TruSeq Custom Amplicon - Extension-Ligation Step

brachysclereid · 11-14-2012, 01:06 PM

Does anyone have a suggestion for an enzyme cocktail to perform the extension-ligation step similar to what is shown in the Illumina TruSeq Custom Amplicon workflow step 2? Note that probe 1 and probe 2 are in the same orientation. This is not a PCR step. It is more of a gap filling step.

Illumina will not tell me what is in the secret sauce aside from it contains a "ligase and a polymerase in proprietary formulation".

nhunkapiller · 12-18-2012, 02:16 PM

I'm also curious about this. Whatever they use, it's likely very important that the polymerase has very low strand displacement activity and NO 5'=>3' exonuclease activity. I suspect the ligase is not thermostabile, given that the on column ligation takes place at 37C (taq ligase is active at 45-65), although the NaOH they use during elution surely inactivates both the polymerase and ligase.

My curiosity is whether these steps can be performed in the absence of any pre or post column purification. A solution that hybridizes, extends, and ligates in a linear but cycle-able way would be ideal. If enough copies of the desired ligated product can be generated, it's possible that dilution could be used to carry the product into direct barcoding PCR, rather than purification. Anyone tried anything like this?

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
custom amplicon sequencing	spacesonics	Illumina/Solexa	0	08-13-2012 12:54 PM
TruSeq Custom Amplicon-diluting oligos?	debbiehughes	Sample Prep / Library Generation	0	05-03-2012 08:42 AM
TruSeq ligation vs old ligation?	jlove	Illumina/Solexa	1	05-01-2012 05:11 AM
AmpliSeq vs TruSeq custom amplicon	epistatic	Ion Torrent	2	02-16-2012 03:18 AM
Is the PCR purification step between ligation and size selection necessary?	skblazer	Sample Prep / Library Generation	7	10-28-2011 04:44 AM

11-14-2012, 01:06 PM	#1
brachysclereid Member Location: California Join Date: Feb 2011 Posts: 32	TruSeq Custom Amplicon - Extension-Ligation Step Does anyone have a suggestion for an enzyme cocktail to perform the extension-ligation step similar to what is shown in the Illumina TruSeq Custom Amplicon workflow step 2? Note that probe 1 and probe 2 are in the same orientation. This is not a PCR step. It is more of a gap filling step. Illumina will not tell me what is in the secret sauce aside from it contains a "ligase and a polymerase in proprietary formulation".

12-18-2012, 02:16 PM	#2
nhunkapiller Junior Member Location: Bay Area Join Date: Dec 2012 Posts: 6	I'm also curious about this. Whatever they use, it's likely very important that the polymerase has very low strand displacement activity and NO 5'=>3' exonuclease activity. I suspect the ligase is not thermostabile, given that the on column ligation takes place at 37C (taq ligase is active at 45-65), although the NaOH they use during elution surely inactivates both the polymerase and ligase. My curiosity is whether these steps can be performed in the absence of any pre or post column purification. A solution that hybridizes, extends, and ligates in a linear but cycle-able way would be ideal. If enough copies of the desired ligated product can be generated, it's possible that dilution could be used to carry the product into direct barcoding PCR, rather than purification. Anyone tried anything like this?