Millions of (same) template copies are exposed to mix of ddNTP (e.g. ddATP) & dNTP (e.g. dATP). DNA poly uses ddATP or dATP to make DNA, & sequences are terminated in different points. ddNTPs terminate, dNTP continues. Electrophoresis: know nt location depending on how long fragments are. Fluorescent dyes can be used: Advantage is that A/C/T/G can be located at the same time with a laser.
Disadvantages over next gen: Need electrophoresis
Advantages over next gen: Cheaper, faster
Next Generation Sequencing
Advantages over Sanger: Can read millions of different templates at once. No need for electrophoresis
Disadvantages: Slower. More expensive
Shears DNA to small fragments. Add oligonucleotide adapters to both ends as primer annealing sites.
Platforms that read products of sequencing:
Has trenches with oligonucleotide primers attached to the bottom. The -OH ends of oligonucleotide primers are on top. Adapters of fragments anneal to primers. Fragments are copied onto oligonucleotide primers using bridge PCR.
Like Sanger in some ways (reading base incorporation).
Has fluorescent-labeled dNTPs, but instead of fully terminating sequence - dye terminators are used to pause sequencing. After color (nt) is read, flo cell is rinsed to remove fluorescent label and rxn continues.
Disadvantages: reliable for only 200 bases because different templates go out of synch
Like Sanger and Illumina (reading base incorporation).
One template in one well. DNA poly is anchored to the bottom of the well. As fluorescent dye bases are added, machine can read each different well/template. Base that fluoresces closest to bottom of well (& DNA poly) is added first! Uses data from Zero Mode Waveguides that only have one template.
Advantages: Does not lose synch like Illumina - goes on longer. Can help piece together data acquired from Illumina.
Disadvantages: Expensive. Fewer templates at once. Worst accuracy.
454 Pyrosequencing Reaction
Amplifies DNA fragments on an water bead in oil (emulsion PCR). Like Illumina, but oligonucleotide primers are on bead.
No fluorescent dye. Trial/error method is used. dNTP is added. If correct, PPi released and sulfurylase produces ATP. Luciferase uses ATP to make light. If wrong base, no light. No light > apyrase degrades dNTP.
Disadvantages: Expensive. Prone to errors when a string of same nt is present (e.g. 8 A's) due to point of saturation of light
Ion Torrent Sequencing Reaction
Similar to 454 (trial and error method). Detects correct base addition due to H+ release (from 3' OH end) using a semiconductor (like a small pH meter). Mg2+ is used to bind PPi so extra H+ concentration can be noticed. Washes the flo cell after each trial. Uses DNA poly sensitive to pH change.