Detection System

Abstract

A method for detecting the presence of a target nucleic acid sequence in a sample, said method comprising: performing nucleic acid amplification on the sample in the presence of (a) a DNA duplex binding agent, (b) a nucleic acid polymerase and (c) a reagent comprising an amplification primer which can hybridise to said target sequence when in single stranded form and which is connected at its 5′ end to a probe which carries a label by way of a chemical linking group, said labelled probe being of a sequence which is similar to that of the said target nucleic acid sequence, such that it can hybridise to a complementary region in an amplification product, and wherein the label is able to absorb fluorescence from or donate fluorescent energy to the DNA duplex binding agent; and monitoring fluorescence of said sample.

1 . A method for detecting the presence of a target nucleic acid sequence in a sample, said method comprising:
 performing nucleic acid amplification on the sample in the presence of (a) a DNA duplex binding agent, (b) a nucleic acid polymerase and (c) a reagent comprising an amplification primer which can hybridise to said target sequence when in single stranded form and which is connected at its 5′ end to a probe which carries a label by way of a chemical linking group, said labelled probe being of a sequence which is similar to that of the said target nucleic acid sequence, such that it can hybridise to a complementary region in an amplification product, and wherein the label is able to absorb fluorescence from or donate fluorescent energy to the DNA duplex binding agent; and monitoring fluorescence of said sample.   
     
     
         2 . A method according to  claim 1 , said method comprising:
 (a) adding to a sample suspected of containing the target nucleic acid sequence, the DNA duplex binding agent, the nucleic acid polymerase and the reagent;   (b) subjecting said sample to conditions under which the primer hybridises to the target nucleic acid sequence and an amplification product comprising the probe is formed;   (c) subjecting said sample to conditions under which the labelled probe hybridises to a complementary region in the amplification product; and   (d) monitoring fluorescence of said sample during at least one of steps (b) and (c).   
     
     
         3 . A method according to  claim 1  wherein the amplification product comprises the probe. 
     
     
         4 . A method according to any one of  claims 1  to  3  wherein the DNA duplex binding agent is an intercalating dye. 
     
     
         5 . A method according to any one preceding claim wherein the DNA duplex binding agent comprises a donor label and the probe comprises the acceptor label. 
     
     
         6 . A method according to any one of  claims 1  to  4  wherein the DNA duplex binding agent comprises an acceptor label and the probe comprises the donor label. 
     
     
         7 . A method according to any one preceding claim wherein the acceptor label is a fluorescent molecule which emits energy at a characteristic wavelength. 
     
     
         8 . A method according to  claim 7  wherein the acceptor label is a rhodamine dye or Cy5. 
     
     
         9 . A method according to any one of  claims 1  to  6  wherein the acceptor label is a dark acceptor. 
     
     
         10 . A method according to  claim 9  wherein the dark acceptor is selected from any one of DABCYL, Methyl Red, a QSY-7 diarylrhodamine dye and 6-(dimethylamino)-2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-1-ethyl quinolinium perchlorate (CAS number 181885-68-7). 
     
     
         11 . A method according to any one of the preceding claims wherein the amplification reaction comprises the polymerase chain reaction (PCR). 
     
     
         12 . A method according to any one of  claims 1  to  8  and  11  wherein the acceptor molecule is a fluorescent molecule and wherein fluorescence of both the donor and the acceptor molecules are monitored and the relationship between the emissions calculated. 
     
     
         13 . A method according to any one of the preceding claims wherein the fluorescent signal from the sample is monitored throughout the amplification reaction and the results used to quantitate the amount of target sequence present in the sample. 
     
     
         14 . A method according to any one of the preceding claims wherein the amplification reaction is performed in the presence of an additional corresponding amplification primer which is not attached to a labelled probe. 
     
     
         15 . A method for detecting nucleic acid amplification comprising: performing nucleic acid amplification on a target polynucleotide in the presence of (a) a nucleic acid polymerase, (b) a DNA duplex binding agent and (c) a reagent comprising an amplification primer which can hybridise to said target sequence when in single stranded form and which is connected at its 5′ end to a probe which carries a second label, by way of a chemical linking group, said labelled probe being of a sequence which is similar to that of the said target sequence, such that it can hybridise to a complementary region in an amplification product, and wherein one of the DNA duplex binding agent or second label comprises a donor label which is able to donate fluorescent energy to the other of the DNA duplex binding agent or second label which comprises an acceptor label able to absorb fluorescent energy from said donor molecule, said primer being capable of hybridising to said target polynucleotide; and monitoring changes in fluorescence during the amplification reaction. 
     
     
         16 . A method according to  claim 15  wherein the amplification is carried out using a pair of primers which are designed such that only the target nucleotide sequence within a DNA strand is amplified. 
     
     
         17 . A method according to any one of the preceding claims wherein the probe is specific either for a splice region of RNA or an intron in DNA, so that only one of amplified RNA or amplified DNA is detected and/or quantitated. 
     
     
         18 . A method for determining a characteristic of a target nucleic acid sequence, said method comprising (a) amplifying said sequence in the presence of a DNA duplex binding agent and a reagent comprising an amplification primer linked by way of a chemical link at its 5′ end to a probe which comprises a sequence which is similar to that of a region of the target sequence and which further comprises a label, where one of said DNA duplex binding agent and the label is a donor label and the other is an acceptor label, the donor label being able to donate fluorescent energy to the acceptor label; so as to form an amplification product incorporating a probe region, (b) subjecting amplification product to conditions under which the probe region thereof will hybridise to the complementary region of the amplification product, and (c) monitoring fluorescence of said sample and determining a particular reaction condition, characteristic of said sequence, at which fluorescence changes as a result of the hybridisation of the probe region to the sample or destabilisation of the duplex formed between the probe region and the target nucleic acid sequence. 
     
     
         19 . A method for detecting a polymorphism and/or allelic variation, said method comprising amplifying a sequence suspected of containing said polymorphism or variation using a method as defined in any one of  claims 1  to  16 , measuring the temperature at which the probe region melts from its complementary sequence within the amplification product using the fluorescent signal generated, and relating this to the presence of a polymorphism or allelic variation. 
     
     
         20 . A kit for use in the method of any one of the preceding claims which kit comprises a reagent comprising an amplification primer linked at its 5′ end by way of a chemical link, to a probe specific for a target nucleotide sequence, wherein the probe comprises a first label which may act as one of either a donor and acceptor label; and a DNA intercalating agent comprising a second label, which second label may act as one of either a donor and acceptor label, wherein the first and second labels form a donor-acceptor pair.