Fingerprint analysis for a plurality of oligonucleotides
View Patent ↗The invention provides a method for evaluating the accuracy of an oligonucleotide sample, specifically a sample containing a variety of oligonucleotides of potentially varying size and sequence. The method provides a fingerprint that can be used to evaluate the accuracy of a multi-oligonucleotide sample whether or not the sample contains differing oligonucleotides that have the same or about the same molecular weight.
1. A method of detecting the presence or absence of a constituent oligonucleotide in an oligonucleotide mixture, the method comprising:
a) obtaining a measurement of a mass spectrum of the oligonucleotide mixture;
b) comparing the measurement of the mass spectrum having a plurality of measured peaks with a calculated mass spectrum having a plurality of calculated peaks, said calculated mass spectrum derived from Formula 1:
MW new =( MW 1 *Intensity 1 +MW 2 *Intensity 2 + . . . MW n *Intensity n )/(Intensity 1 +Intensity 2 + . . . Intensity n )
wherein MW new is a calculated molecular weight of a calculated peak, MW 1 is a first molecular weight of a first subset of one or more oligonucleotides in the oligonucleotide mixture, MW 2 is a second molecular weight of a second subset of one or more oligonucleotides in the oligonucleotide mixture, MW n represents one or more additional molecular weights of one or more additional n subsets in the oligonucleotide mixture, Intensity 1 is a first peak intensity of the first subset of one or more oligonucleotides in the oligonucleotide mixture, Intensity 2 is a second peak intensity of the second subset of one or more oligonucleotides in the oligonucleotide mixture and Intensity n represents one or more additional peak intensities of the one or more additional n subsets in the oligonucleotide mixture; and
c) detecting the presence or absence of the constituent oligonucleotide by correlating one or more of the measured peaks with one or more of the calculated peaks.
2. The method according to claim 1 wherein the mass spectrum is a mass spectrometry trace.
3. The method according to claim 1 wherein the mass spectrum is an electrospray ionization mass spectroscopy trace.
4. The method according to claim 1 wherein the mass spectrum is a matrix assisted laser desorption ionization time-of-flight mass spectrometry.
5. The method according to claim 1 wherein the mass spectrum is an Electrospray Ionization Quadrupole Ion Trap.
6. The method according to claim 1 wherein the mass spectrum is a Matrix Assisted Laser Desorption Ionization Quadrupole Ion Trap.
7. The method according to claim 1 wherein the mass spectrum is a Matrix Assisted Laser Desorption Ionization Quadrupole Time of Flight.
8. The method according to claim 1 wherein the mass spectrum is an Electrospray Ionization Quadrupole Time of Flight.
9. The method of claim 1 wherein the calculated mass spectrum is provided by a processor, wherein said processor accepts a set of given molecular weights of the oligonucleotide mixture to provide a calculated mass spectrum.
10. A kit for an assay utilizing a standard set of oligonucleotides, said kit comprising the standard set of oligonucleotides and a mass spectrum calculated as in claim 1 .
11. A method for predicting a measured peak intensity and a measured peak mass in an electrospray ionization mass spectroscopy trace, the method comprising:
a) assigning each oligonucleotide member in a sample an initial intensity value of one;
b) determining from a set of oligonucleotide members a first sub-set of oligonucleotide members that have an equal molecular weight;
c) combining members of the first sub-set to form a combined first peak intensity using a formula:
I new =I 1 +I 2 + . . . I n
wherein I new is the first combined peak intensity and I 1 and I 2 are intensities of the oligonucleotide members of the first sub-set and I n represents additional oligonucleotide members that may or may not be present in the first sub-set that have an equal molecular weight as I 1 and I 2 ;
d) assigning a first combined peak mass to the first combined peak intensity that correlates with the molecular weight of each member of the first sub-set;
e) determining from the set of oligonucleotide members a second sub-set of oligonucleotide members, said second sub-set of oligonucleotide members having a molecular weight within a range of other members of the second sub-set that when each member of the second sub-set is plotted as a peak in an electrospray ionization mass spectroscopy trace the peaks of members of the second sub-set overlap at greater than 50 percent peak height;
f) combining members of the second sub-set to form a second combined peak intensity using a formula:
I 2 new =I 2 1 +I 2 2 + . . . I 2 n
wherein I 2 new is the second combined peak intensity and I 2 1 and I 2 2 are intensities of the oligonucleotide members of the second sub-set and I 2 n represents additional oligonucleotide members that may or may not be present in the second sub-set;
g) combining the members of the second sub-set to form a second combined peak mass using the formula:
MW new =( MW 1 *I 2 1 +MW 2 *I 2 2 + . . . MW n *I 2 n )/( I 2 1 +I 2 2 + . . . I 2 n )
wherein MW new represents the second combined peak mass, MW 1 and MW 2 are the molecular weights of members of the second sub-set, and MW n represents additional n members that may be present in the second sub-set; and
predicting a measured peak intensity and a measured peak mass in an electrospray ionization mass spectrometry trace from the second combined peak intensity and the second combined peak mass.