ALK gene and EML4 gene detection probe, preparing method thereof and kit
A gene detection and detection probe technology, applied in the field of ALK gene and EML4 gene detection probes and their preparation, can solve the problems of high detection kits and lack of specificity, and achieve the effects of good repeatability and accurate signal counting
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Embodiment 1
[0042] The preparation of embodiment 1ALK gene and EML4 gene detection probe
[0043] The preparation method of the described ALK detection probe of this implementation comprises the following steps:
[0044] Select the clone containing the target gene ALK and both ends of the sequence, such as Figure 1A shown. GSPALK includes two sets of probes:
[0045] The first group of probes includes the first probe, the second probe and the third probe, as shown in the following table, which were purchased from Invitrogen RP11BAC and CTDBAC clone libraries.
[0046] The second group of probes includes the fourth probe, the fifth probe and the sixth probe, as shown in the following table, which were purchased from Invitrogen RP11BAC and CTDBAC clone libraries.
[0047] The following two groups of detection probes were prepared respectively.
[0048] Probe set 1—labeled red R, total length of BAC: 462Kb.
[0049] Probe set 2—labeled green G, total length of BAC: 513Kb.
[0050] ...
Embodiment 2
[0067] Example 2: ALK gene detection, kits for ALK gene and EML4 gene detection
[0068] This embodiment provides two kinds of kits, namely ALK gene detection kit, and ALK gene and EML4 gene detection kits.
[0069] The ALK gene rearrangement detection kit includes two components of a hybridization solution and a DAPI counterstain, wherein the hybridization solution contains two groups of GSPALK probes (two-color) described in Example 1, and a buffer for the hybridization environment (to promote hybridization) Liquid components, COTHumanDNA with closed repeat sequences, etc. DAPI counterstaining agent is mainly used for cell counterstaining after hybridization, in which DAPI will bind to DNA, making the nucleus show blue fluorescence, and the counterstaining agent containing p-phenylenediamine can keep the fluorescence stable.
[0070] The specific formula is as follows:
[0071] Hybridization solution preparation
[0072]
[0073] ALK gene and EML4 gene detection kit in...
Embodiment 3
[0082] Embodiment 3: the detection method of ALK gene and EML4 gene detection kit
[0083] 1. Slide pretreatment
[0084] 1. Place the slides in a constant temperature oven at 65±5°C and bake overnight;
[0085] 2. Take out the slide and place it in xylene (or environmental dewaxing agent) at room temperature for 10 minutes;
[0086] 3. Take out the slide, and put it in another cylinder of room temperature xylene (or environmental dewaxing agent) for 10 minutes;
[0087] 4. Take out the slide, and put it in absolute ethanol at room temperature for 10 minutes to remove residual xylene (or environmental
[0088] dewaxing agent);
[0089] 5. Take out the slide, and put it into 100%, 90%, 70% graded ethanol for rehydration at room temperature for 3 minutes each;
[0090] 6. Take out the slide, put it in sterilized purified water and wash it at room temperature for 3 minutes, and absorb excess water with a lint-free paper towel;
[0091]7. Take out the slide, put the slide int...
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