Saturday, July 27, 2019

DNA sequencing

You can find a good overview in Wikipedia.

A bit more in detail, the most used approach today (2020) by far is Illumina's. Also called SBS (sequence by synthesis), it belongs to the 2nd generation technologies (there are others, like Thermofisher's...), ironically called also NGS (next generation sequencing) because it was once the next generation :). It has its drawbacks (high investment upfront...) but it works.

From an electronics perspective, the detection method here is a high speed camera.

Moving fwd, "A world of opportunities with nanopore sequencing" gives a good overview on the latest (a bit centered on Oxford Nanopore Technologies' solution). These are called "long read", as, unlike Illumina's, it can read a long strand of DNA while Illumina's is limited to ~100bp that then you have to "glue" together with the others to get the whole thing. Nanopores have other advantages (portability, immediate results, less reagents, potentially lower cost...) and some issues, like less accurate (although they seem to be addressing this), but it is not clear they'll be able to displace Illumina. It is a bit like PoC vs centralize lab model.

From an electronics perspective, we measure very small variations on current levels (pA) although some different pore technologies may result on larger currents. If you want to get a bit more insight on the electronics for the nanopore technologies (and still get a good overview of the previous generations), check "Nanopore-CMOS Interfaces for DNA Sequencing" out.

A hardcore Nature paper "Integrated nanopore sensing platform with sub-microsecond temporal resolution" on nanopore electronics noise.

Just a last note. DNA sequencing cannot be replaced on some areas, for some tasks. Nevertheless, one does not need to sequence the whole DNA to do things like finding if an illness is coming from a given virus (see qPCR), if someone is the father of the baby, or to find out that one is the carrier of a "cancer gene"... There are more cost effective methods to do that, still based on DNA/RNA.

Books:
  1. "Molecular Cloning: a Laboratory Manual" is not something that you want to read (I haven't, that's just the index, not online) just for basic learning. Instead is mentioned on the first article as the in depth reference for the basic techniques used for DNA manipulation/preparation...
  2. Single-Channel Recording. Sakmann, B.; Neher, E. Springer; 2009
  3. Ion Channels of Excitable Membranes

Thursday, July 18, 2019

Sizes

Always wondered how big were all these things biologists talk about? :)
  1. DNA base pair (bp): ~3.4 Å (340 pm) of length along the strand. Weight: roughly 618 or 643 daltons for DNA and RNA respectively
  2. DNA chain: ~2 nm diameter, helical pitch 3.4-3.6 nm, 50 nm persistence length (stiffness) [1]
    • See Wikipedia for physical description of the helix. 
  3. Human DNA: The haploid human genome (23 chromosomes) is estimated to be about 3.2 billion bases long and to contain 20,000–25,000 distinct protein-coding genes.
    1. One genome copy weights about 3pg
    2. Each somatic cell has 6.16 pg of DNA (sperm and egg cells have 3.08 pg).
    3. The total amount of related DNA base pairs on Earth is estimated at 5.0×1037 and weighs 50 billion tonnes. In comparison, the total mass of the biosphere has been estimated to be as much as 4 TtC (trillion tons of carbon).
  4. Protein: hemoglobin: 6.5 nm
  5. Virus:
  6. Cell:
    1. (Red blood cell) 10um diameter of the "disc". Contains ~280M hemoglobin molecules.
    2. (White blood cell)
    3. Neuron
    4. Epithelial cell
    5. Endothelial cell
  7. Blood vessel:
  8. Schematic representation of the vessels of the cardiovascular system, with the inner diameter, average blood-flow speed, and Reynolds number from Berger et al. [2].
  9. Almost every cell, in almost every tissue of a vertebrate, is located within 50–100 μm of a capillary [4]. Check [5] for some introductory but nice reading on capillaries. Can we get a 3D image of the capillary network in a volume?
  10. Blood drop: 25 ul
  11. Hair:
  12. Mosquito: 3-6 mm. 5 mg. Fly at ~1-2 km/h. Wing frequency: 500 bps
  13. Hypodermic needle: there is a range of gauges:
    • 21-gauge (0.8 mm/0.5 mm outer/inner diameters) needles are most commonly used for drawing blood for testing purposes, and 16- or 17-gauge needles (~1.5 mm outer diameter) are most commonly used for blood donation.
    • The smallest hypodermic needle typically used (30 gauge) is about 320 μm in outer diameter and 160 μm in inner diameter.
    • Thinking to copy something from the mosquito? :) [3] Newer article here.
  14. Organ sizes:
    1. Brain size: about 1 liter: 10x10x10 cm^3. 10^11, i.e., 100B neurons.


A nice diagram with some of these:





References:
[2] Original: Berger SA, Goldsmith W, Lewis ER. 1996. Introduction to Bioengineering. Oxford, UK: Oxford Univ. Press. Extracted from: Microrobots for Minimally Invasive Medicine, Nelson et al.
[3] Fabrication of a micro needle for a trace blood test
[4] Molecular Biology of the Cell, Bruce Alberts et al.

Reimbursement/costs

For the US, can't tell private costs but one can search for Medicare/Medicaid reimbursements . For instance: Search physician fee schedu...