Multi-site methylation kit for detecting colorectal cancer-related genes and its application
A colorectal cancer and kit technology, applied in the medical field, can solve problems such as inability to detect multiple gene loci, and achieve the effect of facilitating subsequent detection and improving sensitivity and specificity
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Embodiment 1
[0058] The present invention proposes a multi-site methylation kit for detecting colorectal cancer-related genes and its application. The fecal genome extraction kit is used for DNA separation and extraction to obtain the fecal genome. The steps are as follows:
[0059] 1. Use a special feces collection box to collect the feces of hospital patients, which contains a fecal genome preservation solution, and the preservation solution contains 0.1mol / L EDTA and 0.5mol / L TRIS, and its function is to protect the genomic DNA from the nucleic acid in the feces enzymatic degradation.
[0060] 2. In this example, the feces sample was fully shaken and mixed in the sample preservation solution, and then 1.5ml of the mixed solution was taken into a 2.0ml EP tube, and 1 / 9 of the 40% SDS lysate of the sample was added, and 8 μL of proteinase K was added. After shaking and mixing, lyse at 70°C for 10 minutes.
[0061] 3. Centrifuge at 12000rpm for 8min, transfer 900μL supernatant to a new EP...
Embodiment 2
[0079] This embodiment provides a multi-site methylation kit and application for detecting colorectal cancer-related genes. The primer combination includes the first primer pair, the second primer pair, the third primer pair, the fourth primer pair and the fifth primer pair. A pair of primers, the base sequence of the first primer pair is shown in SEQ ID NO.1-2, the base sequence of the second primer pair is shown in SEQ ID NO.3-4, the third primer The base sequence of the pair is shown in SEQ ID NO.5-6, the base sequence of the fourth primer pair is shown in SEQ ID NO.7-8, and the base sequence of the fifth primer pair is shown in SEQ ID NO Shown in .9-10; Described probe comprises first probe, second probe, the 3rd probe, the 4th probe and the 5th probe, the base sequence of described first probe is as SEQ ID Shown in NO.11, the sequence of the second probe is shown in SEQ ID NO.12, the sequence of the third probe is shown in SEQ ID NO.13, and the sequence of the fourth prob...
Embodiment 3
[0111] The invention provides a multi-site methylation kit for detecting colorectal cancer-related genes and its application. The kit also includes a sample preservation solution, a nucleic acid extraction solution, a PCR reaction solution, a positive quality control product, a negative quality control product, One or more of internal standard controls and standard reagents.
[0112] The kit also includes one or more of a dissolution buffer, a methylation conversion reagent, a binding buffer, and a washing buffer, which can perform CT conversion on the above-mentioned purified DNA.
[0113] The dissolution buffer includes 1-5mol / L NaOH and 5-20mmol / L hydrogen, preferably 3mol / L NaOH and 10mmol / L hydrogen have the best effect.
[0114] The methylation conversion reagents include but are not limited to salts containing sulfite groups such as sodium bisulfite, sodium sulfite, ammonium bisulfite and ammonium sulfite, with a concentration of 1-10mol / L, preferably ammonium bisulfite...
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