×

Primer extension mass spectroscopy nucleic acid sequencing method

  • US 5,849,542 A
  • Filed: 05/10/1996
  • Issued: 12/15/1998
  • Est. Priority Date: 11/17/1993
  • Status: Expired due to Fees
First Claim
Patent Images

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.

View all claims
  • 3 Assignments
Timeline View
Assignment View
    ×
    ×