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Old 11-14-2012, 01:06 PM   #1
brachysclereid
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Default 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".
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Old 12-18-2012, 02:16 PM   #2
nhunkapiller
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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?
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