Software breakthrough radically boosts the speed of nanopore DNA sequencers

Post by dnautics » Fri, Dec 18, 2020, 7:58 AM UTC

epgui wrote: post #35

lbotos wrote: post #31

Earlier quoted context omitted.

I'm not an expert, but I think the PCR page will help: https://en.wikipedia.org/wiki/Polymerase_chain_reaction basically for PCR (enzyme based) it's sort of "unzipped" down the middle and the base pairs match a specific way which is how it's duplicated. The rest I can't answer but I hope this was as fun a read as it was for me the first time. It really is insane to think about.

Biochemist here! The DNA polymerase reaction can only take place when the gene is unwound from the histone in the first place. Therefore the PCR wiki page will not provide you with the answer... The key is the "DNA purification" prep step where you digest the histones with proteases. See here: https://en.wikipedia.org/wiki/DNA_extraction

Post by epgui » Fri, Dec 18, 2020, 8:10 AM UTC

dnautics wrote: post #41

epgui wrote: post #35

Earlier quoted context omitted.

Biochemist here! The DNA polymerase reaction can only take place when the gene is unwound from the histone in the first place. Therefore the PCR wiki page will not provide you with the answer... The key is the "DNA purification" prep step where you digest the histones with proteases. See here: https://en.wikipedia.org/wiki/DNA_extraction

This is not really right. The high duplex denaturing temperature of the initial thermocycle will usually also denature histones and unwind them from the dna. That's why thermocycle procedures for raw samples have a long initial denaturation (5-10 min) and thermocycle procedures for prepurified DNA need not have such a long initial incubation at 95 C.

Post by HPsquared » Fri, Dec 18, 2020, 9:50 AM UTC

msandford wrote: post #12

Michael Schatz has been involved in a number of interesting computational biology projects. I worked in a bioinformatics lab ~10-12 years ago and I remember using his fast short read aligner to help speed things up. Back then the nanopore devices were still just rumors. Computers have Moore's law with a base or time constant of 12-24mo. Batteries seem to have the same with a base of about 10 years (slow Moore's law I…

Post by PunchTornado » Fri, Dec 18, 2020, 12:46 PM UTC

ivirshup wrote: post #23

PunchTornado wrote: post #11

Earlier quoted context omitted.

to me nanopore seq is not that promising. it has been here for a long time now and the benefits didn't convince a lot of customers to adopt it. there are 2 main use cases now in precision medicine: rare disease and cancer. for both you need high precision reads, which nanopore doesn't provide.

Who are the customers here? The waiting list for new nanopore sequencers is quite long at the moment. Our neighboring lab has been waiting on a promethION for about a year, and only just got it because their last one bit the dust putting them on top of the list.

Post by epgui » Fri, Dec 18, 2020, 7:28 PM UTC

burning_hamster wrote: post #46

dnautics wrote: post #41

Earlier quoted context omitted.

This is not really right. The high duplex denaturing temperature of the initial thermocycle will usually also denature histones and unwind them from the dna. That's why thermocycle procedures for raw samples have a long initial denaturation (5-10 min) and thermocycle procedures for prepurified DNA need not have such a long initial incubation at 95 C.

Did you guys all skip the DNA extraction practical at university? 1) Proteinases are indeed not necessary. 2) The step that removes any DNA bound proteins and in fact denatures the vast majority of proteins is the salting out. DNA is negatively charged. Proteins bind DNA by being positively charged. When you add a lot of salt, you add a lot of ions that compete for those ionic bonds. Proteins fall of the DNA, and wil…