DNA Sequencing Flashcards
Describe the Sanger sequencing method, including capillary Sanger sequencing
It involves the use of ddNTPs, which lack the 3’ -OH needed to form phosphodiester bond with the next dNTP. The templates are denatured in high temp, and primers are added, with dNTPs and ddNTPs in the solution, separated into 4 solutions with a different type of ddNTP in each. The results are visualised using gel electrophoresis, and the bases sequenced. Next-gen Sanger method involves using fluorescent-labelled ddNTP, therefore can be run in tube. Capillary electrophoresis is used to run it, and the laser used to pick up the different fluorescent, represented on the chromatogram.
Describe the Maxim and Gilbert Sequencing method
1st proposed sequencing method.
Involves the use of chemistry to sequence DNA.
Dimethyl sulphate reacts with purines (A&G) and hydrazine reacts with pyrimidines (C&T), by cleaving their glycosidic bonds. Piperdine used to catalyse phosphodiester bond cleavage. Formic acid used to distinguish between A&G and 2M NaCl used for C&T
Explain the Phred Quality score
It represents the probability of an incorrect base call (logarithmically linked to a probability of an incorrect base call), and is a measure of base call accuracy. Ranges from 0-93, but rarely > 50. E.g. 10 = 1/10 errors, accuracy = 90%; 20 = 1/100, accuracy = 99%; 30 = 1/1000, accuracy = 99.9% etc
Outline the disadvantages of Sanger sequencing
Not high throughput, only 1 template per reaction
High cost per base
How do you design the initial primer for the unknown DNA sequence?
Digest using RE, clone into plasmid. since plasmid sequence prior to insert is known, can design primer and amplify using PCR.
How are long DNA sequences >1000bp sequence?
Primer walking or Shotgun sequencing.
Describe Contiguous/shotgun sequencing
DNA is randomly fragmented (digested by RE), and cloned into plasmid, thus sequenced. The sequence is removed and assembled into contiguous sequences (contigs).
How large is the human genome?
3.2 billion bp
Outline the principles of NGS
Next-Gen sequencing involves the following steps:
- DNA fragmentation
- Ligation of Adaptors (on fragment ends)
- Clonal amplification on solid surface (beads or glass- flow cell)
- Usage of primers (complimentary to adaptor, DNA polymerase, dNTPs- fluorescently labelled)
- Sequencing by synthesis
Explain ion-torrent sequencing
Uses semi-conducting chip, containing millions of wells
Same DNA fragment covered on a bead, and is amplified across the bead with 1 bead per well. There are therefore millions of wells with millions of different DNA fragments. The technique works by flooding the well every 15 secs with a different dNTP. If it binds, it releases a H ion, and therefore changes the pH of the well, measured by a pH meter at the bottom, which converts it to a voltage. The different voltage changes indicate a specific dNTP. Therefore different bases are called, and the DNA is sequenced.
Describe 454 DNA sequencing
DNA is fragmented and mixed with capture beads specific to each adaptor (ligased onto DNA fragment). PCR reagents and emulsion oil is added into the tube, where the DNA fragments are amplified in each water droplet. The beads are then loaded onto a PicoTiter plate, with Sulfurylase and Luciferase added as enzyme beads. Sequencing-by-synthesis is achieved when the luciferase oxidises the luciferin when a dNTP attaches, producing fluorescence
Outline Primer Walking
Primer walking involves the consecutive design of new primers to walk or extend into new sequences
