Daniel:Notebook/ComboLock/2016-12-9: Difference between revisions

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[[Image:C_Probes-BadCircularization.png|center|400x200px|frame|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.]]
[[Image:C_Probes-BadCircularization.png|center|400x200px|frame|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.]]


Instead, we now introduce the '''mirror''' C probes. These C probes have their 3' and 5' ends reversed such that the 3' end is the genome matching region and the 5' end is the adapter region.
This actually makes quite a bit of sense since the probability of only the padlock binding is much higher than the probability of both the latch and padlock binding.
 
To address this problem, we now introduce the '''mirror''' C probes. These C probes have their 3' and 5' ends reversed such that the 3' end is the genome matching region and the 5' end is the adapter region.
 
[[Image:C_Probes-MirrorHybrid.png|center|400x200px|frame|Mirror C probes. C probe in red, mirror C probe in blue, and template in black. Vertical line indicates 5' end, arrow indicates 3' end.]]
 
Notice that because of the new orientation a polymerase that has no latch to work off will fall off the second C probe, rendering it impossible to circularize (hopefully).
 
[[Image:C_Probes-Mirror-Polymerization.png|center|400x200px|frame|Mirror C probes polymerization. C probe in red, mirror C probe in blue, padlock in green, polymerization product in magenta, and template in black. Vertical line indicates 5' end, arrow indicates 3' end.]]
 
To this end I have ordered two control mirror C probes to test (for the positive control oligo). I have also had a second idea based off the Landegren (Weibrecht) paper. The idea is to add a ligation event that will connect together the two C probes (via the latch). This will provide a stable template for the padlock probe that becomes latch independent. First, we look at just the latch binding.
 
[[Image:Latch_Binding.png|center|400x200px|frame|Mirror C probes polymerization. C probe in red, mirror C probe in blue, latch in dashed green, and template in black. Vertical line indicates 5' end, arrow indicates 3' end.]]
 
Now I introduce the "Lock oligo", which is reverse complement to the latch oligo. Rather than incubating the latch and padlock at the same time, we incubate the lock and latch at the same time which ligate the C probes together. It then no longer matters if the latch remains bound to the adapter regions, the padlock has a contiguous template to work off.
 


[[Image:C_Probes-MirrorHybrid.pngcenter|400x200px|frame|Mirror C probes. C probe in red, mirror C probe in blue, and template in black. Vertical line indicates 5' end, arrow indicates 3' end.]]


==Protocol==
==Protocol==

Revision as of 18:59, 9 December 2016

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.

File:C Probes-Original.png
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.

File:C Probes-BadCircularization.png
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.

This actually makes quite a bit of sense since the probability of only the padlock binding is much higher than the probability of both the latch and padlock binding.

To address this problem, we now introduce the mirror C probes. These C probes have their 3' and 5' ends reversed such that the 3' end is the genome matching region and the 5' end is the adapter region.

File:C Probes-MirrorHybrid.png
Mirror C probes. C probe in red, mirror C probe in blue, and template in black. Vertical line indicates 5' end, arrow indicates 3' end.

Notice that because of the new orientation a polymerase that has no latch to work off will fall off the second C probe, rendering it impossible to circularize (hopefully).

File:C Probes-Mirror-Polymerization.png
Mirror C probes polymerization. C probe in red, mirror C probe in blue, padlock in green, polymerization product in magenta, and template in black. Vertical line indicates 5' end, arrow indicates 3' end.

To this end I have ordered two control mirror C probes to test (for the positive control oligo). I have also had a second idea based off the Landegren (Weibrecht) paper. The idea is to add a ligation event that will connect together the two C probes (via the latch). This will provide a stable template for the padlock probe that becomes latch independent. First, we look at just the latch binding.

File:Latch Binding.png
Mirror C probes polymerization. C probe in red, mirror C probe in blue, latch in dashed green, and template in black. Vertical line indicates 5' end, arrow indicates 3' end.

Now I introduce the "Lock oligo", which is reverse complement to the latch oligo. Rather than incubating the latch and padlock at the same time, we incubate the lock and latch at the same time which ligate the C probes together. It then no longer matters if the latch remains bound to the adapter regions, the padlock has a contiguous template to work off.


Protocol