Primer extension mass spectroscopy nucleic acid sequencing method
First Claim
1. A method of sequencing a target nucleic acid by using primer extension mass spectroscopy to generate an observed mass spectrum wherein the observed mass spectrum is generated by the steps of:
- a) hybridising at least one oligonucleotide primer to the target nucleic acid so as to form at least one primer-target hybrid,b) Subjecting each primer-target hybrid to polymerase catalysed chain extension conditions in the presence of four chain-extending nucleotides and of four chain-terminating nucleotide analogues, so as to generate a nested set of primer extension products,c) Subjecting each nested set of primer extension products to minimal fragmentation mass spectroscopy so as to generate said observed mass spectrum,wherein the following values are known with certainty;
CM-dA-, CM-dC-, CM-dG-, CM-dT-, CM-ddA-, CM-ddC-, CM-ddG-, CM-ddT-, and CM0 (the mass of the primer used),wherein the calculated value CM0 is used in order to calibrate inter-peak mass difference values (OIPMD) of the observed mass spectrum,base calling cycles are carried out using the calibrated OIPMD values such that;
each called base allows mass calculation for the peak in the observed mass spectrum corresponding to said called base, and this calculated mass is then used as a further calibration point for subsequent rounds of base calling.
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Abstract
A method of sequencing a target nucleic acid using primer extension mass spectroscopy to generate an observed mass spectrum, on which base calling cycles are carried out using calibrated inter-peak mass difference values such that: each called base allows mass calculation for the base-called peak in the observed mass spectrum; this calculated mass is then used as a further calibration point for subsequent rounds of base calling. A reaction mixture of all four base-specific chain extension nucleotides and four chain termination nucleotide analogues. A method of performing mass spectroscopy, which method comprises subjecting molecular ions which have been chemically charged in a predetermined manner to time-of-flight or Fourier transform mass spectroscopy.
194 Citations
17 Claims
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1. A method of sequencing a target nucleic acid by using primer extension mass spectroscopy to generate an observed mass spectrum wherein the observed mass spectrum is generated by the steps of:
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a) hybridising at least one oligonucleotide primer to the target nucleic acid so as to form at least one primer-target hybrid, b) Subjecting each primer-target hybrid to polymerase catalysed chain extension conditions in the presence of four chain-extending nucleotides and of four chain-terminating nucleotide analogues, so as to generate a nested set of primer extension products, c) Subjecting each nested set of primer extension products to minimal fragmentation mass spectroscopy so as to generate said observed mass spectrum, wherein the following values are known with certainty; CM-dA-, CM-dC-, CM-dG-, CM-dT-, CM-ddA-, CM-ddC-, CM-ddG-, CM-ddT-, and CM0 (the mass of the primer used), wherein the calculated value CM0 is used in order to calibrate inter-peak mass difference values (OIPMD) of the observed mass spectrum, base calling cycles are carried out using the calibrated OIPMD values such that; each called base allows mass calculation for the peak in the observed mass spectrum corresponding to said called base, and this calculated mass is then used as a further calibration point for subsequent rounds of base calling. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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- 16. A reaction mixture containing all four base specific chain extension nucleotides which are not labelled so as to be distinguishable from one another, and four chain termination nucleotide analogues which are not labelled so as to be distinguishable from one another.
Specification