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( a ) Initiation of strand synthesis Primer 5 ' 3 ' 111 3 ' 5 ' 3 ' 5 ' 3 ' 3 ' 3 ' 3 ' 3 ' 5 ' 3 ' 3 ' 5 ' 3 ' Template DNA TTT 5 ' www . E TTT 5 ' uu TTT 5 ' ( c ) Strand synthesis terminates when a ddNTP is added 5 ' 3 ' ddA ddA TTT 5 ' سس EF TTT 5 ' ddA TTT 5 ' ( b ) A dideoxynucleotide O- O- Ꮕ Sanger's Method 8-4 = 0 6-2 = 0 0 = 0-9 -O - P - O - P - O - P - O -O - CH , H ddA H C The ' A ' family H * O Position where the -OH of a dNTP is replaced by -H ddA ddA Base H C C I H Haemophilus influenzae Chocolate agar DNA Sequencing H. influenzae genomic DNA DNA fragments of various sizes Lane 1 : Sonicated H. influenzae DNA Lane 2 : DNA markers Purify DNA from the gel DNA fragments - 1.6-2.0 kb Sonicate Agarose gel electrophoresis 1 7 Prepare a clone library Obtain end - sequences of DNA inserts RT End - sequences Construct sequence contigs Shotgun Sequencing