Daniel:Notebook/ComboLock/2016-12-9

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Revision as of 18:31, 9 December 2016 by >Djacobse (→‎Lock Oligo Protocol)
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Lock Oligo Protocol

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This protocol covers a new method using ideas taken from Weibrecht et al New Biotechnology 2012 (Landegren lab). The main idea I see is that they almost never use polymerase, but prefer to instead use ligation events as the predominant form. Based on the results from the extended latch experiment, the RCA-based cell test, and the sequencing from the first circularization test, it seems the probes do not circularize correctly. Note the original orientation of the C probes with the 5' end being the genome matching region and the 3' end being the latch/padlock adapter.

C probes in the orientation of their original design. Template (mRNA/control oligo) is in black, C probes in red. Vertical line indicates 5' end, arrow indicates 3' end.

When subjected to polymerization, if the latch is not present this yields a product that follows the backbone of the second C probe. See image below for clarification.

C probes gap-filled when padlock binds (but not latch). C probes in red, padlock probe in green and polymerase-added bases in magenta. Vertical line indicates 5' end, arrow indicates 3' end.

Protocol