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DNA Sequencing

DNA Sequencing. Timothy G. Standish, Ph. D. OH. Phosphate. NH 2. HO. O. P. Base. N. N. O. N. N. CH 2. 5’. O. 4’. 1’. Sugar. 3’. 2’. OH. H. H. 2’3’-dideoxynucleotide monophosphate. Dideoxynucleotides. 2’-dideoxynucleotide monophosphate.

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DNA Sequencing

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  1. DNA Sequencing Timothy G. Standish, Ph. D.

  2. OH Phosphate NH2 HO O P Base N N O N N CH2 5’ O 4’ 1’ Sugar 3’ 2’ OH H H 2’3’-dideoxynucleotide monophosphate Dideoxynucleotides 2’-dideoxynucleotide monophosphate • DNA Sequencing using the Sanger method involves the use of 2’3’-dideoxynucleotide triphosphates in addition to regular 2’-deoxynucleotide triphosphates • Because 2’3’-dideoxynucleotide triphosphates lack a 3’ hydroxyl group, and DNA polymerization occurs only in the 3’ direction, once 2’3’-dideoxynucleotide triphosphates are incorporated, primer extension stops

  3. CH3 O H OH HN N OH NH2 O P HO O O CH2 N N O O N N CH2 O OH P O OH NH2 B A S E S H O N H2N H O H P HO O N O N N NH 2’3’dideoxynucleotide O SUGAR-PHOSPHATE BACKBONE N N O H2O NH2 N O O CH2 N O CH2 N O HN N O HO P H2N H O O H H P HO O O CH2 O O CH2 O O HO P OH H HO 2’3’dideoxy-nucleotidesTerminateDNAReplication

  4. DNA Sequencing • In DNA sequencing reactions all the basic components needed to replicate DNA are used • 4 reactions are set up, each containing: • DNA Polymerase • Primer • Template to be sequenced • dNTPs • A small amount of one ddNTP • ddATP, ddCTP, ddGTP, ddTTP • As incorporation of ddNTPs terminates DNA replication, a series of fragments is produced all terminating with the ddNTP that was added to each reaction

  5. Cloned fragment Primer Primer Binding sites DNA Sequencing Plasmid (or phage) with cloned DNA fragment

  6. Pol. Pol. Pol. Pol. 5’TTATCGTACCATGACTAGA 5’TTATCGTACCATGA 5’TTATCGTA 5’TTATCGTACCATGACTAGATGCGATA Let me Through! Oh come on! Not Again! Agggg…. 5’TTATCGTACCA 5’TTATCGTACCATGACTA 5’TTATCGTACCATGACTAGATGCGA The ddATP Reaction 3’AATAGCATGGTACTGATCTTACGCTAT5’ 5’TTATCG 5’TTATCGTA 5’TTATCGTACCATGA 5’TTATCGTACCATGACTAGA 5’TTATCGTACCATGACTAGATGCGATA

  7. ddATP ddCTP ddGTP ddTTP Read 5’ to 3’ from bottom to top DNA Sequencing • Products from 4 reactions each containing a small amount of a dideoxynucleotide are loaded onto a gel • Polyacrlyamide gels capable of separating fragments differing in size by only one base • High concentrations of urea are used to prevent formation of double-stranded DNA or secondary structures • Because polymerization goes 5’ to 3’ shortest fragments are 5’ compared to longer fragments which are in the 3’ direction

  8. A C G T DNA SequencingWhat A SequencingAutorad ActuallyLooks Like • To read the autorad it is important to start at the bottom and work up so that it is read in the 5’ to 3’ direction 5’CTAGAGGATCCCCGGGTACCGAGCT...3’

  9. The End

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