Kit for detecting methylation of colorectal cancer related gene multi-sites and application of kit
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 kit for detecting colorectal cancer-related gene multi-site methylation and its application. The fecal genome extraction kit is used for DNA separation and extraction to obtain a fecal genome, and the steps are as follows:
[0059] 1. Use a special fecal collection box to collect feces from hospital patients, which contains fecal genome preservation solution, and the preservation solution contains 0.1mol / L EDTA and 0.5mol / L TRIS, its function is to protect the genomic DNA from the nucleic acid in the feces. Enzymatic degradation.
[0060] 2. In this example, 1.5ml of the mixture was taken into a 2.0ml EP tube after the fecal sample was fully shaken and mixed in the sample preservation solution, and 1 / 9 of the 40% SDS lysis solution of the sample was added, and 8 μL of proteinase K was added. After shaking and mixing, it was lysed at 70 °C for 10 min.
[0061] 3. Centrifuge at 12000rpm for 8min, transfer 900μL of supernatant to a new EP ...
Embodiment 2
[0079] This embodiment provides a kit and application for detecting multi-site methylation of colorectal cancer-related genes. The primer combination includes a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, and a fifth primer pair. primer pair, 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 pair 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. 9-10; the probe includes a first probe, a second probe, a third probe, a fourth probe and a fifth probe, and the base sequence of the first probe is as shown in SEQ ID As 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 probe is sho...
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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