Sequence for kidney cancer diagnosis and evaluation, kit and application
A diagnostic kit and kit technology, applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as endless DNA primers
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Embodiment 1
[0014] In this example, using the nimblegen chip (Roche Company, NimbleGen Sequence CaptureHuman Exome 2.1M Array) and the HiSeq high-throughput sequencing system (IUumina Company, Hiseq 2000), 3 cases of kidney cancer patients obtained from Peking University Shenzhen Hospital Samples underwent exome sequencing.
[0015] The details of the samples from 3 renal cancer patients are shown in Table 1.
[0016] Table 1 Details of samples from 2 patients with renal cell carcinoma.
[0017]
[0018] 1. Fragmentation of genomic DNA
[0019] Genomic DNA was extracted from the samples of each patient using the kit QIAamp DNA Mini Kit (Qiagen, 51306), and the obtained genomic DNA was fragmented into fragments with a size of about 200 bp using the ultrasonic method (Covaris, S-2).
[0020] 2. End repair of DNA fragments and 3' connection of "A" bases According to the manufacturer's instructions, use T4 DNAPolymerase (Enzymatics, P708L), Klaneow Fragment (Enzymatics, P706L) and T4Poly...
Embodiment 2
[0026] Processing and analysis of exome sequencing data of samples
[0027] The exome sequencing data of each sample were processed and analyzed as follows to verify the significantly mutated genes positively related to the ubiquitin-mediated proteolytic pathway in the RCC samples, as well as the mutated sites in these genes.
[0028] 1. As described in Example 1, obtain the exome sequencing data of each sample;
[0029] 2. For the exome sequencing data of each sample, remove the sequencing fragments (reads) containing the adapter sequence (adapter connector) in step 4 of Example 1; Sequencing fragment deduplication processing, this step is to remove some data redundancy caused by sequencing.
[0030] Firstly, the specific linker sequence is obtained according to the report of the off-line data of each single lane, and the sequence fragments containing the linker sequence are removed by traversing each sequencing fragment of the off-line data.
[0031] Secondly, in the off-m...
Embodiment 3
[0050] PCR-sanger verification
[0051] Method steps:
[0052] Take the following sites as an example and verify by PCR-sanger sequencing:
[0053] HERC2 K11 G.chr15:37186472C>T C.3695G>A CGT=>CAT Arg => His missense
[0054] 1) PCR-sanger verification experiment reaction system:
[0055] Use 0.5ul Taq HS (5U / ul) (TaKaRa, DR007B), 2ul 10 PCR
[0056] Buffer (Mg2+Plus) (TaKaRa, DR007B), 2ul dNTP Mixture (each 2.5mM) (TaKaRa, DR007B), lul genomic DNA, upstream and downstream primers (SEQ ID NO: 1 and SEQ ID NO: 2) each lul and 12.5 ul self-made double distilled water, mixed to prepare a 20ul reaction system.
[0057] 2) PCR-sanger verification experiment reaction conditions:
[0058] A common PCR instrument (Applied Biosystems, Veriti 96-Well Thermal Cycler) was used to carry out the PCR reaction, and the reaction conditions were as follows:
[0059] 95℃4min
[0060] 95℃40s
[0061] 62℃30s
[0062] 72℃50s
[0063] 72℃10min
[0064] 35 cycles. ...
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