A nucleic acid typing standard with fluorescent label and its preparation method and application
A fluorescent dye labeling and standard technology, which is applied to the nucleic acid typing standard with fluorescent label and its preparation and application fields, can solve the problems of unsuccessful typing and the inability to guarantee the resolution of a single base, and achieves low production cost. , Improve the internal standard calibration ability, the effect of high amplification efficiency
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Embodiment 1
[0074] Embodiment 1, design and preparation of primers
[0075] Plasmid pBR322 is shown in SEQ ID NO:22.
[0076] Design upstream primers. Use fluorescent dye LIZ to label the upstream primer (for practical operation, fluorescent dyes such as FAM, HEX, ROX, and TAMRA can be used).
[0077] Upstream primer (SEQ ID NO: 1): 5'-TCCTCTACGCCGGACGCA-3'.
[0078] Design initial downstream primers based on the length of the expected amplified fragment. The effect of the downstream primers was verified by the preliminary experiment, and the 5' end of the downstream primers was modified (to improve the specificity of the primers, avoid complex secondary structures, hairpin structures, and dimers between primers), and obtain the modified downstream primers. The modified downstream primers are shown in Table 1.
[0079] Table 1
[0080]
Embodiment 2
[0081] Embodiment 2, the preparation of standard substance
[0082] 1. Using plasmid pBR322 as a template, 20 PCR systems were used for PCR amplification.
[0083] In PCR system 1, adopt the upstream primer shown in SEQ ID NO:1 and the downstream primer shown in SEQ ID NO:2; In PCR system 2, adopt the upstream primer shown in SEQ ID NO:1 and SEQ ID NO:3 The downstream primers shown; and so on; in the PCR system 20, the upstream primer shown in SEQ ID NO:1 and the downstream primer shown in SEQ ID NO:21 are used.
[0084] ABI9700 PCR amplification instrument was used.
[0085] Each PCR amplification system was the same, with a volume of 10 μl, see Table 2 for details.
[0086] Table 2
[0087]
[0088] Each PCR amplification program is the same, see Table 3 for details.
[0089] table 3
[0090]
[0091] 2. The PCR amplification product contains protein and salt ions, which will cause the degradation of DNA fragments, which is not conducive to long-term storage, so i...
Embodiment 3
[0104] Example 3, the nucleic acid typing standard prepared by the present invention is used as an internal standard to analyze the accuracy of gene fragments
[0105] The "Allelic Ladder" in the human DNA typing kit was used as a sample. Mix 1 μl of sample, 8.7 μl of formamide and 0.3 μl of the nucleic acid typing standard prepared in Example 2, then use the human DNA typing kit and load the mixture according to the instructions, and repeat 96 times of denaturing polyacrylamide gel electrophoresis. Analyze each value of each genotype of each locus in "Allelic Ladder", take the mean and calculate the standard deviation (SD), and the value of SD is less than 0.1 bases. Using Genemapper-IDX analysis, the R2 of the best fit second order curve in Size calling curve>0.99999. R 2 Reflects the linear relationship of the typing standard, R 2 The closer it is to 1, the higher the linear relationship and the higher the accuracy of the typing standards. Theoretically, within a specif...
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