Primer extension mass spectroscopy nucleic acid sequencing metho

Details Primer extension mass spectroscopy nucleic acid sequencing metho Primer extension mass spectroscopy nucleic acid sequencing metho

1996-05-10

1998-12-15

Jones, W. Gary

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 912, 536 231, 536 243, 364498, 422 8201, 250281, 250287, C12P 1934, C07H 2104, G06G 758, B01D 5944

Patent

active

058495428

DESCRIPTION:

BRIEF SUMMARY
This application is a 371 of PCT/B94/02527 filed Nov. 17,1994.


INTRODUCTION
hybridisation of an oligonucleotide primer to target nucleic acid. A polymerase is then used to extend this complex into four separate nested sets of extension products terminating in dideoxy base analogues. These nested sets are resolved by size using gel electrophoresis. Various labelling and detection strategies can be employed to read the sequence from the base-specific nested sets. Conventional nucleic acid sequencing is highly skilled, very laborious and the rate of data acquisition is limited by the time consuming gel electrophoresis step. the stringent hybridisation of many oligonucleotide probes (such as all the possible 8 mers) to target nucleic acid. Although elegant, such schemes are critically dependent on the ability to discriminate perfect matches from mismatches. Such discrimination is currently far from a reality. SBH is a compromise between the size of the experiment (65,536 8 mers need to be synthesised to make the complete set) and the quality of the data used to reconstruct the target sequence. The longer the sequence data obtained from each hybridisation, the easier the target sequence is to reconstruct but the larger the number of hybridisations that need to be carried out.
This invention directly addresses a great many of the abovementioned problems.
In one aspect the invention provides 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: acid so as to form at least one primer-target hybrid, 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, fragmentation mass spectroscopy so as to generate the observed mass spectrum, CM.sup.-ddC-, CM.sup.-ddG-, CM.sup.-ddT-, and CM0 (the mass of the primer used), difference values (OIPMD) of the observed mass spectrum, that: observed mass spectrum, subsequent rounds of base calling.
In another aspect the invention provides, as a reagent suitable for use in this method though also having other uses, 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.
In another aspect the invention provides, as a technique suitable for use in this method though also having other uses, 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. This aspect is described below under the heading "Chemically-created Molecular Ions".


BRIEF DESCRIPTION OF DRAWINGS

Reference is directed to the accompanying drawings in which:
FIG. 1 is a Mass Spectrograph,
FIG. 2 is a Base Calling Program, and
FIG. 3 is a Flow Diagram for the Program of FIG. 2.


PRIMER EXTENSION MASS SPECTROSCOPIC (PEMS) SEQUENCING

PEMS sequencing according to this invention differs from Sanger sequencing and sequencing by hybridisation in that it generates extension products from primers (with all four dNTPs and all four ddNTPs together in one reaction) and thence allows the sequence to be directly read by the observed mass difference between sequential peaks in a mass spectrum. Many hybridisation events may be spatially arrayed, extended in parallel and then sequentially analysed by sequential desorption/ionisation followed by mass spectroscopy.
With the PEMS method, the amount of sequence data obtained from each initial hybridisation is greater than that of just the oligonucleotide used for hybridisation. The amount of sequence data obtained from each initial hybridisation is potentially of the order of 100 or more bases (dependent upon the resolution of the mass spectrometer used for sequence de

REFERENCES:
patent: 5108892 (1992-04-01), Burke et al.
patent: 5221518 (1993-06-01), Mills
patent: 5228644 (1993-07-01), Beavis et al.
Huth-Fehre, T., et al., "Matrix-Assisted Laser Desorption Mass Spectrometry of Oligodeoxythymidylic Acids", Rapid Commun. Mass Spectrom., 6:209-213 (1992).
Jacobson, Bruce K., et al., "Applications of Mass Spectrometry to DNA Sequencing", GATA, 8(8):223-229 (1991).
Nelson, Randall W., et al., "Time-of-Flight Mass Spectrometry of Nucleic Acids by Laser Ablation and Ionization from a Frozen Aqueous Matrix", Rapid Commun. Mass Spectrom., 4(9):348-351 (1990).
Nordhoff, E., et al., "Ion Stability of Nucleic Acids in Infrared Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry", Nucl. Acids Res., 21(15):3347-3357 (1993).

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