PCR primers for detecting structural variants of focal adhesion kinase and its detection method and application
A technology of focal adhesion kinase and structural variation, applied in biochemical equipment and methods, microbiological determination/testing, DNA/RNA fragments, etc., can solve problems affecting sensitivity and accuracy
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Embodiment 1
[0060] Whole-genome sequencing of the tumor tissue and adjacent normal tissue of a male smoking lung cancer patient #711 found that the internal sequence tandem repeat expansion of the focal adhesion kinase gene FAK-ITD was specifically present in the tumor tissue of this patient. The RNA of lung cancer patient #711 was extracted by the Trizol method, and then 2ug of the RNA was reverse-transcribed with oligo(dT)15 to generate the first strand of cDNA. Take 1ul of cDNA from the tumor tissue of lung cancer patient #711, dilute it 20 times with PCR-grade water, mix well, and take 2ul as a template for PCR amplification. Using primers to amplify FITD-RITD, the PCR reaction system is as follows:
[0061]
[0062] The total reaction volume was 20ul. Vortex and mix well. The reaction conditions are: pre-denaturation at 95°C for 5 minutes; cycle amplification at 95°C for 30s, 60°C for 30s, and 72°C for 30s. After 35 cycles, continue to extend at 72°C for 5 minutes.
[0063] Afte...
Embodiment 2
[0065] When sequencing the FAK coding sequences of the tumor tissues of 4 lung cancer patients, it was found that alternatively spliced exons Box 6 and Box 7 were inserted and expressed behind exon 14 and exon 15 respectively (see Figure 2A ). The PCR forward sequencing peak showed that a sequencing doublet appeared after exon 14, and the reverse sequencing peak showed that a sequencing doublet appeared before exon 16. There were no double peaks in the sequencing profile of the corresponding paracancerous control tissue. Using the cDNA of the tumor tissues and normal adjacent tissues of 4 patients as templates, PCR amplification was carried out with primers Fplus-Rplus, Fi6-Rplus, and Fplus-Ri7, respectively. The PCR reaction system and reaction conditions are the same as in Implementation 1. PCR products were detected by 3% agarose gel electrophoresis. The results of electrophoresis showed that among the products amplified by Fplus-Rplus, 3 bands were amplified in the t...
Embodiment 3
[0067] Using the cDNA of lung cancer cell lines Calu-6, A549, NCI-H460, NCI-H1975, EPLC-32M1 and L78 as templates, primers Fplus-Rplus were used for reverse transcription PCR amplification, and the reaction system and conditions were the same as in Example 2. After amplification, it was detected by 3% agarose gel electrophoresis. The results showed that in the lung cancer cell line Calu-6, there were 3 amplified bands, the molecular weight of which was consistent with the size of the amplification product of Fplus-Rplus in Example 2, and there was only one wild-type FAK band in the remaining 5 lung cancer cell lines. Amplified bands (see Figure 3A ); the amplified product of the lung cancer cell line Calu-6 was subjected to Sanger sequencing, and the results were consistent with Example 2: double peaks appeared after exon 14 in the forward sequencing peak, indicating that Box 6 was expressed; the reverse sequencing peak was in A double peak before exon 16 indicated Box 7 exp...
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