Molecule that carries the information for growth, development, functioning, and reproduction of living organisms and viruses.
Composed of 4 units called nucleotides (or bases): A, C, G, T.
Human Genome contains 3 billion bases; SARS-COV-2 contains about 30,000 bases.
The structure of DNA is a double helix.
A binds to T, and C binds to G.
DNA Example
Single-stranded DNA sequence example: G A A C T T A A T T A A
The double-stranded version is:
GAACTTAATTAA
CTTGAATTAATT
Polymerase Chain Reaction (PCR)
When heated to around 95°C, the two DNA strands separate.
A short, specific, complementary DNA (primer) is added to either end:
GAACTTAAGTAA
CTTAAGTAA CTTGAATTCATT
Cooling it down allows the enzyme polymerase to fill in the rest, creating two identical copies of the original DNA.
GAACTTAAGTAA
CTTGAATTCATT
GAACTTAAGTAA
CTTGAATTCATT
Repeating the cycle multiple times results in billions of copies, making the specific DNA easily detectable.
PCR COVID testing uses 3 sets of primers targeting 3 different regions of the SARS-COV-2 genome.
Further learning is available in the Labster lab on the module Minerva page, UNIT 1.1.
Why DNA Sequencing?
Comparative Genomics
Sequencing of many animal species.
Structure and function of the human genome.
Genome evolution.
Human Genetic Variation
Genomic contribution to disease.
Agriculturally important species.
Microbial Communities
Medical
Environmental
Food
Bioterrorism
Cost per Human Genome
Decreasing cost over time:
The cost has decreased significantly from 2001 to 2021
This information is available at genome.gov/sequencingcosts.
Next-Generation Sequencing
Massively Parallel
Higher throughput.
Lower cost.
Faster.
More accurate.
Shorter read lengths.
Sequencing by Synthesis
DNA is sheared into 200bp pieces.
The sheared DNA is attached to the surface of a flow cell.
The attached DNA is amplified via PCR.
Sequencing by synthesis involves using fluorescently labeled nucleotides.
Further learning is available in the Labster lab on the module Minerva page.
DNA sequencing enabled by electronics
Industrial capabilities of electronics allow for:
Highly parallel processing.
Low cost.
Small footprint.
Low power consumption.
Elimination of the need for optics/lasers.
MOSFET
There is a diagram of a MOSFET
MOSFET vs ISFET
ISFET (ion-sensitive FET).
SiO2 layer is sensitive to pH (concentration of H+ in solution).
The pH of the solution affects the source-drain current.
ISFET is a great pH sensor.
ISFET sequencing
When DNA polymerase fills in a new nucleotide, an H+ ion is released.
Further learning is available in the Ion Torrent video on Minerva under Unit 1.1.
ISFET Reading
An integrated semiconductor device enabling non-optical genome sequencing.
Piet Bergveld - 40 years of ISFET technology: From neuronal sensing to DNA sequencing, Electronics Letter, 2011.
Third Generation Sequencing
Nanopore Sequencing (Oxford Nanopores).
SMRT sequencing (Pacific Biosciences) - not discussed.
Third Generation Sequencing Advantages
Longer reads.
Portable (Oxford Nanopore).
Speed (Oxford Nanopore).
Nanopore Sequencing
A diagram is provided with labels such as Ag/AgCl electrodes, Cis, Trans, PMMA, Electrolyte, DNA, and Nanopore.
Challenges
The width of single-stranded DNA is 1.5 nm. Creating nanopores this small is challenging.
DNA travels through the nanopore very quickly (less than 10μs), causing a modulation in the current of few tens of pA. Reading single bases is challenging.
Nanopores
MspA
α-hemolysin
How to slow down DNA
A motor protein sits on top of the nanopore and ratchets DNA down the nanopore with controlled speed (like DNA polymerase).
Nanopore Reading
Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision, Nature Biotech, 2012.
Three decades of nanopore sequencing, Nature Biotech, 2016.