Detection of nucleic acid amplification
a nucleic acid and target sequence technology, applied in the field of detection and measurement of amplification of nucleic acid target sequences, can solve the problems of high background signal level, low background signal level, time-consuming and cumbersome, etc., and achieve the effect of facilitating detection or localization of amplification products, facilitating detection or localization of background signals, and facilitating detection and localization
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example 1
[0031]The real-time detection of amplification of the instant invention was compared to conventional post-amplification detection of amplified target sequences. Fragments of the IS6110 sequence of Mycobacterium tuberculosis (M.tb) were amplified in SDA reactions performed essentially as described by Walker, et al. (1992, Nuc. Acids Res.), except that each 60 μL reaction mixture contained 0.2 μg of human placental DNA and varying amounts of genomic M.tb DNA. Amplification primer sequences (S1 and S2) and bumper primer sequences (B1 and B2) were also as in Walker, et al. (1992, Nuc. Acids Res.) For the amplification reactions incorporating signal primers, the 32P-labeled signal primer 32P-CGTTATCCACCATAC (SEQ ID NO:1) was added to the reactions prior to amplification at a final concentration of 60 nM. Predicted secondary amplification products produced in these reactions were 35 and 56 nucleotides in length. For post-amplification detection of amplified target sequences, one-tenth of ...
example 2
[0033]SDA reactions were performed generally as previously described (Walker, 1993, PCR—Methods and Applications 3. 1) in 50 mM KiPO4 (pH 7.5), 0.1 mg / mL bovine serum albumin, 0.5 mM dUTP, 0.2 mM each dGTP, dCTP and dATPαS, 7 mM MgCl2, 11% (v / v) glycerol, the indicated concentrations of amplification primers, 25 nM bumper primers, 50 ng human placental DNA, the indicated amount of exonuclease deficient Klenow (United States Biochemicals), 150 units HincII (New England Biolabs). Reactions were run for the indicated time at 41° C. SDA reactions contained varying amounts of M.tb DNA, which contains the IS6110 target sequence for amplification. The S1 amplification primer sequence, the B1 bumper primer sequence and the B2 bumper primer sequence used were as described by Walker, et al. (1992, Nuc. Acids Res., supra). The S2 amplification primer (SEQ ID NO:2) had the target binding sequence and HincII site disclosed by these authors, but comprised a different sequence at the 5′ end. The a...
example 3
[0047]Two signal primers, one modified to facilitate capture and one modified to facilitate detection, were used to generate secondary amplification products in an SDA reaction. In this experiment, one signal primer had an affinity ligand (Q′, three biotin moieties) attached to its 5′ end and the second signal primer was 5′-end labeled with a reporter group (R, a 32P-containing phosphate group). Thus, double-stranded secondary amplification products which comprised both the reporter group and the affinity ligand (such as structure #3 and structure #9 of FIG. 1A and FIG. 1B) could be captured and detected. In this example, streptavidin coated magnetic beads were used to capture and separate the secondary amplification products, which were then detected by scintillation counting.
[0048]Biotinylated signal primers were prepared as follows. Oligonucleotide SEQ ID NO:7 was synthesized on an Applied Biosystems DNA Synthesizer Model 380B, using standard phosphoramidite chemistry. The instru...
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