Nanopores for sequencing DNA - question on beads?

[rwooduk]

Hi, maybe someone here could help explain this, I understand the concept of a nanopore; a graphene sheet with a hole burned in it, force DNA through it and measure the conductance to determine the base. But I'm unclear how micron sized beads help the process. Please find attached a slide from my lecture notes, any explanation of how the beads help sequencing, or what they are doing to the DNA would be really appreciated. The notes say the optical tweezers (that keep the bead in place) can manupulate the DNA, but why is there 3 strands of DNA attached to the bead? what is happening?

thanks for any help

 

[Ygggdrasil]

In general, nanopore seqeuencing does not require the DNA to be attached to a bead. One of the problems with nanopore sequencing, however, is that the DNA often goes through the pore too fast to get enough data to accurately identify each base. While there have been many attempts to solve this problem, the paper you cite attaches the DNA to a bead in order to use optical tweezers to measure the force pulling the DNA through the bead as well as slow the rate at which the DNA moves through the pore.

There are multiple DNAs attached to the bead because it's difficult to make beads with only one DNA molecule attached (presumably the authors did some controls to show that the nanopore only has one molecule of DNA going through at a time).

 

[rwooduk]

In general, nanopore seqeuencing does not require the DNA to be attached to a bead. One of the problems with nanopore sequencing, however, is that the DNA often goes through the pore too fast to get enough data to accurately identify each base. While there have been many attempts to solve this problem, the paper you cite attaches the DNA to a bead in order to use optical tweezers to measure the force pulling the DNA through the bead as well as slow the rate at which the DNA moves through the pore.

There are multiple DNAs attached to the bead because it's difficult to make beads with only one DNA molecule attached (presumably the authors did some controls to show that the nanopore only has one molecule of DNA going through at a time).

Many thanks, that explain why the beads are used. I'm not sure on your second point, does the DNA go through the nanopore, attach itself to the bead and then get dragged through the nanopore when the optical tweezers are adjusted in such a way to create a gradient force on the bead, i.e. cause the bead to move dragging the DNA through the nanopore? and are you saying that the change in pulling force is measured and will be different for each base? so is the voltage across the base as it passes through the nanopore not measured in this instance?

thanks again

 

[Ygggdrasil]

From the methods section of the paper you cite: (http://www.nature.com/nphys/journal/v2/n7/full/nphys344.html)

Beads with DNA are flushed into the cell, trapped with the optical tweezers setup, and brought close to the nanopore. When initially trapping the DNA-coated bead, the voltage across the nanopore is applied such that no DNA will enter the nanopore. After trapping one bead, 5

l of buffer without beads is flushed through the cell, which has a volume of 1

l.

You can probably find the answer to your other questions by reading through the paper, but without having read it carefully myself, I would not expect the pulling force to vary for each base.

 

[rwooduk]

From the methods section of the paper you cite: (http://www.nature.com/nphys/journal/v2/n7/full/nphys344.html)

You can probably find the answer to your other questions by reading through the paper, but without having read it carefully myself, I would not expect the pulling force to vary for each base.

excellent, thanks very much for your replies!