Amersham Pharmacia Biotech UK Limited

General Electric Company

Nycomed Amersham Plc

Amersham International Plc

Exiqon A/s

Clifford Leslie Smith

David Loakes

Alan L Hamilton

Adrian Christopher Simmonds

Daniel M Brown

Shiv R Kumar

Mark McDougall

Satyam Nampalli

Fergal Hill

John Richard Nelson

Robert Scott Duthie

Sonali Jagdish Shah

William Jonathan Cummins

David Williams

Carl Fuller

Patrick Finn

Adrian Simmons

Methods and kits for efficient amplification of nucleic acids are provided. The methods comprise in-vitro amplification of a nucleic acid template employing partially constrained primers having terminal mismatch primer-dimer structure. The methods also comprise in-vitro amplification of a nucleic acid template employing partially constrained primers having nucleotide analogues. The methods enhance efficiency of nucleic acid amplification reaction by reducing non-specific amplification reactions.

Methods and kits for reducing non-specific nucleic acid amplification

Nucleoside analogues or base analogues having structure (I), where X&equals;O or NH or S, Y&equals;N or CHR or CR , W&equals;N or NR or CHR or CR or S, n&equals;1 or 2; each R is independently H or O or alkyl or alkenyl or alkoxy or aryl or a reporter moiety; where necessary (i.e. when Y and/or W is N or CR where R is not O) a double bond is present between Y and W or W and W, and Q is H or a sugar or a sugar analogue or a nucleic acid backbone or backbone analogue.

Base analogues

Nucleoside analogues have structure (2) wherein Q is H or a sugar moiety or sugar analogue or a modified sugar or a nucleic backbone or backbone analogue, W is an alkylene or alkenylene chain of 0-5 carbon atoms, any of which may carry a substituent R , X is O or N or NR or CR , X&prime; is O or S or N, provided that when X&prime; is O or S, then X is C, Y is CH or N, R is NH or SMe or SO Me or NHNH , each of R and R is independently H or F or alkyl or alkenyl or aryl or acyl or a reporter moiety, R is independently H or alkyl or alkenyl or aryl or acyl or a reporter moiety, and R is H or &boxH;O or F or alkyl or aryl or a reporter moiety.

Tricyclic base analogues

DNA and RNA diagnostics based on mass spectrometry, e.g. Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry, Electrospray (ES) mass spectrometry, Ion Cyclotron Resonance (ICR) mass spectrometry, Fourier Transform mass spectrometry, or combinations thereof, where fully or partially LNA modified DNA probes are used in order to enhance stability and resolution. The invention in particular relates to a process for detecting a target nucleic acid sequence of a nucleic acid molecule or for detecting a mutation in a nucleic acid sequence of a nucleic acid molecule, wherein (a) the nucleic acid molecule or (b) a part of the nucleic acid molecule or (c) an oligonucleotide complementary to the sequence or at least a sub-sequence of the nucleic acid molecule is analysed by mass spectrometry in order to obtain direct or indirect information about the target nucleic acid sequence or mutation, and wherein the process involves the hybridisation of an LNA modified oligonucleotide to the nucleic acid molecule.

Use of LNA in mass spectrometry

Nucleoside analogues containing the degenerate base analogue P and derivatives thereof are provided with reporter moieties preferably comprising signal moieties. The nucleoside analogues are useful for labeling DNA or RNA or for incorporating in oligonucleotides.

Nucleoside analogues

Nucleoside analogues in which a group M replaces the natural base where M is (1) or (2) or (3), where each of X,, X,and X,are C or N, each of R,and R,is the same or different and each is H, NO,, CO, COR,, OR,, CN, O, CON(R,),, COOR,, SO,R,, SO,R,, SR,, NHCHO, (CH,),(R,),, halogen, or a reporter moiety, each of R,and R,is H or hydrocarbyl or a reporter moiety, and n is 0-4. The analogues are substrates for polymerase and terminal transferase enzymes.

Nucleoside analogue method

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Tring, United Kingdom

Clifford Leslie Smith