Comprehensive Notes on DNA Sequencing

DNA Sequencing: A Nanotechnology Perspective

DNA Molecule

  • Carries the information for growth, development, functioning, and reproduction of all known living organisms (and viruses).

DNA Composition

  • Molecule that carries the information for the growth, development, functioning, and reproduction of all known living organisms.
  • Composed of 4 units called nucleotides (or bases): A, C, G, T.
  • Human Genome contains 3 billion bases (SARS-COV-2 about 30,000).

DNA Structure

  • The structure of DNA is a double helix.
  • A binds to T, and C binds to G.

DNA Sequence Example

  • Single-stranded DNA sequence example: G A A C T T A A T T A A
  • Double-stranded version:
    GAACTTAATTAA
    CTTGAATTAATT

Polymerase Chain Reaction (PCR)

  • Warming DNA to approximately 95°C separates the two strands.
    • GAACTTAATTAA
    • CTTGAATTAATT
  • Add a short, specific bit of complementary DNA at either end (a primer).
    • GAACTTAAGTAA
    • CTTAA CTTGAATTCATT
  • Cool it down, and the enzyme polymerase fills in the rest, resulting in two identical, “photocopied” versions of the original DNA.
    • GAACTTAAGTAA
    • CTTGAATTCATT
    • GAACTTAAGTAA
    • CTTGAATTCATT
  • Repeat the cycle a few times to create billions of copies, making the specific DNA bit easy to detect.
  • PCR COVID testing relies on 3 sets of primers targeting 3 different regions of the SARS-COV-2 genome.
  • Further learning about PCR is available in the Labster lab.

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 applications.
  • Environmental applications.
  • Food applications.
  • Bioterrorism applications.

Cost per Human Genome

  • The cost of sequencing a human genome has drastically decreased over the years.
  • In the early 2000s, it cost approximately 100,000,000100,000,000. By 2021, the cost has fallen significantly to around 10001000.
  • This decrease is due to technological advancements and is represented graphically, showing a trend similar to Moore's Law.

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 is performed by synthesis using fluorescently labeled nucleotides.

DNA Sequencing Enabled by Electronics

  • Industrial capabilities.
  • Electronics is highly parallel.
  • Low-cost.
  • Small footprint.
  • Low power consumption.
  • No need for optics/lasers.

MOSFET

  • Depicts a diagram of a MOSFET (Metal-Oxide-Semiconductor Field-Effect Transistor) with labeled components such as VGate, Source, Drain, SiO2, n+, and p-Si.
  • Drain Characteristics: A graph illustrating the relationship between Drain Current (ID) and Drain-Source Voltage (VDS) at various gate voltages (VGS).

MOSFET vs ISFET

  • MOSFET (Dry) vs DNA sequencing (Wet).
  • ISFET (ion-sensitive FET)

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 and DNA Sequencing

  • ISFET is a great pH sensor, but how do we sequence DNA?

ISFET Sequencing

  • When DNA polymerase fills in a new nucleotide, a H+ ion is released.

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).

Third Generation Sequencing Features

  • Longer reads.
  • Portable (Oxford Nanopore).
  • Speed (Oxford Nanopore).

Nanopore Sequencing

  • Illustrates the process of nanopore sequencing.

Challenges of Nanopore Sequencing

  1. Width of single-stranded DNA is 1.5 nm. How do you make nanopores this small?
  2. DNA travels through the nanopore really fast (less than 10μs10 \mu s) and causes a modulation in the current of few tens of pA. How do you read single bases?

Nanopores

  • MspA.
  • α-hemolysin.

Slowing Down DNA

  • A motor protein sits on top of the nanopore and ratchets DNA down the nanopore with a controlled speed.
  • Nanopore Motor Protein (like DNA polymerase).