Direct nucleic acid detection in bodily fluids
A technology for body fluids and target nucleic acids, applied in sugar derivatives, organic chemistry, fermentation, etc., can solve the problems of PCR application carrying pollution and other problems
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Embodiment 1
[0292] Designing 10-fold reactions (manual): Examination of the INVADER test
[0293] The following experimental examples describe the manual design of amplification primers for multiplex amplification reactions, and the subsequent detection of amplicons using the INVADER assay.
[0294] Ten target sequences were selected from a set of pre-validated SNP-containing sequences available in TWT's internal oligonucleotide order entry database. Each target sequence contains a single nucleotide polymorphism (SNP) for which an INVADER assay has been predesigned. The INVADER assay oligonucleotides were designed using INVADER CREATOR software (Third Wave Technologies, Inc. Madison, WI), so in this example the footprint region is defined as the INVADER "footprint", or by INVADER and probe oligonucleotides. Covered bases whose optimal positioning is used to detect target bases, in this case single nucleotide polymorphisms. For the analysis of the amplification primer design, each of the...
Embodiment 2
[0321] Design of 101-fold PCR using application software
[0322] Use the TWT Oligo order entry database to obtain 144 sequences with a length of less than 200 nucleotides, where SNPs are marked with brackets to indicate the SNP position of each sequence (such as NNNNNN[N (wt) / N (mt)]NNNNNNNN). To expand the sequence data flanking the SNP of interest, the sequence was extended to approximately 1 kB in length (500 nts on each side of the SNP) using BLAST analysis. Of the 144 starting sequences, 16 could not be expanded by BLAST, resulting in a final set of 128 sequences extended to approximately 1 kB in length. These extended sequences are provided to users in Excel format along with the following information for each sequence: (1) TWT number, (2) short name identifier, and (3) sequence. This Excel file was converted to a comma-separated format and used as an input file for Primer Designer INVADER CREATOR v1.3.3. software (this version of the program does not screen primers...
Embodiment 3
[0326] Applying INVADER Test to Determine the Amplification Multiple of PCR
[0327] The INVADER assay can be used to monitor the progress of amplification during a PCR reaction, ie, to determine the fold amplification F that reflects the amplification efficiency of a particular amplicon in the reaction. In particular, the INVADER assay allows the determination of the number of molecules present at any point in a PCR reaction with reference to a standard curve generated by quantifying reference DNA molecules. The amplification factor F was determined as the ratio of the PCR product concentration after amplification to the initial target concentration. This example demonstrates the effect of different primer concentrations on the fold amplification of the assay.
[0328] The PCR reaction was performed for a variable number of cycles in increments of 5, ie 5, 10, 15, 20, 25, 30 cycles, allowing the progress of the reaction to be assessed by measuring the accumulated product wit...
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