System, kit and application for detecting the 5`utr region of fxs syndrome-related gene fmr1
A kit and gene technology, applied in the field of molecular biology, can solve the problems of sensitive and efficient technical means, and achieve the effect of high accuracy and simple test.
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Embodiment 1
[0075] Extract genomic DNA from normal women, add 12.5 μL of 2X Buffer Mix to tube A, 0.9 μL of the first primer (SEQ ID Nos 2), 0.9 μL of the second primer (SEQ ID Nos 36), liquid 7-deza-dGTP (1 / 300) μL, 2.5mM dNTPs 4μL, 2.5mM guanine and cytosine 1μL each, liquid betaine 2μL, Phusion® High-Fidelity DNA Polymerase enzyme 1U, human genomic DNA 20ng, and finally make up to 25μL with ultrapure water;
[0076] Reaction conditions: 98°C for 5min; 30 cycles of 98°C for 20s, 62°C for 30s, and 68°C for 4min; 68°C for 10min;
[0077] Take 1 μL of the amplification product, add 1 μL of GeneScan™ 1000 ROX™ dye Size Standard, and then add 10 μL of HIDI. After mixing, use a spectrally calibrated conventional capillary electrophoresis instrument (such as ABI3500XL) for fragment analysis program electrophoresis;
[0078] Test results such as figure 2 shown;
[0079] The test results of the sample in this picture: two main peaks, one is 296bp, representing the CGG number of 29; the other ...
Embodiment 2
[0082] To extract the genomic DNA of female FXS patients, add 12.5 μL of 2X Buffer Mix to tube A, 0.9 μL of the first primer (SEQ ID Nos 2), 0.9 μL of the second primer (SEQ ID Nos 36), liquid 7-deza-dGTP (1 / 300 ) μL, 2.5mM dNTPs 4μL, 2.5mM guanine and cytosine 1μL each, liquid betaine 2μL, Phusion® High-Fidelity DNA Polymerase enzyme 1U, human genomic DNA 20ng, and finally make up to 25μL with ultrapure water;
[0083] Reaction conditions: 98°C for 5min; 30 cycles of 98°C for 20s, 62°C for 30s, and 68°C for 4min; 68°C for 10min;
[0084]Take 1 μL of the amplification product, add 1 μL of GeneScan™ 1000 ROX™ dye Size Standard, and then add 10 μL of HIDI. After mixing, use a spectrally calibrated conventional capillary electrophoresis instrument (such as ABI3500XL) for fragment analysis program electrophoresis;
[0085] Test results such as image 3 shown;
[0086] The test results of the sample in this picture: two main peaks, one is 318bp, representing a CGG number of 36; t...
Embodiment 3
[0088] Example 3: A tube and B tube are used together
[0089] To extract the genomic DNA of female FXS patients, add 12.5 μL of 2X Buffer Mix to tube A, 0.9 μL of the first primer (SEQ ID Nos 2), 0.9 μL of the second primer (SEQ ID Nos 36), liquid 7-deza-dGTP (1 / 300 ) μL, 2.5mM dNTPs 4μL, 2.5mM guanine and cytosine 1μL each, liquid betaine 2μL, Phusion® High-Fidelity DNA Polymerase enzyme 1U, human genomic DNA 20ng, and finally make up to 25μL with ultrapure water;
[0090] Reaction conditions: 98°C for 5min; 30 cycles of 98°C for 20s, 62°C for 30s, and 68°C for 4min; 68°C for 10min;
[0091] After tube A is amplified, take out 1 μL tube A amplification product and dilute it 300 times;
[0092] Add 12.5 μL of 2X Buffer Mix to tube B, 0.5 μL of the first primer (SEQ ID NOs 17), 0.5 μL of the second primer (SEQ ID NOs 36), 7-deza-dGTP (1 / 150) μL, 3.5 μL of 2.5mM dNTPs , 0.25 μL each of 2.5 mM guanine and cytosine, 2 μL of liquid betaine, 1 U of Phusion® High-Fidelity DNA Poly...
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