Primers, probes and methods for nucleic acid amplification

Abstract

Homogenous detection during or following PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled primers and probes, improves reproducibility and quantification. Low-temperature homogeneous detection during or following non-symmetric PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled mismatch-tolerant probes permits analysis of complex targets. Sequencing sample preparation methods following LATE-PCR amplifications reduce complexity and permit “single-tube” processing.

1 .- 24 . (canceled) 
     
     
         25 . A double-stranded nucleic acid hybridization probe for a preselected nucleic acid target sequence comprising:
 (a) a first oligonucleotide comprising a first sequence perfectly complementary to said target sequence and having a calculated melting temperature (T m ) with respect to said target sequence of 30-55° C.;   (b) a second oligonucleotide comprising a second sequence that is complementary to said first sequence but is shorter than said first sequence by up to ten nucleotides;   (c) a fluorophore label attached to said first sequence that is excitable by fluorescence emission from a fluorescent DNA dye but is not excitable at the maximum absorption wavelength of said dye; and   (d) a non-fluorescent quencher attached to said second sequence.   
     
     
         26 . An oligonucleotide set comprising (a) a pair of LATE-PCR primers for amplifying a preselected target sequence and (b) a probe according to  claim 25 . 
     
     
         27 . A low-temperature, mismatch tolerant, single-stranded oligonucleotide hybridization probe having a first calculated melting temperature with respect to its perfect complement of not more than 60° C., said probe including a stem-loop hairpin having a stem whose calculated melting temperature is at least 10° C. lower than said first melting temperature but not lower than 30° C., said probe comprising a fluorophore capable of absorption of fluorescence energy emitted by a fluorescent DNA dye and means to quench fluorescence that would result from exposing said probe to said dye and exciting the dye in the absence of a target for the probe. 
     
     
         28 . A low-temperature linear oligonucleotide hybridization probe
 (a) that is mismatch tolerant at temperatures in the range of 30-60° C.;   (b) that forms secondary structure in the temperature range of 30° C. to 50° C.; and   (c) is dual-labeled with
 (i) a fluorophore that is excitable by fluorescence emission from at least one fluorescent DNA dye but is not excitable at the wavelength appropriate for exciting said dye; and 
 (ii) a non-fluorescence quencher located so as to substantially quench fluorescence associated with said secondary structure but not florescence associated with a probe-target hybrid. 
   
     
     
         29 . The probe according to  claim 28 , wherein said secondary structure is a stem formed by complementary terminal nucleotides, said stem having a T m  greater than the T m  of any other secondary structure the probe would form in the absence of the stem. 
     
     
         30 . A sequential DNA amplification-sequencing method comprising:
 a) amplifying at least one DNA target by a LATE-PCR process to generate an amplification product containing copies of at least one excess primer strand and at least one limiting primer strand;   b) cleaning up the amplification product by diluting it by a factor of at least five and   c) sequencing the copies of at least one of said strands in the cleaned-up amplification product.   
     
     
         31 . The method of  claim 30 , wherein sequencing is dideoxy cycle sequencing. 
     
     
         32 . The method of  claim 31 , wherein said amplification product is diluted by a factor of at least twenty. 
     
     
         33 . The method of  claim 30 , wherein said amplification product is diluted by a factor of at least twenty. 
     
     
         34 . The method of  claim 30 , wherein an excess primer strand is sequenced. 
     
     
         35 . The method of  claim 30 , wherein a limiting primer strand is sequenced. 
     
     
         36 . The method of  claim 30 , wherein the act of diluting is performed in two steps. 
     
     
         37 . The method of  claim 30 , wherein diluting comprises combining the amplification product with a mixture of sequencing reagents. 
     
     
         38 . The method of  claim 30 , wherein the at least one target comprises at least two targets. 
     
     
         39 . The method of  claim 30 , wherein a one limiting primer strand is sequenced utilizing its complementary excess primer as a sequencing primer and wherein other excess primers in the cleaned-up amplification product are blocked. 
     
     
         40 . The method of  claim 30 , wherein the at least one target comprises at least two variant sequences. 
     
     
         41 . The method of  claim 30 , wherein the LATE-PCR amplification comprises monitoring the adequacy of production of single-stranded product strand, wherein the monitoring comprises determining the ratio of single-stranded product to double-stranded product. 
     
     
         42 . A kit comprising primers and the probe of  claim 25 . 
     
     
         43 . The kit according to  claim 38 , further comprising amplification reagents. 
     
     
         44 . A homogeneous detection method for detecting allelic variants of at least one nucleic acid target sequence that includes amplification of said at least one target by a non-symmetric nucleic sequence acid amplification process that generates single-stranded amplification products and includes at least one primer-annealing temperature above a first detection temperature, comprising:
 (a) forming an amplification reaction mixture that includes said at least one nucleic acid target sequence and a mismatch-tolerant probe for the single-stranded amplification products of said at least one target sequence that binds to progressively more mismatched sequences as the temperature is lowered, said probe hybridizing to one allelic variant at said first detection temperature but hybridizing to other allelic variants at detection temperatures below said first detection temperature;   (b) amplifying said nucleic acid target sequence by said non-symmetric amplification process;   (c) detecting fluorescence emission from said probe at said first detection temperature and at one or two lower detection temperatures that are below said first detection temperature and that are temperatures at which the probe binds to said other allelic variants; and   (d) determining the allelic make-up of said at least one target sequence by normalizing the probe's fluorescence emission as a ratio of emissions at said first detection temperature and said at least one lower detection temperature.   
     
     
         45 . The method of  claim 44 , wherein said one or two lower detection temperatures consist of one temperature. 
     
     
         46 . The method of  claim 45 , wherein said step of detecting is performed following completion of the amplification reaction as an end-point detection. 
     
     
         47 . The method of  claim 44 , wherein said one or two lower detection temperatures consist of two temperatures, a low temperature and an intermediate temperature, and said ratio is determined by the formula (Fs−Ft)/(Fb−Ft), where Ft is the fluorescence at said first detection temperature, Fb is the fluorescence at said low temperature, and Fs is the fluorescence at said intermediate temperature. 
     
     
         48 . The method of  claim 47 , wherein said step of detecting is performed following completion of the amplification reaction as an end-point detection. 
     
     
         49 . The method of  claim 44 , wherein said step of detecting is performed following completion of the amplification reaction as a melt analysis. 
     
     
         50 . The method of  claim 44 , wherein said at least one target sequence is at least two target sequences, and there is a one detector probe for each sequence.