Isolate genomic DNA and break Into fragments. Deposit the beads Into a picotiter plate. Only one bead can fit Into each well. Fragment of genomic DNA Covalently attach oligonucleotide adaptors to the 5' and 3' ends of the DNA. Adaptors Denature the DNA Into single strands and attach to beads vla the red adaptors. Note: Only one DNA strand is attached to a bead. Add sequencing reagents: Primers, DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine-5'- phosphosulfate, and luciferin. Sequentially flow solutions contalning À, T, G, or CInto the wells. In the example below, T has been added to the wells. PP, (pyrophosphate) Is released when Tis Incorporated Into the growing strand. Thymine nucleotides -DNA strand (shown In black) Is complementary to adaptor shown in red. * Adaptor Bead Emulsify the beads so there is only one bead per droplet. The droplets also contain PCR reagents that amplify the DNA. Primer (complementary to the adaptor shown in green) PP. + Adenosine-5'- phosphosulfate ATP sulfurylase ATP + luciferin Luciferase Light Light is detected by a camera In the sequencing machine. Unincorporated nucleotides and ATP are degraded Бy aругase. FIGURE 23.14 Pyrosequencing, an example of a newer highthroughput sequencing technology.

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