Methods of DNA Sequencing Flashcards
1
Q
how has the cost of DNA sequence changed
A
published in 2001 and has drastically dropped
prior 2007, they used Sanger sequencing
after 2007, they used massive parallel sequencing (bringing sequencing cost down drastically)
2
Q
describe the Sanger method
A
- can read up to 1000 bases
- 1 read per run
- method of sequencing is termination
- expensive but accurate
3
Q
describe Illumina method
A
- involved in parallel sequencing
- can read up to 100 bases
- 10^9-10^10 reads per run
- using real time incorporation method
- cheap and accurate
4
Q
describe pacific bioscience
A
- reads 15,000 bases
- 10^6 reads per run
- real time incorporation method
- cheap and low accuracy
5
Q
describe nanopore method
A
- reads up to 100,000bases
- 10^6 reads per run
- changes in current method
- cheap and low accuracy
6
Q
describe real time incorporation method
A
- set up a flow cell when you have the DNA fragment that you’re sequencing, involved in
- nucleotides are split, one to have a fluorescence group, and the other is to have a 3’ hydroxyl blocked
- so then the nucleotide will be incorporated into the DNA but wont allow more because the 3’ is blocked and brings a fluorescent group
(this occurs for Illumina)
7
Q
how does pacific biosciences use real time incorporation
A
- DNA polymerase will incorporate labeled dNTPs which is at the very end of the phosphate
- system recognizes the bases through the dNTP’s entering
8
Q
how are Illumina and pacific biosciences different in their approaches
A
illumina specializes in short-read raw sequence data while pacific bioscience focuses on long-read raw sequence data
