DNA Sequencing Notes

DNA Sequencing: A (nano)technology perspective

DNA

  • 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
  1. DNA is sheared into 200bp pieces.
  2. The sheared DNA is attached to the surface of a flow cell.
  3. The attached DNA is amplified via PCR.
  4. 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).
  • SiO2SiO_2 layer is sensitive to pH (concentration of H+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+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

  1. Nanopore Sequencing (Oxford Nanopores).
  2. 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
  1. The width of single-stranded DNA is 1.5 nm. Creating nanopores this small is challenging.
  2. DNA travels through the nanopore very quickly (less than 10μs10 \mu s), 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
  1. Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision, Nature Biotech, 2012.
  2. Three decades of nanopore sequencing, Nature Biotech, 2016.