DNA sequencing technologies
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The generation of sequencing that is firsy done purely on relatively pure RNA species
First generation sequencing
conducted the first whole nucleic acid sequence (alanin tRNA from Saccharomyces cerevisiae) using ribonuclease treatments
Robert Holley and Colleagues
developed a related technique based on the detection of radiolabelled partial-digestion fragments after two-dimensional fractionation
Fred Sanger and Colleagues
first complete protein-coding gene sequence (coat protein of bacteriophage MS2)
Walter Fiers et. al
the persons who used DNA polymerase to fill the ends in with radioactive nucleotides, supplying each nucleotide one at a time and measuring incorporation to deduce sequence
Ray Wu and Dale Kaiser
in which only a single type of nucleotide is present, thus all extensions will end with that base
“plus” reaction
in which three are used, which produces sequences up to the position before the next missing nucleotide
‘minus reaction’
What is the first genome that is used by Sanger et al to sequence its DNA
bacteriophage ϕX174
Difference of Maxam and Gilbert Technique from Coulson and Sanger technique
GM use radiolabelled DNA, while CS use DNA polymerase
What characterized 1st Generation Sequencing?
Uses radioactivity or fluorescence, slab gels, and separates molecules by length. It requires amplification of many identical molecules. (e.g., Sanger, Maxam-Gilbert).
What characterized 2nd Generation Sequencing?
High throughput, fluorescence-based, capillary-based or massive parallel arrays, and automation. Often uses “Sequence-by-Synthesis” or “Sequence-by-Ligation.” (e.g., Illumina, Pyrosequencing)
What is the main limitation of Sanger sequencing compared to NGS?
It has low throughput and is expensive per base, though it is very accurate for short reads (up to ~900bp).
What is the mechanism of Sanger Sequencing?
Chain-termination. It uses DNA polymerase to synthesize a new strand until a specific nucleotide stops the process.
What specific molecule is used to stop the DNA chain in Sanger sequencing?
ddNTPs (dideoxynucleotides).
Why do ddNTPs terminate the DNA chain?
They lack the 3’ hydroxyl (OH) group required to form a phosphodiester bond with the next nucleotide.
What improvements were made to the original Sanger method?
• Replacement of radioactive labels with fluorescence. • Capillary electrophoresis instead of slab gels. • Shotgun sequencing and automation.
What is Pyrosequencing?
A “sequence-by-synthesis” method that measures light produced by the release of pyrophosphate (PPi).
Which two enzymes are crucial for Pyrosequencing detection?
ATP Sulfurylase (converts PPi to ATP) and Luciferase (uses ATP to produce light).
What is a major disadvantage/error type in Pyrosequencing?
Homopolymers (e.g., AAAAA). It is difficult to determine the exact number of bases based solely on light intensity.
How does Ion Torrent sequencing work?
It measures pH change caused by the release of an H+ ion whenever a nucleotide is incorporated. (No optical signals/light used).
How does Illumina sequencing work (General mechanism)?
Reversible terminator sequencing. Fluorescently labeled nucleotides are added, imaged, and then the terminator is removed to allow the next base to bind. Uses Bridge Amplification.
What is SOLiD sequencing technology based on?
Sequencing by Ligation (using ligase instead of polymerase).
What does SMRT stand for (PacBio)?
Single Molecule Real-Time sequencing.
How does PacBio SMRT sequencing work?
Uses a Zero-Mode Waveguide (ZMW). A polymerase is fixed at the bottom, and fluorescent bursts are detected in real-time as bases are added