ultra long fragments capturing

Thuur · 11-04-2015, 11:20 PM

Is it possible to capture ultra long fragments, for example >200kb. For this I need to extract High molecular weight DNA from Human blood. But then I want to capture these long fragments for example chromosome 6 short arm or a specific 200k fragment, Hopefully there is a technique without PCR.

Anybody has experience with this?

tangjj · 06-19-2019, 01:34 AM

Hi,have you successfully captured ultra long fragments? Recently I want to capture a 200kb fragment from high molecular weight DNA. I wonder if you have any successful experience.

pmiguel · 06-21-2019, 07:44 AM

Old school method involved embedding cells in agarose and lysing/prepping the DNA in situ. Initially these were just little slabs of agarose. But later moved more to higher surface area collects of agarose "beads".
Anyway, then they get run on a pulsed field gel for size selection.
Anyone using agarose for their large fragment preps these days?

--
Phillip

JTH-Genome · 06-27-2019, 11:05 AM

I agree with Phillip. Use the agarose plugs for the lysis. It might be old school, but it is making a comeback! You can use the BioNano protocol for high MW DNA. It's one of the best protocols around.

https://bionanogenomics.com/support-...isolation-kit/
https://bionanogenomics.com/products/bionano-prep-kits/

Or you can do your own homebrew version with agarose plugs.

ychang · 05-29-2020, 08:44 PM

https://sagescience.com/wp-content/u...ER-BO-ZHOU.pdf
I think this is the best method for your purpose.

ajthomas · 06-01-2020, 09:41 AM

There are a couple of options. The first is the SageHLS (suggested by ychang, above) https://sagescience.com/products/sagehls/.
The second is CAS9-targeted sequencing on the MinION. If you don't have a SageHLS, or know someone nearby with one, this might be the better option, as the SageHLS requires fresh cells to start, and shipping them to someone to do the capture might be a challenge. If you can access a SageHLS, that's probably a better way to try first.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Cufflinks 2.0.2 segmentation fault on long fragments	plger	RNA Sequencing	3	01-06-2015 03:41 PM
Capturing >60nt fragments	smarajit	Sample Prep / Library Generation	5	02-15-2013 09:50 AM
Comparison of sequence capturing	polyhedron	General	4	06-20-2012 07:31 AM
PubMed: Solution hybrid selection with ultra-long oligonucleotides for massively para	Newsbot!	Literature Watch	0	02-03-2009 06:00 AM

11-04-2015, 11:20 PM	#1
Thuur Junior Member Location: NL Join Date: Nov 2015 Posts: 1	ultra long fragments capturing Is it possible to capture ultra long fragments, for example >200kb. For this I need to extract High molecular weight DNA from Human blood. But then I want to capture these long fragments for example chromosome 6 short arm or a specific 200k fragment, Hopefully there is a technique without PCR. Anybody has experience with this?

06-27-2019, 11:05 AM	#4
JTH-Genome Junior Member Location: USA, NC Join Date: Apr 2019 Posts: 6	BioNano I agree with Phillip. Use the agarose plugs for the lysis. It might be old school, but it is making a comeback! You can use the BioNano protocol for high MW DNA. It's one of the best protocols around. https://bionanogenomics.com/support-...isolation-kit/ https://bionanogenomics.com/products/bionano-prep-kits/ Or you can do your own homebrew version with agarose plugs.

06-19-2019, 01:34 AM	#2
tangjj Junior Member Location: china Join Date: Aug 2017 Posts: 1	Hi,have you successfully captured ultra long fragments? Recently I want to capture a 200kb fragment from high molecular weight DNA. I wonder if you have any successful experience.

06-21-2019, 07:44 AM	#3
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Old school method involved embedding cells in agarose and lysing/prepping the DNA in situ. Initially these were just little slabs of agarose. But later moved more to higher surface area collects of agarose "beads". Anyway, then they get run on a pulsed field gel for size selection. Anyone using agarose for their large fragment preps these days? -- Phillip

05-29-2020, 08:44 PM	#5
ychang Member Location: philly Join Date: Oct 2010 Posts: 17	https://sagescience.com/wp-content/u...ER-BO-ZHOU.pdf I think this is the best method for your purpose.

06-01-2020, 09:41 AM	#6
ajthomas Senior Member Location: Utah Join Date: Mar 2010 Posts: 166	There are a couple of options. The first is the SageHLS (suggested by ychang, above) https://sagescience.com/products/sagehls/. The second is CAS9-targeted sequencing on the MinION. If you don't have a SageHLS, or know someone nearby with one, this might be the better option, as the SageHLS requires fresh cells to start, and shipping them to someone to do the capture might be a challenge. If you can access a SageHLS, that's probably a better way to try first.