Multi-sequence capture system
Systems, devices, and methods for capturing single source-specific biological material from a multi-source aggregate of biological material are disclosed and discussed. A capture system is generated using reversible chain-blocking to make capture substrates having substrate-linked populations of capture molecules specific for molecules of interest. Incubating such capture substrates in the presence of only a single source of biological material facilitates the association of molecules of interest from the same source. Capture substrate-specific barcode sequences coupled to the capture molecules allow multisource aggregate processing and subsequent grouping to retain the source-specific information following downstream processing.
1. A method of making a multi-probe capture system, comprising:
synthesizing a plurality of barcode sequences extending from a capture substrate, where the plurality of barcode sequences includes a specific identifier to the capture substrate;
protecting a first portion of the plurality of barcode sequences with a first protecting group; and
protecting a second portion of the plurality of barcode sequences with a second protecting group, where the second protecting group is stable under conditions for deprotection of the first protecting group, stable under conditions for oligonucleotide synthesis, and is capable of deprotection under conditions compatible with oligonucleotides.
2. The method of claim 1 , further comprising:
deprotecting the first portion of the plurality of barcode sequences by removing the first protecting group;
synthesizing a plurality of first oligonucleotide primers extending from the first portion of the plurality of barcode sequences;
deprotecting the second portion of the plurality of barcode sequences by removing the second protecting group; and
synthesizing a plurality of second oligonucleotide primers extending from the second portion of the plurality of barcode sequences.
3. The method of claim 2 , further comprising capping terminal ends of the plurality of first oligonucleotide primers prior to deprotecting the second portion of the plurality of barcode sequences.
4. The method of claim 2 , wherein the plurality of first oligonucleotide primers and the plurality of second oligonucleotide primers are sequenced 5′ to 3′.
5. The method of claim 2 , wherein protecting the first and second portions of the plurality of barcode sequences cormprises:
reacting the plurality of barcode sequences with a mixture including:
first nucleoside building blocks including the first protecting group; and
second nucleoside building blocks including the second protecting group, wherein the first portion of the plurality of barcode sequences is a result of reaction with the first nucleoside building blocks and the second portion of the plurality of barcode sequences is a result of reaction with the second nucleoside building blocks.
6. The method of claim 5 , wherein the first and second nucleoside building blocks are nucleoside phosphoramidites.
7. The method of claim 6 , wherein the first protecting group is dimethoxytrityl (DMT).
8. The method of claim 6 , wherein the second protecting group is levulinyl (Lev).
9. The method of claim 5 , wherein the mixture comprises the first nucleoside building blocks and the second nucleoside building blocks in a ratio of from 1:100 to 100:1.
10. The method of claim 5 , wherein the mixture comprises the first nucleoside building blocks and the second nucleoside building blocks in a ratio of from 1:10 to 10:1.
11. The method of claim 5 , wherein the mixture comprises the first nucleoside building blocks and the second nucleoside building blocks in a ratio of from 1:4 to 4:1.
12. The method of claim 2 , wherein the plurality of first oligonucleotide primers and the plurality of second oligonucleotide primers are configured to capture a specific first mRNA and a specific second mRNA having a functional relationship with one another.
13. The method of claim 2 , wherein one of the plurality of first oligonucleotide primers and the plurality of second oligonucleotide primers is configured to capture a specific mRNA, and the other of the plurality of first oligonucleotide primers and the plurality of second oligonucleotide primers is configured to capture nonspecific mRNA.