EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit
A detection kit and fluorescence quantitative technology, applied in the field of molecular biology, can solve the problems of difficulty in popularization, high cost and time-consuming
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Embodiment 1
[0108] Example 1: Detection of EML4-ALK subtype 1 (E13; A20) fusion gene A pair of probes and primers designed to specifically detect the EML4-ALK subtype 1 (E13; A20) fusion gene:
[0109] Variant 1 - forward primer: 5'-GAGTCATGCTTATATGGAGCAAAACT-3' (SEQ ID NO.1);
[0110] Variant 1 - reverse primer: 5'-TCAGCTTGTACTCAGGGCTCTG-3' (SEQ ID NO.2);
[0111] Variant 1-Taqman-probe: 5'-FAM-CCTAAAGTGTACCGCCGGAAGCACC-TAMRA-3' (SEQ ID NO.3);
[0112] Then optimize the reaction system for FQ-PCR detection: 15 μl reaction system, 0.15 μl (20 μM) of forward and reverse primers, 0.2 μl (20 μM) of each probe, 2.5 μl of sample template DNA (100-300 ng / μl), 2 *Taqman universal PCR.
[0113] Master Mix (purchased from Applied Bio, USA) 7.5 μl, ddH 2 O4.3 μl.
[0114] PCR reaction conditions: pre-denaturation at 95°C for 30 sec; and 65 cycles of amplification reaction at 95°C for 5 seconds and 58°C for 33 seconds. At the same time, in the fluorescence quantitative test, while testing the s...
Embodiment 2
[0115] Example 2: Detection of EML4-ALK subtype 2 (E20; A20) fusion gene
[0116] Design a pair of probes and primers that can specifically detect the EML4-ALK subtype 2 (E20; A20) fusion gene:
[0117] Variant 2 - forward primer: 5'-AAGTATATAATGTCTAACTCGGGAGACTATGA-3' (SEQ ID NO.4);
[0118] Variant 2 - reverse primer: 5'-AGCAGTAGTTGGGGTTGTAGTCG-3' (SEQ ID NO.5);
[0119] Variant 2-Taqman-probe: 5'-FAM-TTGTACTTGTACCGCCGGAAGCACCA-TAMRA-3' (SEQ ID NO.6);
[0120] Then optimize the reaction system for FQ-PCR detection: 15 μl reaction system, 0.15 μl (20 μM) of forward and reverse primers, 0.2 μl (20 μM) of each probe, 2.5 μl of sample template DNA (100-300 ng / μl), 2 *Taqman universalPCR
[0121]Master Mix (purchased from Applied Bio, USA) 7.5 μl, ddH 2 O4.3 μl.
[0122] PCR reaction conditions: pre-denaturation at 95°C for 30 sec; and 65 cycles of amplification reaction at 95°C for 5 seconds and 58°C for 33 seconds. At the same time, in the fluorescence quantitative test, ...
Embodiment 3
[0123] Example 3: Detection of EML4-ALK subtype 3a (E6a; A20) fusion gene
[0124] Design a pair of probes and primers that can specifically detect EML4-ALK subtype 3a (E6a; A20) fusion gene:
[0125] Variant 3a - forward primer: 5'-ACCAAAACTGCAGACAAGCATAAAG-3' (SEQ ID NO. 7);
[0126] Variant 3a - reverse primer: 5'-AGCTTGCTCAGCTTGTACTCAGG-3' (SEQ ID NO. 8);
[0127] Variant 3a-Taqman-probe: 5'-FAM-TGTCATCATCAACCAAGTGTACCGCCG-TAMRA-3' (SEQ ID NO.9);
[0128] Then optimize the reaction system for FQ-PCR detection: 15 μl reaction system, 0.15 μl (20 μM) of forward and reverse primers, 0.2 μl (20 μM) of each probe, 2.5 μl of sample template DNA (100-300 ng / μl), 2 *Taqman universal PCR
[0129] Master Mix (purchased from Applied Bio, USA) 7.5 μl, ddH 2 O4.3 μl.
[0130] PCR reaction conditions: pre-denaturation at 95°C for 30 sec; and 65 cycles of amplification reaction at 95°C for 5 seconds and 58°C for 33 seconds. At the same time, in the fluorescence quantitative test, w...
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