREMENT Template dna Primer DNA polymerase Pool of normal nucleotides Dideoxynucleotides

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DISADVANTAGES : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 23 DISADVANTAGES very time consuming A highly experienced and skilled scientist was able to determine the sequence of only 1000-2000 nucleotides per week replaced by an automated method

RECENT INNOVATIONS IN CHAINTERMINATION SEQUENCING : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 24 RECENT INNOVATIONS IN CHAINTERMINATION SEQUENCING Thermal cycle sequencing Automated DNA sequencing Pyrosequencing Sequencing by hybridization

Thermal cycle sequencing : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 25 Thermal cycle 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.

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AUTOMATED DNA SEQUENCING : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 27 AUTOMATED DNA SEQUENCING Leroy Hood and colleagues,1986 fluorescent labels, laser induced fluorescence detection, and computerized base calling

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PYROSEQUENCING : 

6/11/2009 Comp 590/Comp 790-90 Fall 2008 30 PYROSEQUENCING A novel DNA sequencing method in which addition of a nucleotide to the end of a growing polynucleotide is detected directly by conversion of the released pyrophosphate into a flash of chemiluminescence's. not require electrophoresis enzyme sulfurylase nucleotidase enzyme

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6/11/2009 Comp 590/Comp 790-90 Fall 2008 33 Thank u