Method of oligonucleotide analysis
Described herein, among other things, is a method of estimating efficiency of an oligonucleotide synthesis reaction. In some embodiments, the method comprises subjecting the products of one or more oligonucleotide synthesis reactions to LC-MS to produce a series of mass spectra, analyzing the mass spectra, and estimating the overall efficiency of an oligonucleotide synthesis reaction and/or the efficiency of addition of one or more of G, A, T or C individually in an oligonucleotide synthesis reaction.
1. A method of estimating efficiency of an oligonucleotide synthesis reaction, comprising:
(a) subjecting the products of one or more oligonucleotide synthesis reactions to liquid chromatography-mass spectrometry (LC-MS) to produce a series of mass spectra;
(b) plotting an extracted ion chromatogram for m/z values in the mass spectra of (a) that correspond to: i. the full length oligonucleotide; and ii. individual n−1 reaction products corresponding to a missing G, A, T, or C;
(c) separately integrating peaks corresponding to the full length oligonucleotide and the individual n−1 reaction products in the extracted ion chromatogram, thereby providing the amount of the full length oligonucleotide and ii. the amount of the n−1 reaction products, either collectively or individually, in the one or more oligonucleotide synthesis reactions; and
(d) estimating, using the results of (c): i. the efficiency of an oligonucleotide synthesis reaction and/or ii. the efficiency of addition of one or more of G, A, T or C individually in an oligonucleotide synthesis reaction.
2. The method of claim 1 , wherein the method is done on a single oligonucleotide synthesis reaction, wherein the full length oligonucleotide of the reaction comprises G, A, T and C.
3. The method of claim 1 , wherein the method is done on multiple oligonucleotide synthesis reactions, wherein: (i) the full length oligonucleotide of each reaction lacks a G, A, T or C, or any combination thereof, and (ii) collectively, the full length oligonucleotides comprise G, A, T and C.
4. The method of claim 1 , further comprising calculating the overall efficiency of an oligonucleotide synthesis reaction.
5. The method of claim 1 , further comprising calculating the overall cycle yield of an oligonucleotide synthesis reaction.
6. The method of claim 1 , further comprising calculating the singe base deletion rate of an oligonucleotide synthesis reaction.
7. The method of claim 1 , further comprising calculating the individual cycle yield for one or more G, A, T and C in an oligonucleotide synthesis reaction.
8. The method of claim 1 , further comprising calculating the individual base deletion rate for one or more of G, A, T and C in an oligonucleotide synthesis reaction.
9. The method of claim 1 , wherein the one or more oligonucleotide synthesis reactions are capping-free reactions.
10. The method of claim 1 , wherein the one or more oligonucleotide synthesis reactions comprise capping.
11. The method of claim 1 , wherein the one or more oligonucleotides are 10 to 60 nucleotides in length.
12. The method of claim 1 , wherein the mass spectra of (a) are obtained at a rate in the range of 0.1/sec to 1000/sec.
13. The method of claim 1 , wherein the liquid chromatography is high performance liquid chromatography (HPLC).
14. The method of claim 1 , wherein the mass spectrometry is done by electrospray ionization-time of flight mass spectrometry (ESI-TOF).
15. A method comprising;
(a) using the method of claim 1 to analyze a first oligonucleotide synthesis reaction and a second oligonucleotide synthesis reaction;
(b) comparing the results obtained in step (a).
16. The method of claim 15 , wherein the first and second oligonucleotide synthesis reactions are performed under different conditions.
17. The method of claim 15 , wherein the first and second oligonucleotide synthesis reactions are performed at different times.