CN110684848A - 用于遗传性肿瘤胚系突变检测的多重pcr引物组及试剂盒 - Google Patents
用于遗传性肿瘤胚系突变检测的多重pcr引物组及试剂盒 Download PDFInfo
- Publication number
- CN110684848A CN110684848A CN201911021454.6A CN201911021454A CN110684848A CN 110684848 A CN110684848 A CN 110684848A CN 201911021454 A CN201911021454 A CN 201911021454A CN 110684848 A CN110684848 A CN 110684848A
- Authority
- CN
- China
- Prior art keywords
- dna
- artificial primer
- primer
- pcr
- artificial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000035772 mutation Effects 0.000 title claims abstract description 68
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 60
- 210000004602 germ cell Anatomy 0.000 title claims abstract description 26
- 230000002068 genetic effect Effects 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 title abstract description 13
- 238000007403 mPCR Methods 0.000 claims abstract description 29
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 108020004414 DNA Proteins 0.000 claims description 288
- 239000011324 bead Substances 0.000 claims description 41
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 238000012408 PCR amplification Methods 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 9
- 102000036365 BRCA1 Human genes 0.000 claims description 8
- 101150072950 BRCA1 gene Proteins 0.000 claims description 8
- 230000003321 amplification Effects 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 108700020463 BRCA1 Proteins 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 108010004586 Ataxia Telangiectasia Mutated Proteins Proteins 0.000 claims description 4
- 102000052609 BRCA2 Human genes 0.000 claims description 4
- 108700020462 BRCA2 Proteins 0.000 claims description 4
- 101150008921 Brca2 gene Proteins 0.000 claims description 4
- 108010009392 Cyclin-Dependent Kinase Inhibitor p16 Proteins 0.000 claims description 4
- 102100033996 Double-strand break repair protein MRE11 Human genes 0.000 claims description 4
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 4
- 102100033254 Tumor suppressor ARF Human genes 0.000 claims description 4
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 102100033793 ALK tyrosine kinase receptor Human genes 0.000 claims description 3
- 102100034580 AT-rich interactive domain-containing protein 1A Human genes 0.000 claims description 3
- 102100034540 Adenomatous polyposis coli protein Human genes 0.000 claims description 3
- 102100033816 Aldehyde dehydrogenase, mitochondrial Human genes 0.000 claims description 3
- 102100034044 All-trans-retinol dehydrogenase [NAD(+)] ADH1B Human genes 0.000 claims description 3
- 102100035683 Axin-2 Human genes 0.000 claims description 3
- 101700002522 BARD1 Proteins 0.000 claims description 3
- 102100028048 BRCA1-associated RING domain protein 1 Human genes 0.000 claims description 3
- 102100035631 Bloom syndrome protein Human genes 0.000 claims description 3
- 102100025423 Bone morphogenetic protein receptor type-1A Human genes 0.000 claims description 3
- 102100034808 CCAAT/enhancer-binding protein alpha Human genes 0.000 claims description 3
- 102100028914 Catenin beta-1 Human genes 0.000 claims description 3
- 108091007854 Cdh1/Fizzy-related Proteins 0.000 claims description 3
- 102000038594 Cdh1/Fizzy-related Human genes 0.000 claims description 3
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 claims description 3
- 102100035186 DNA excision repair protein ERCC-1 Human genes 0.000 claims description 3
- 108010086291 Deubiquitinating Enzyme CYLD Proteins 0.000 claims description 3
- 102100023387 Endoribonuclease Dicer Human genes 0.000 claims description 3
- 101000924266 Homo sapiens AT-rich interactive domain-containing protein 1A Proteins 0.000 claims description 3
- 101000780453 Homo sapiens All-trans-retinol dehydrogenase [NAD(+)] ADH1B Proteins 0.000 claims description 3
- 101000874569 Homo sapiens Axin-2 Proteins 0.000 claims description 3
- 101000934638 Homo sapiens Bone morphogenetic protein receptor type-1A Proteins 0.000 claims description 3
- 101000945515 Homo sapiens CCAAT/enhancer-binding protein alpha Proteins 0.000 claims description 3
- 101000916173 Homo sapiens Catenin beta-1 Proteins 0.000 claims description 3
- 101000876529 Homo sapiens DNA excision repair protein ERCC-1 Proteins 0.000 claims description 3
- 101001095815 Homo sapiens E3 ubiquitin-protein ligase RING2 Proteins 0.000 claims description 3
- 101000907904 Homo sapiens Endoribonuclease Dicer Proteins 0.000 claims description 3
- 101001057193 Homo sapiens Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 1 Proteins 0.000 claims description 3
- 101000896484 Homo sapiens Mitotic checkpoint protein BUB3 Proteins 0.000 claims description 3
- 101000896657 Homo sapiens Mitotic checkpoint serine/threonine-protein kinase BUB1 Proteins 0.000 claims description 3
- 101000794228 Homo sapiens Mitotic checkpoint serine/threonine-protein kinase BUB1 beta Proteins 0.000 claims description 3
- 101000945735 Homo sapiens Parafibromin Proteins 0.000 claims description 3
- 101000740048 Homo sapiens Ubiquitin carboxyl-terminal hydrolase BAP1 Proteins 0.000 claims description 3
- 208000026350 Inborn Genetic disease Diseases 0.000 claims description 3
- 101000740049 Latilactobacillus curvatus Bioactive peptide 1 Proteins 0.000 claims description 3
- 102100027240 Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 1 Human genes 0.000 claims description 3
- 108010009513 Mitochondrial Aldehyde Dehydrogenase Proteins 0.000 claims description 3
- 102100021718 Mitotic checkpoint protein BUB3 Human genes 0.000 claims description 3
- 102100021691 Mitotic checkpoint serine/threonine-protein kinase BUB1 Human genes 0.000 claims description 3
- 102100030144 Mitotic checkpoint serine/threonine-protein kinase BUB1 beta Human genes 0.000 claims description 3
- 102100034743 Parafibromin Human genes 0.000 claims description 3
- 102100028286 Proto-oncogene tyrosine-protein kinase receptor Ret Human genes 0.000 claims description 3
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 claims description 3
- 102100024250 Ubiquitin carboxyl-terminal hydrolase CYLD Human genes 0.000 claims description 3
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 3
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 3
- 208000016361 genetic disease Diseases 0.000 claims description 3
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 claims description 3
- 102100027452 ATP-dependent DNA helicase Q4 Human genes 0.000 claims description 2
- 102100035886 Adenine DNA glycosylase Human genes 0.000 claims description 2
- 101710195848 Centrosomal protein CEP57L1 Proteins 0.000 claims description 2
- 102100031213 Centrosomal protein of 57 kDa Human genes 0.000 claims description 2
- 101710147964 Centrosomal protein of 57 kDa Proteins 0.000 claims description 2
- 108010043471 Core Binding Factor Alpha 2 Subunit Proteins 0.000 claims description 2
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 claims description 2
- 102000004480 Cyclin-Dependent Kinase Inhibitor p57 Human genes 0.000 claims description 2
- 108010017222 Cyclin-Dependent Kinase Inhibitor p57 Proteins 0.000 claims description 2
- 102100036252 Cyclin-dependent kinase 4 Human genes 0.000 claims description 2
- 102100038017 DIS3-like exonuclease 2 Human genes 0.000 claims description 2
- 102100021122 DNA damage-binding protein 2 Human genes 0.000 claims description 2
- 102100031866 DNA excision repair protein ERCC-5 Human genes 0.000 claims description 2
- 108010035476 DNA excision repair protein ERCC-5 Proteins 0.000 claims description 2
- 102100028849 DNA mismatch repair protein Mlh3 Human genes 0.000 claims description 2
- 102100034157 DNA mismatch repair protein Msh2 Human genes 0.000 claims description 2
- 102100037700 DNA mismatch repair protein Msh3 Human genes 0.000 claims description 2
- 102100021147 DNA mismatch repair protein Msh6 Human genes 0.000 claims description 2
- 102100035481 DNA polymerase eta Human genes 0.000 claims description 2
- 102100029094 DNA repair endonuclease XPF Human genes 0.000 claims description 2
- 102100039116 DNA repair protein RAD50 Human genes 0.000 claims description 2
- 102100034484 DNA repair protein RAD51 homolog 3 Human genes 0.000 claims description 2
- 102100034483 DNA repair protein RAD51 homolog 4 Human genes 0.000 claims description 2
- 101100226017 Dictyostelium discoideum repD gene Proteins 0.000 claims description 2
- 102100036367 Dual 3',5'-cyclic-AMP and -GMP phosphodiesterase 11A Human genes 0.000 claims description 2
- 102000012804 EPCAM Human genes 0.000 claims description 2
- 101150084967 EPCAM gene Proteins 0.000 claims description 2
- 101150097734 EPHB2 gene Proteins 0.000 claims description 2
- 101150105460 ERCC2 gene Proteins 0.000 claims description 2
- 102100031785 Endothelial transcription factor GATA-2 Human genes 0.000 claims description 2
- 102100031968 Ephrin type-B receptor 2 Human genes 0.000 claims description 2
- 102100029055 Exostosin-1 Human genes 0.000 claims description 2
- 102100029074 Exostosin-2 Human genes 0.000 claims description 2
- 108010067741 Fanconi Anemia Complementation Group N protein Proteins 0.000 claims description 2
- 102000016627 Fanconi Anemia Complementation Group N protein Human genes 0.000 claims description 2
- 102100034553 Fanconi anemia group J protein Human genes 0.000 claims description 2
- 102100034552 Fanconi anemia group M protein Human genes 0.000 claims description 2
- 102100027909 Folliculin Human genes 0.000 claims description 2
- 102100028122 Forkhead box protein P1 Human genes 0.000 claims description 2
- 102100029974 GTPase HRas Human genes 0.000 claims description 2
- 102100031885 General transcription and DNA repair factor IIH helicase subunit XPB Human genes 0.000 claims description 2
- 102100035184 General transcription and DNA repair factor IIH helicase subunit XPD Human genes 0.000 claims description 2
- 102100032530 Glypican-3 Human genes 0.000 claims description 2
- 102100022057 Hepatocyte nuclear factor 1-alpha Human genes 0.000 claims description 2
- 102100038720 Histone deacetylase 9 Human genes 0.000 claims description 2
- 102100038970 Histone-lysine N-methyltransferase EZH2 Human genes 0.000 claims description 2
- 102100032742 Histone-lysine N-methyltransferase SETD2 Human genes 0.000 claims description 2
- 102100029239 Histone-lysine N-methyltransferase, H3 lysine-36 specific Human genes 0.000 claims description 2
- 102100021088 Homeobox protein Hox-B13 Human genes 0.000 claims description 2
- 101000580577 Homo sapiens ATP-dependent DNA helicase Q4 Proteins 0.000 claims description 2
- 101001000351 Homo sapiens Adenine DNA glycosylase Proteins 0.000 claims description 2
- 101000884385 Homo sapiens Arylamine N-acetyltransferase 1 Proteins 0.000 claims description 2
- 101000951062 Homo sapiens DIS3-like exonuclease 2 Proteins 0.000 claims description 2
- 101001041466 Homo sapiens DNA damage-binding protein 2 Proteins 0.000 claims description 2
- 101000577867 Homo sapiens DNA mismatch repair protein Mlh3 Proteins 0.000 claims description 2
- 101001134036 Homo sapiens DNA mismatch repair protein Msh2 Proteins 0.000 claims description 2
- 101001027762 Homo sapiens DNA mismatch repair protein Msh3 Proteins 0.000 claims description 2
- 101000968658 Homo sapiens DNA mismatch repair protein Msh6 Proteins 0.000 claims description 2
- 101001094607 Homo sapiens DNA polymerase eta Proteins 0.000 claims description 2
- 101000865085 Homo sapiens DNA polymerase theta Proteins 0.000 claims description 2
- 101000743929 Homo sapiens DNA repair protein RAD50 Proteins 0.000 claims description 2
- 101001132271 Homo sapiens DNA repair protein RAD51 homolog 3 Proteins 0.000 claims description 2
- 101001132266 Homo sapiens DNA repair protein RAD51 homolog 4 Proteins 0.000 claims description 2
- 101000591400 Homo sapiens Double-strand break repair protein MRE11 Proteins 0.000 claims description 2
- 101001072029 Homo sapiens Dual 3',5'-cyclic-AMP and -GMP phosphodiesterase 11A Proteins 0.000 claims description 2
- 101001066265 Homo sapiens Endothelial transcription factor GATA-2 Proteins 0.000 claims description 2
- 101001034811 Homo sapiens Eukaryotic translation initiation factor 4 gamma 2 Proteins 0.000 claims description 2
- 101000918311 Homo sapiens Exostosin-1 Proteins 0.000 claims description 2
- 101000918275 Homo sapiens Exostosin-2 Proteins 0.000 claims description 2
- 101000848171 Homo sapiens Fanconi anemia group J protein Proteins 0.000 claims description 2
- 101000848187 Homo sapiens Fanconi anemia group M protein Proteins 0.000 claims description 2
- 101001060703 Homo sapiens Folliculin Proteins 0.000 claims description 2
- 101001059893 Homo sapiens Forkhead box protein P1 Proteins 0.000 claims description 2
- 101000584633 Homo sapiens GTPase HRas Proteins 0.000 claims description 2
- 101000920748 Homo sapiens General transcription and DNA repair factor IIH helicase subunit XPB Proteins 0.000 claims description 2
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 claims description 2
- 101001045751 Homo sapiens Hepatocyte nuclear factor 1-alpha Proteins 0.000 claims description 2
- 101001032092 Homo sapiens Histone deacetylase 9 Proteins 0.000 claims description 2
- 101000882127 Homo sapiens Histone-lysine N-methyltransferase EZH2 Proteins 0.000 claims description 2
- 101000654725 Homo sapiens Histone-lysine N-methyltransferase SETD2 Proteins 0.000 claims description 2
- 101000634050 Homo sapiens Histone-lysine N-methyltransferase, H3 lysine-36 specific Proteins 0.000 claims description 2
- 101001041145 Homo sapiens Homeobox protein Hox-B13 Proteins 0.000 claims description 2
- 101000580021 Homo sapiens Inactive rhomboid protein 2 Proteins 0.000 claims description 2
- 101001046526 Homo sapiens Killin Proteins 0.000 claims description 2
- 101001038435 Homo sapiens Leucine-zipper-like transcriptional regulator 1 Proteins 0.000 claims description 2
- 101000738901 Homo sapiens PMS1 protein homolog 1 Proteins 0.000 claims description 2
- 101000692768 Homo sapiens Paired mesoderm homeobox protein 2B Proteins 0.000 claims description 2
- 101000987581 Homo sapiens Perforin-1 Proteins 0.000 claims description 2
- 101000605639 Homo sapiens Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Proteins 0.000 claims description 2
- 101000601770 Homo sapiens Protein polybromo-1 Proteins 0.000 claims description 2
- 101000631899 Homo sapiens Ribosome maturation protein SBDS Proteins 0.000 claims description 2
- 101000655897 Homo sapiens Serine protease 1 Proteins 0.000 claims description 2
- 101000777277 Homo sapiens Serine/threonine-protein kinase Chk2 Proteins 0.000 claims description 2
- 101000628562 Homo sapiens Serine/threonine-protein kinase STK11 Proteins 0.000 claims description 2
- 101000639975 Homo sapiens Sodium-dependent noradrenaline transporter Proteins 0.000 claims description 2
- 101000702606 Homo sapiens Structure-specific endonuclease subunit SLX4 Proteins 0.000 claims description 2
- 101000951145 Homo sapiens Succinate dehydrogenase [ubiquinone] cytochrome b small subunit, mitochondrial Proteins 0.000 claims description 2
- 101000685323 Homo sapiens Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial Proteins 0.000 claims description 2
- 101000874160 Homo sapiens Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial Proteins 0.000 claims description 2
- 101000934888 Homo sapiens Succinate dehydrogenase cytochrome b560 subunit, mitochondrial Proteins 0.000 claims description 2
- 101000772267 Homo sapiens Thyrotropin receptor Proteins 0.000 claims description 2
- 101000637950 Homo sapiens Transmembrane protein 127 Proteins 0.000 claims description 2
- 101001087416 Homo sapiens Tyrosine-protein phosphatase non-receptor type 11 Proteins 0.000 claims description 2
- 101001026573 Homo sapiens cAMP-dependent protein kinase type I-alpha regulatory subunit Proteins 0.000 claims description 2
- 102100027537 Inactive rhomboid protein 2 Human genes 0.000 claims description 2
- 102000001626 Kazal Pancreatic Trypsin Inhibitor Human genes 0.000 claims description 2
- 108010093811 Kazal Pancreatic Trypsin Inhibitor Proteins 0.000 claims description 2
- 102100022260 Killin Human genes 0.000 claims description 2
- 102100040274 Leucine-zipper-like transcriptional regulator 1 Human genes 0.000 claims description 2
- 108700019589 MRE11 Homologue Proteins 0.000 claims description 2
- 229910015837 MSH2 Inorganic materials 0.000 claims description 2
- 102000013760 Microphthalmia-Associated Transcription Factor Human genes 0.000 claims description 2
- 108010050345 Microphthalmia-Associated Transcription Factor Proteins 0.000 claims description 2
- 108010074346 Mismatch Repair Endonuclease PMS2 Proteins 0.000 claims description 2
- 102100025725 Mothers against decapentaplegic homolog 4 Human genes 0.000 claims description 2
- 101710143112 Mothers against decapentaplegic homolog 4 Proteins 0.000 claims description 2
- 102100037482 PMS1 protein homolog 1 Human genes 0.000 claims description 2
- 108010011536 PTEN Phosphohydrolase Proteins 0.000 claims description 2
- 102000014160 PTEN Phosphohydrolase Human genes 0.000 claims description 2
- 102100026354 Paired mesoderm homeobox protein 2B Human genes 0.000 claims description 2
- 102000012850 Patched-1 Receptor Human genes 0.000 claims description 2
- 108010065129 Patched-1 Receptor Proteins 0.000 claims description 2
- 102100028467 Perforin-1 Human genes 0.000 claims description 2
- 102100038332 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Human genes 0.000 claims description 2
- 102100037516 Protein polybromo-1 Human genes 0.000 claims description 2
- 102000001195 RAD51 Human genes 0.000 claims description 2
- 108010068097 Rad51 Recombinase Proteins 0.000 claims description 2
- 102100029981 Receptor tyrosine-protein kinase erbB-4 Human genes 0.000 claims description 2
- 101710100963 Receptor tyrosine-protein kinase erbB-4 Proteins 0.000 claims description 2
- 102100028750 Ribosome maturation protein SBDS Human genes 0.000 claims description 2
- 102100025373 Runt-related transcription factor 1 Human genes 0.000 claims description 2
- 108700028341 SMARCB1 Proteins 0.000 claims description 2
- 101150008214 SMARCB1 gene Proteins 0.000 claims description 2
- 102100025746 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 Human genes 0.000 claims description 2
- 102100032491 Serine protease 1 Human genes 0.000 claims description 2
- 102100031075 Serine/threonine-protein kinase Chk2 Human genes 0.000 claims description 2
- 102100026715 Serine/threonine-protein kinase STK11 Human genes 0.000 claims description 2
- 102100033929 Sodium-dependent noradrenaline transporter Human genes 0.000 claims description 2
- 102100031003 Structure-specific endonuclease subunit SLX4 Human genes 0.000 claims description 2
- 102100038014 Succinate dehydrogenase [ubiquinone] cytochrome b small subunit, mitochondrial Human genes 0.000 claims description 2
- 102100023155 Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial Human genes 0.000 claims description 2
- 102100035726 Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial Human genes 0.000 claims description 2
- 102100031715 Succinate dehydrogenase assembly factor 2, mitochondrial Human genes 0.000 claims description 2
- 108050007461 Succinate dehydrogenase assembly factor 2, mitochondrial Proteins 0.000 claims description 2
- 102100025393 Succinate dehydrogenase cytochrome b560 subunit, mitochondrial Human genes 0.000 claims description 2
- 101150057140 TACSTD1 gene Proteins 0.000 claims description 2
- 102100033455 TGF-beta receptor type-2 Human genes 0.000 claims description 2
- 102100029337 Thyrotropin receptor Human genes 0.000 claims description 2
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 claims description 2
- 102100032072 Transmembrane protein 127 Human genes 0.000 claims description 2
- 102100033019 Tyrosine-protein phosphatase non-receptor type 11 Human genes 0.000 claims description 2
- 108700031763 Xeroderma Pigmentosum Group D Proteins 0.000 claims description 2
- 102100037490 cAMP-dependent protein kinase type I-alpha regulatory subunit Human genes 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 101150071637 mre11 gene Proteins 0.000 claims description 2
- 108010073629 xeroderma pigmentosum group F protein Proteins 0.000 claims description 2
- 102100037480 Mismatch repair endonuclease PMS2 Human genes 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 36
- 238000012217 deletion Methods 0.000 abstract description 9
- 230000037430 deletion Effects 0.000 abstract description 9
- 108700020796 Oncogene Proteins 0.000 abstract description 6
- 102000043276 Oncogene Human genes 0.000 abstract description 6
- 238000012165 high-throughput sequencing Methods 0.000 abstract description 5
- 239000012634 fragment Substances 0.000 abstract description 4
- 230000036541 health Effects 0.000 abstract description 4
- 238000012163 sequencing technique Methods 0.000 abstract description 4
- 238000003780 insertion Methods 0.000 abstract description 3
- 230000037431 insertion Effects 0.000 abstract description 3
- 201000011510 cancer Diseases 0.000 description 30
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 206010064571 Gene mutation Diseases 0.000 description 10
- 201000010099 disease Diseases 0.000 description 10
- 206010006187 Breast cancer Diseases 0.000 description 9
- 208000026310 Breast neoplasm Diseases 0.000 description 9
- 238000012360 testing method Methods 0.000 description 8
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 7
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 7
- 201000002528 pancreatic cancer Diseases 0.000 description 7
- 208000008443 pancreatic carcinoma Diseases 0.000 description 7
- 206010061535 Ovarian neoplasm Diseases 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000001717 pathogenic effect Effects 0.000 description 6
- 206010033128 Ovarian cancer Diseases 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 108700040618 BRCA1 Genes Proteins 0.000 description 4
- 208000008051 Hereditary Nonpolyposis Colorectal Neoplasms Diseases 0.000 description 4
- 201000005027 Lynch syndrome Diseases 0.000 description 4
- 206010029260 Neuroblastoma Diseases 0.000 description 4
- 208000011580 syndromic disease Diseases 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 206010018338 Glioma Diseases 0.000 description 3
- 206010051922 Hereditary non-polyposis colorectal cancer syndrome Diseases 0.000 description 3
- 101100520007 Homo sapiens PHOX2B gene Proteins 0.000 description 3
- 101150033433 Msh2 gene Proteins 0.000 description 3
- 101150097597 PHOX2B gene Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 201000008519 polycystic kidney disease 1 Diseases 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 102000000872 ATM Human genes 0.000 description 2
- 108091007743 BRCA1/2 Proteins 0.000 description 2
- 108700010154 BRCA2 Genes Proteins 0.000 description 2
- -1 CDKN 11 Proteins 0.000 description 2
- 101100491149 Caenorhabditis elegans lem-3 gene Proteins 0.000 description 2
- 208000026372 Congenital cystic kidney disease Diseases 0.000 description 2
- 230000005971 DNA damage repair Effects 0.000 description 2
- RNPABQVCNAUEIY-GUQYYFCISA-N Germine Chemical compound O1[C@@]([C@H](CC[C@]23C)O)(O)[C@H]3C[C@@H](O)[C@@H]([C@]3(O)[C@@H](O)[C@H](O)[C@@H]4[C@]5(C)O)[C@@]12C[C@H]3[C@@H]4CN1[C@H]5CC[C@H](C)C1 RNPABQVCNAUEIY-GUQYYFCISA-N 0.000 description 2
- 101150107124 MED12 gene Proteins 0.000 description 2
- 208000000172 Medulloblastoma Diseases 0.000 description 2
- 108010026664 MutL Protein Homolog 1 Proteins 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- RNPABQVCNAUEIY-UHFFFAOYSA-N germine Natural products O1C(C(CCC23C)O)(O)C3CC(O)C(C3(O)C(O)C(O)C4C5(C)O)C12CC3C4CN1C5CCC(C)C1 RNPABQVCNAUEIY-UHFFFAOYSA-N 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000007918 pathogenicity Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 208000030761 polycystic kidney disease Diseases 0.000 description 2
- 208000015768 polyposis Diseases 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- 101150028074 2 gene Proteins 0.000 description 1
- 101710083984 AH receptor-interacting protein Proteins 0.000 description 1
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 102100039723 Aurora kinase A-interacting protein Human genes 0.000 description 1
- 208000024400 Blepharophimosis-intellectual disability syndrome, Ohdo type Diseases 0.000 description 1
- 108091009167 Bloom syndrome protein Proteins 0.000 description 1
- 101150012716 CDK1 gene Proteins 0.000 description 1
- 101150107019 CEP1 gene Proteins 0.000 description 1
- 101100170001 Caenorhabditis elegans ddb-1 gene Proteins 0.000 description 1
- 208000037051 Chromosomal Instability Diseases 0.000 description 1
- 206010008805 Chromosomal abnormalities Diseases 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 208000026292 Cystic Kidney disease Diseases 0.000 description 1
- 102100037969 DIS3-like exonuclease 1 Human genes 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 201000003727 FG syndrome Diseases 0.000 description 1
- 208000033890 FG syndrome type 1 Diseases 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010058991 Hepatic vein occlusion Diseases 0.000 description 1
- 208000033640 Hereditary breast cancer Diseases 0.000 description 1
- 208000017095 Hereditary nonpolyposis colon cancer Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000779641 Homo sapiens ALK tyrosine kinase receptor Proteins 0.000 description 1
- 101000924577 Homo sapiens Adenomatous polyposis coli protein Proteins 0.000 description 1
- 101000951181 Homo sapiens DIS3-like exonuclease 1 Proteins 0.000 description 1
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 1
- 101100029888 Homo sapiens PKD1 gene Proteins 0.000 description 1
- 101100224483 Homo sapiens POLE gene Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 208000001242 Lujan-Fryns syndrome Diseases 0.000 description 1
- 101150114927 MUC1 gene Proteins 0.000 description 1
- 102000008071 Mismatch Repair Endonuclease PMS2 Human genes 0.000 description 1
- 102000013609 MutL Protein Homolog 1 Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010061876 Obstruction Diseases 0.000 description 1
- 201000003048 Ohdo syndrome Diseases 0.000 description 1
- 101150056230 PKD1 gene Proteins 0.000 description 1
- 101150049489 PKHD1 gene Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 208000036878 aneuploidy Diseases 0.000 description 1
- 231100001075 aneuploidy Toxicity 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 208000011654 childhood malignant neoplasm Diseases 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 208000025581 hereditary breast carcinoma Diseases 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 208000004840 megacolon Diseases 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 238000009806 oophorectomy Methods 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 201000006038 polycystic kidney disease 4 Diseases 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了用于遗传性肿瘤胚系突变检测的多重PCR引物组及试剂盒。多重PCR引物组至少包括SEQ ID NO.：1～276所示的引物序列。本发明的多重PCR引物之间无干扰，可以通过一次将引物混合进行多重PCR，扩增出所述要的多种基因，大大减少了PCR的操作量，进而大幅降低了整体的测序成本。在本发明的一些实例中，可以一次扩增出胚系遗传的152个核心CPG致癌基因的全部外显子区域以及常发生突变的内含子区域，包括点突变、小片段缺失和***等突变类型，极大地方便了后续的高通量测序操作。通过对高通量测序数据进行进一步的解读，可以对待测者的肿瘤发病风险进行评估，使其避免相关的不健康生活方式，降低肿瘤发生风险，并指导个性化健康管理。
Description
技术领域
本发明涉及一种用于遗传性肿瘤胚系突变检测的多重PCR引物组及试剂盒。
背景技术
据估计，人类中约有5％～10％肿瘤是由于致癌基因的种系变异(胚系突变)遗传而发生的。胚系突变又叫生殖细胞突变，通常身上所有细胞都带有突变，具有家族遗传性。发生胚系突变的人群中，发生肿瘤的风险要比普通人群发生肿瘤的风险高数十倍或数百倍。父母双亲中，如果其中一方携带致病基因突变并传给了下一代，其后代与普通人相比的患癌风险将大大增加。
1990年，研究者发现了一种直接与遗传性乳腺癌有关的基因，命名为乳腺癌1号基因(Brest Cancer Susceptibility Gene 1，BRCA1)。1994年，又发现另外一种与乳腺癌有关的基因，称为BRCA2。研究发现，BRCA1/2是两种具有抑制恶性肿瘤发生的基因，在调节人体细胞的复制、遗传物质DNA损伤修复、细胞的正常生长等方面有着重要的作用。肿瘤细胞的一个明显特征是能够无限制的复制增殖，而BRCA1等抑癌基因可抑制肿瘤细胞的增殖，促进其凋亡，达到抑癌的效果。此外，大多数肿瘤都表现出染色体的不稳定性，如染色体结构异常、非整倍体及染色体的增加或缺失。DNA损伤修复可以使异常的细胞转化为正常细胞或进入细胞凋亡过程。而BRCA1基因的突变导致BRCA1编码的蛋白质从细胞核进入细胞质中，丧失了与DNA结合的能力，从而失去其正常的抑制增殖、促进凋亡和DNA损伤修复等功能。研究发现，拥有BRCA1基因突变的家族倾向于具有高乳腺癌发生率，通常发生在较年轻时，病人的两侧***都患癌几率大，且可能同时患有卵巢癌。
随着分子生物技术蓬勃发展，部分基因突变已被证实为特定癌症的风险因子。其中，比较典型和广为人知的便是BRCA基因突变与乳腺癌和卵巢癌的关系。在过去的研究中，BRCA1和BRCA2基因突变，已被证明与较高的乳腺癌(breast cancer)及卵巢癌(ovariancancer)罹患风险有关。大约5％-10％的乳腺癌病例和10％-18％的卵巢癌病例可归因于胚系BRCA1和BRCA2基因突变。
《自然》中<Realising the Promise of Cancer Predisposition Genes.Naturevolume 505，pages 302–308(16January 2014)>介绍种系突变导致癌症高风险或中度风险增加的基因称为癌症易感性基因(CPG)，超过100个致癌CPG已被确定，在许多领域提供了重要的科学解释，特别是癌症病因机制。
《新英格兰医学杂志》中<Germline Mutations in Predisposition Genes inPediatric Cancer.NEJM.2015Dec 10；373(24):2336-2346>对1120名20岁以下的儿童及青少年癌症患者的健康组织与肿瘤组织进行DNA序列分析，研究结果表明，在儿童及青少年癌症患者中，8.5％的患者中存在致病性或可能致病性的癌症易感基因胚系突变。对于大部分患者，家族病史并不能预测是否存在癌症易感性，即使家族史中没有出现过癌症，儿童也可能携带癌症易感基因。此研究提供了目前最全面的儿童癌症易感基因突变情况，发现肿瘤患儿的癌症易感基因突变率比人们认为的要高，这些突变将增加患癌症的风险。有助于人们进一步了解癌症发生机制。
《美国医学会杂志》中<Association Between Inherited Germline Mutationsin Cancer Predisposition Genes and Risk of Pancreatic Cancer.JAMA.2018；319(23):2401–2409.>对2000-2016年间的3046例胰腺癌病人进行基因测试，将21个癌基因的检测结果与来自123000例无胰腺癌的病人的基因测试结果进行了比较。结果显示6个基因与胰腺癌风险增加存在明显关系：BRCA1、BRCA2、CDKN2A、TP53、MLH1和ATM。5.5％的病人体内的这些基因存在突变，其中包括5.2％的无胰腺癌家族史的病人。因此他们推荐所有胰腺癌病人都进行基因测试，而不管是否有胰腺癌家族史，使得引起胰腺癌的遗传因素背后的分子机制更加清晰。
《细胞》中<Pathogenic Germline Variants in 10,389Adult Cancers.Cell,173(2),355-370>研究分析了TCGA中33个癌种共计10389例样本的致病性变异的图谱，发现致病胚系变异或疑似胚系变异占比8％，在致癌基因中鉴定了33个变异，然后通过在其他癌种群体的验证和RET等位基因的实验，调查了这些变异的功能性后果。最后，又发现了18个拷贝数缺失和47个基于多条功能性证据的VUSs。本研究呈现了关于罕见的胚系易感变异的最大***性分析的结果，也提供了解决癌症功能性问题的强大基础支持。
过去多由医生建议具癌症家族病史的高风险族群进行相关基因检测。但根据《American Journal of Preventive Medicine》日前刊登美国德克萨斯大学医学分部的最新文章中阐述，根据统计结果，自身不具备相关癌症(乳腺癌或卵巢癌)病史、但仍愿意主动进BRCA基因突变检测的女性比例，出现显著增加。在发展为癌症之前，早期发现更多的BRCA1/2突变携带者，并采取适当的预防性治疗措施(例如***切除术和卵巢切除术)，可以降低乳腺癌和卵巢癌的发生率。对于已经诊断为乳腺癌、且BRCA突变阳性的患者，预防性治疗可以降低另一种癌症的发病风险。
美国***与人类服务部发布的2020年健康人标准表明，遗传性突变导致的疾病将显著影响患者与其家庭的生活。在2020年的目标中，基因组医学第一次被置于优先列表中，强调了解家族史与基因史的重要性。
现代生命科学研究告诉我们，“除外伤以外，一切疾病都与基因有关”。有大量资料表明，癌症是一种基因病，先天性的家族遗传及后天的基因突变都可以使基因发生改变，从而导致癌症的发生。随着基因与基因组学研究不断发展，基因检测正引导预测医学的发展，并应用于疾病预防、个性化医疗等各个方面。
然而遗传性肿瘤胚系突变的数量众多，常规的测序检测成本高昂，为降低成本，往往需要减少检测的基因数量。开发出一种低成本的遗传性肿瘤胚系突变检测技术具有非常重要的意义。
发明内容
本发明的目的在于克服现有技术的不足，提供一种用于遗传性肿瘤胚系突变检测的多重PCR引物组及试剂盒。
本发明所采取的技术方案是：
本发明的第一个方面，提供：一种用于遗传性肿瘤胚系突变的多重PCR引物组，所述多重PCR引物组至少包括如下引物序列：
表中，SEQ ID NO.从上到下，从左到右依次为SEQ ID NO.：1～276。
在一些引物的实例中，所述多重PCR引物组还包括检测如下遗传病相关的引物组：
ADH1B、AIP、ALDH2、ALK、APC、ARID1A、ATM、AXIN2、BAP1、BARD1、BLM、BMPR1A、BRCA1、BRCA2、BRIP1、BUB1、BUB1B、BUB3、CDC73、CDH1、CDK4、CDKN1C、CDKN2A、CEBPA、CEP57、CHEK2、CTNNB1、CYLD、DDB2、DICER1、DIS3L2、EGFR、EPCAM、EPHB2、ERBB4、ERCC1、ERCC2、ERCC3、ERCC4、ERCC5、EXT1、EXT2、EZH2、FANCA、FANCB、FANCC、FANCD2、FANCE、FANCF、FANCG、FANCI、FANCL、FANCM、FH、FLCN、FOXP1、GATA2、GPC3、HDAC9、HNF1A、HOXB13、HRAS、KIT、KLLN、KRAS、LZTR1、MAX、MEN1、MET、MITF、MLH1、MLH3、MPL、MRE11、MRE11A、MSH2、MSH3、MSH6、MUTYH、NAT1、NBN、NF1、NF2、NSD1、PALB2、PBRM1、PDE11A、PHOX2B、PIK3CA、PMS1、PMS2、POLE、POLH、PRF1、PRKAR1A、PRSS1、PTCH1、PTEN、PTPN11、RAD50、RAD51、RAD51C、RAD51D、RB1、RECQL4、RET、RHBDF2、RUNX1、SBDS、SDHA、SDHAF2、SDHB、SDHC、SDHD、SETD2、SLX4、SMAD4、SMARCB1、SPINK1、STK11、SUFU、TGFBR2、TMEM127、TP53、TSC1、TSC2、TSHR、VHL、WRN、WT1、XPA、XPC。
本发明的第二个方面，提供：一种用于遗传性肿瘤胚系突变的多重PCR试剂盒，其使用的多重PCR引物组如本发明第一个方面所述。
本发明的第三个方面，提供：一种多重PCR的方法，其使用的多重PCR引物组如本发明第一个方面所述。
在一些多重PCR的实例中，PCR反应体系中，dNTP的初始浓度为0.3mM，Mg2+的浓度为2.5mM，每50μL反应体系中的聚合酶为5U。
在一些多重PCR的实例中，PCR反应体系中，引物的浓度为0.15μM。
在一些多重PCR的实例中，PCR扩增体系如下：
| 试剂名称 | 体积(μL) |
| 样品DNA | 4.5 |
| 捕获引物混合液 | 8 |
| 文库扩增反应液 | 12.5 |
| 总量 | 25 |
。
在一些多重PCR的实例中，PCR扩增的反应条件为：
在一些多重PCR的实例中，还包括PCR产物的纯化，所述纯化包括：
1)在PCR反应完成后的体系内，每一管加入9μL AMPure XP磁珠，混匀，静置；
2)吸附磁珠至溶液澄清，吸取上清至新的0.2ml PCR管中；
3)在所述新的PCR管中加入15μL磁珠，混匀，静置，吸附磁珠至溶液澄清，弃上清，留磁珠；
4)在PCR管中加入100μL 70％乙醇，充分悬浮磁珠后，吸附磁珠至溶液澄清，弃上清，留磁珠，挥干乙醇；
5)加入15μL洗脱液，充分悬浮磁珠，洗脱DNA，其中，所述洗脱液为10mM Tris-HCl，pH 7.5-8.0，温度为55～65℃。
在一些多重PCR的实例中，所述洗脱液的温度为60℃。
本发明的有益效果是：
本发明的多重PCR引物之间无干扰，可以通过一次将引物混合进行多重PCR，扩增出所述要的多种基因，大大减少了PCR的操作量，进而大幅降低了整体的测序成本。
在本发明的一些实例中，可以一次扩增出胚系遗传的152个核心CPG致癌基因(参考包括MSK-IMPACT中的468个基因)的全部外显子区域以及常发生突变的内含子区域，包括点突变、小片段缺失和***(Indel)等突变类型。极大地方便了后续的高通量测序操作。通过对高通量测序数据进行进一步的解读，可以对待测者的肿瘤发病风险进行评估，使其避免相关的不健康生活方式，降低肿瘤发生风险，并指导个性化健康管理。
具体实施方式
下面结合实施例，进一步说明本发明的技术方案。
引物设计
由于需要在一个体系中完成152个基因序列的扩增，需要设计适合的特异性引物，保证整个扩增引物池的有效扩增。为解决极多重PCR引物设计问题，在上述设计原则的基础上，我们特别应用了以下条件：
1)由于遗传性肿瘤胚系突变基因检测受检样品普遍是血卡样品，DNA存在不同程度降解情况。我们要求扩增产物的长度峰度值在200bp附近。尽可能保证能在降解DNA中获取最大量的信息。
2)引物的设计流程首先使用权威软件Primer3(MIT,Cambridge,MA)设计出预选引物组合m1，然后通过UCSC Genome Browser对m1组合进行本种群的目标扩增区域预测，确认没有发生目标区域的脱靶，从而把m1组合优化为m2组合。然后使用发明人的MendelGene-PdesignV1.2软件把m2组合里面的不适合引物剔除，具体方法是通过全基因组BLAST搜索来避免重复序列，通过结构预测来避免高度折叠区域，再通过配对测试来剔除可能的引物二聚体。
3)引物的位点特异性序列设计得比一般PCR引物稍长一点(24–35bases)，解链温度稍高一点(平均65℃以上)，并且GC含量控制在50–60％。
4)保证每个单一引物浓度都低于单重PCR中的浓度，否则将会使反应中的总引物浓度会就会太高。例如如果该单一引物在传统单重PCR反应中使用的引物浓度为0.2μM，那么我们就会尝试使用0.15μM浓度(引物浓度范围通常在0.05μM至0.4μM之间)。
5)一般多重PCR需要更多的dNTP、镁离子和聚合酶。我们找到其中一种比较好的组合是0.3mM dNTP(而非标准的0.2mM)、2.5mM Mg2+(而非1.2至2mM)以及每50μL反应体系5units聚合酶(而非1.25units)。
基于以上设计步骤，本方案成功设计出共计3000对引物对，且确保引物对之间互不干扰，能一次性完成152个基因的外显子和包括致病的内含子区域进行捕获建库。
第一轮PCR扩增体系配置
按照表1，在0.2ml PCR管中配制第一轮PCR扩增体系。将已加入DNA模板的PCR扩增体系，涡旋混匀并短暂离心。
表1第一轮PCR扩增体系
第一轮PCR扩增
按照表2中参数设置PCR反应条件，将上一步配制好的体系放入PCR仪中，开始程序。
表2第一轮PCR扩增条件
PCR产物纯化
在进行该步实验之前，将洗脱液预热至60℃可以进一步提高得率。洗脱液为10mMTris-HCl，pH7.5-8.0。
1>在上述反应体系中的每一管加入9ul AMPure XP磁珠,用50ul移液器上下吹打10次充分混匀。室温静置2分钟。
2>磁力架吸附磁珠，5分钟，直至溶液澄清为止。
3>小心吸取上清至新的0.2ml PCR管中，避免吸到磁珠；弃磁珠。(用于去除PCR管中超过1Kb的大片段)。
4>在3>中新的PCR管中加入15ul磁珠，用50ul移液器上下吹打10次充分混匀。室温静置2分钟。
5>磁力架吸附磁珠，5分钟，直至溶液澄清为止。用移液器小心吸取上清，弃去上清，留磁珠。
6>加入100ul 70％乙醇，用磁力架反复在不同的两面来回吸附磁珠以充分悬浮磁珠。磁力架吸附磁珠，5分钟，直至溶液澄清为止。用移液器小心去除上清，避免吸到磁珠。
7>室温放置5分钟左右直至乙醇挥发干净。此步绝不可省略，否则残余的乙醇会严重影响得率和后续实验。
8>加入15ul洗脱液，充分悬浮磁珠，室温静置2分钟以洗脱DNA。
9>磁力架吸附磁珠，5分钟，直至溶液澄清。将上清14ul转移至一新的0.2ml PCR管中用于后续实验。
第二轮PCR扩增体系配置
在本步骤中引入Illumina测序相应接头标签，不同的样品须选择不同的标签引物。
按照表3，在0.2ml PCR管中配制第二轮PCR扩增体系。然后，将配置好的PCR扩增体系涡旋混匀并短暂离心。
表3第二轮PCR扩增体系
*:不同样本的标签引物必须不一样。做好样本和标签引物对照记录。
第二轮PCR扩增
按照表4中参数设置PCR反应条件，将上一步配制好的体系放入PCR仪中，开始程序。
表4第二轮PCR扩增条件
将上步中的PCR体系放入PCR仪中，开始程序。该步骤用时约20分钟。
产物纯化回收
7.1>第一轮回收：
①PCR产物中加21ul AMPure XP磁珠，用50ul移液器上下吹打10次以充分混匀。室温静置2分钟。
②磁力架吸附磁珠，5分钟，直至溶液澄清为止。
③移液器小心吸取上清，抛弃上清，留磁珠。
④加入100ul 70％乙醇，用磁力架反复在不同的两面来回吸附磁珠以充分悬浮磁珠便洗涤。
⑤磁力架吸附磁珠，2分钟，直至溶液澄清为止。用移液器小心去除上清，避免吸到磁珠。
⑥室温放置，直至乙醇挥发干净。
⑦加入20ul洗脱液，充分悬浮磁珠，室温静置2min以洗脱DNA。
⑧将磁珠用磁力架吸附，所得到的DNA溶液上清吸至一新的0.2ml PCR管中
7.2>第二轮回收：
步骤同第一轮回收，但在第一步中加入AMPure XP磁珠体积为12ul。所得到的20ulDNA溶液可直接用于后续试验或置于-20℃保存。
实验数据
本次实验对肿瘤患者及有肿瘤家庭史的人群的人类全外显子区域进行高通量测序。实验针对人类基因组的编码基因区域进行检测，检测包括基因编码区单核苷酸位点变异(SNV)、小片段***/缺失(INDEL)等突变类型。
相关的检测基因列表：ADH1B、AIP、ALDH2、ALK、APC、ARID1A、ATM、AXIN2、BAP1、BARD1、BLM、BMPR1A、BRCA1、BRCA2、BRIP1、BUB1、BUB1B、BUB3、CDC73、CDH1、CDK4、CDKN1C、CDKN2A、CEBPA、CEP57、CHEK2、CTNNB1、CYLD、DDB2、DICER1、DIS3L2、EGFR、EPCAM、EPHB2、ERBB4、ERCC1、ERCC2、ERCC3、ERCC4、ERCC5、EXT1、EXT2、EZH2、FANCA、FANCB、FANCC、FANCD2、FANCE、FANCF、FANCG、FANCI、FANCL、FANCM、FH、FLCN、FOXP1、GATA2、GPC3、HDAC9、HNF1A、HOXB13、HRAS、KIT、KLLN、KRAS、LZTR1、MAX、MEN1、MET、MITF、MLH1、MLH3、MPL、MRE11、MRE11A、MSH2、MSH3、MSH6、MUTYH、NAT1、NBN、NF1、NF2、NSD1、PALB2、PBRM1、PDE11A、PHOX2B、PIK3CA、PMS1、PMS2、POLE、POLH、PRF1、PRKAR1A、PRSS1、PTCH1、PTEN、PTPN11、RAD50、RAD51、RAD51C、RAD51D、RB1、RECQL4、RET、RHBDF2、RUNX1、SBDS、SDHA、SDHAF2、SDHB、SDHC、SDHD、SETD2、SLX4、SMAD4、SMARCB1、SPINK1、STK11、SUFU、TGFBR2、TMEM127、TP53、TSC1、TSC2、TSHR、VHL、WRN、WT1、XPA、XPC。
本发明实验结果：
第一例实验受检者为兄妹两人，家系信息如下：
两位受检者共检测到可疑突变8个：如下表所示。综合ClinVar和OMIM[1]数据库可知，患者携带2个遗传性肿瘤易感综合征的POLE基因突变，并携带肝硬化、免疫缺陷性肝静脉闭塞病和遗传性肝内胆汁阻塞疾病的相关突变，因此可能导致临床指征的发生和发展。
2)第二例实验受检者临床为诊断髓母细胞瘤，经本发明检测后发现致病位点，并对父母进行验证，检测结果及分析如下：
1>受检者共检测到可疑突变3个，分别为c.A505G点突变(MSH2基因，NM_000251转录本)，c.A6139G点突变(MED12基因，NM_005120转录本)和c.765_779del缺失突变(PHOX2B基因，NM_003924转录本)，其中c.A505G点突变和c.765_779del缺失突变为杂合突变，c.A6139G点突变为纯合突变；MSH2基因和PHOX2B基因为肿瘤遗传风险基因；3个突变均已被ClinVar数据库[3][4][5]收录，该数据库根据ACMG[2]基因突变指南，判定c.A505G点突变为可能良性的变异(Likely benign)，c.A6139G点突变为临床意义不明的变异(Uncertainsignificance)，c.765_779del缺失突变为良性/可能良性的变异(Benign/Likelybenign)。
2>ClinVar和OMIM数据库指明：
①c.A505G点突变(MSH2基因，NM_000251转录本)关联的肿瘤疾病为Lynch综合征(Lynch syndrome，即)和透克氏症(Turcot syndrome/Mismatch repair cancersyndrome)。Lynch综合征即遗传性非息肉病性结直肠癌；而透克氏症又名胶质瘤息肉病综合征，主要特征是具有家族遗传性大肠息肉症的患者併发中枢神经***的肿瘤的发生，通常表现为神经胶质瘤(患者临床诊断所患的髓母细胞瘤是神经胶质瘤的一种病理分类[6])。
②c.A6139G点突变(MED12基因，NM_005120转录本)关联的疾病为智力障碍类疾病(Lujan-Fryns syndrome,Ohdo syndrome,Opitz-Kaveggia syndrome)；
3>c.765_779del缺失突变(PHOX2B基因，NM_003924转录本)关联疾病为成神经细胞瘤易感性2型(Neuroblastoma,susceptibility to,2)和成神经细胞瘤伴巨结肠(Neuroblastoma with Hirschsprung disease)。
3)第三例实验受检者是胎儿B超显示多囊肾，利用本发明对流产组织进行检测后发现致病位点，检测结果及分析如下：
1>受检者在PKD1基因上检测到c.G10102A和c.G3931A杂合突变，未被ClinVar数据库收录，InterVar软件根据ACMG[2]基因突变指南，判定两个点突变为临床意义不明。OMIM数据库指明该两个突变的相关疾病为多囊性肾病1型(Polycystic kidney disease 1)。
2>受检者在MUC1基因上检测到c.C460G杂合突变，未被ClinVar数据库收录，InterVar软件根据ACMG[2]基因突变指南，判定两个点突变为临床意义不明。OMIM数据库指明该两个突变的相关疾病为髓样多囊性肾病1型(Medullary cystic kidney disease 1)。
3>受检者在PKHD1基因上检测到c.T8335G和c.G325A杂合突变，均被ClinVar数据库收录，数据库根据ACMG[2]基因突变指南，判定两个点突变分别为临床意义不明和争议致病性。Clinvar数据库指明该两个突变的相关疾病为多囊性肾病4型(Polycystic kidneydisease 4)。
从实验结果发现，本发明一次检测胚系遗传的152个核心CPG致癌基因(参考包括MSK-IMPACT中的468个基因)的全部外显子区域以及常发生突变的内含子区域，均检测出遗传性肿瘤相关基因的胚系致病突变，检测结果符合受检者临床表现及其他实验室检查结果的临床诊断。
<110>广州万德基因医学科技有限公司
<120>用于遗传性肿瘤胚系突变检测的多重PCR引物组及试剂盒
<160>276
<210>1 <211>22 <212>DNA <213>人工引物 <400>1 cctggaaaggccactttgtaag22
<210>2 <211>25 <212>DNA <213>人工引物 <400>2 gtcgattgattagagcctagtccag25
<210>3 <211>22 <212>DNA <213>人工引物 <400>3 cctggaaaggccactttgtaag22
<210>4 <211>25 <212>DNA <213>人工引物 <400>4 gtcgattgattagagcctagtccag25
<210>5 <211>21 <212>DNA <213>人工引物 <400>5 tggccagaaccaccatctttc21
<210>6 <211>25 <212>DNA <213>人工引物 <400>6 cttaacttgtttacagcgatgccaa25
<210>7 <211>29 <212>DNA <213>人工引物 <400>7 tcaagttcactttcttccatttctatgct29
<210>8 <211>25 <212>DNA <213>人工引物 <400>8 tacctggtactgattatggcactca25
<210>9 <211>33 <212>DNA <213>人工引物 <400>9 gaccaagatttttggcaaaactataagataagg33
<210>10 <211>26 <212>DNA <213>人工引物 <400>10 caagtttctcttcaggaggaaaagca26
<210>11 <211>32 <212>DNA <213>人工引物 <400>11 ctgaacataaaaacaacaattacgaaccaaac32
<210>12 <211>29 <212>DNA <213>人工引物 <400>12 ggtttgcctaaattcctagtttgtagttc29
<210>13 <211>28 <212>DNA <213>人工引物 <400>13 cttcagagaattctttgccacgtatttc28
<210>14 <211>32 <212>DNA <213>人工引物 <400>14 tgaaagtctctttaggtgattctcttattctg32
<210>15 <211>32 <212>DNA <213>人工引物 <400>15 caggaagtcagtttgaatttactcagtttaga32
<210>16 <211>23 <212>DNA <213>人工引物 <400>16 gtcatttttcaacaggccagcaa23
<210>17 <211>25 <212>DNA <213>人工引物 <400>17 atttgcgttgaggaacttgtgacta25
<210>18 <211>22 <212>DNA <213>人工引物 <400>18 catccaatgcctcgtaacaacc22
<210>19 <211>26 <212>DNA <213>人工引物 <400>19 caaccaaagtctttgttccacctttt26
<210>20 <211>27 <212>DNA <213>人工引物 <400>20 aactgaaaggcaaaaattcatcacaca27
<210>21 <211>27 <212>DNA <213>人工引物 <400>21 ttaaccacacccttaagatgagctcta27
<210>22 <211>26 <212>DNA <213>人工引物 <400>22 gttgtgacatcccttgataaaccttg26
<210>23 <211>20 <212>DNA <213>人工引物 <400>23 atccactaggactgctccca20
<210>24 <211>29 <212>DNA <213>人工引物 <400>24 tgcaacataagtactaatgtgtggtttga29
<210>25 <211>27 <212>DNA <213>人工引物 <400>25 agatatattcctccaattcaggaccca27
<210>26 <211>28 <212>DNA <213>人工引物 <400>26 agcaaaacacctgcagatctaatagaaa28
<210>27 <211>35 <212>DNA <213>人工引物 <400>27 taacaataaaaacatcaaaaagacattttagccat35
<210>28 <211>26 <212>DNA <213>人工引物 <400>28 acagttccagtagtcctactttgaca26
<210>29 <211>32 <212>DNA <213>人工引物 <400>29 ggaaggaaagaattttgcttaagatatcagtg32
<210>30 <211>30 <212>DNA <213>人工引物 <400>30 agcttctcaaagtatttcattttcttggtg30
<210>31 <211>25 <212>DNA <213>人工引物 <400>31 ccctagagtgctaacttccagtaac25
<210>32 <211>28 <212>DNA <213>人工引物 <400>32 acaaatgcacctggttcttttactaagt28
<210>33 <211>22 <212>DNA <213>人工引物 <400>33 taaggcaggaggactgcttcta22
<210>34 <211>35 <212>DNA <213>人工引物 <400>34 gaaaataactctcctgaacatctaaaagatgaagt35
<210>35 <211>29 <212>DNA <213>人工引物 <400>35 atcagcaaactgaaaaacctcttcttaca29
<210>36 <211>29 <212>DNA <213>人工引物 <400>36 ggaaacatcatctgcttgatccattttag29
<210>37 <211>25 <212>DNA <213>人工引物 <400>37 gtggcttcttcatttcagggtatca25
<210>38 <211>25 <212>DNA <213>人工引物 <400>38 tcaaagctacagaattctgtgtggt25
<210>39 <211>26 <212>DNA <213>人工引物 <400>39 gcaagcaatttgaaggtacagttgaa26
<210>40 <211>26 <212>DNA <213>人工引物 <400>40 acttattggatgtacctctgcagaag26
<210>41 <211>30 <212>DNA <213>人工引物 <400>41 aacgagaataaatcaaaaatttgccaaacg30
<210>42 <211>28 <212>DNA <213>人工引物 <400>42 tgaagtttccaaactaacatcacaaggt28
<210>43 <211>35 <212>DNA <213>人工引物 <400>43 ggttgtgctttttaaatttcaattttatttttgct35
<210>44 <211>31 <212>DNA <213>人工引物 <400>44 gtcataaaagccatcagtattgtagacaaac31
<210>45 <211>20 <212>DNA <213>人工引物 <400>45 ggaaggccatggaatctgct20
<210>46 <211>33 <212>DNA <213>人工引物 <400>46 gtggattttgcttctctgatataaactaacttt33
<210>47 <211>31 <212>DNA <213>人工引物 <400>47 gttgactttttgcaaatgtttaacataggtg31
<210>48 <211>28 <212>DNA <213>人工引物 <400>48 ctgcaaatgctatcgatttcttgatcac28
<210>49 <211>24 <212>DNA <213>人工引物 <400>49 gaagacttctgaggctacagtagg24
<210>50 <211>21 <212>DNA <213>人工引物 <400>50 agccttcatccggagagtgta21
<210>51 <211>27 <212>DNA <213>人工引物 <400>51 agatgatgtcagcaaacctaagaatgt27
<210>52 <211>22 <212>DNA <213>人工引物 <400>52 ccagtcctgccaatgagaagaa22
<210>53 <211>30 <212>DNA <213>人工引物 <400>53 ggctaggattgacaaattctttaagttcac30
<210>54 <211>29 <212>DNA <213>人工引物 <400>54 gcaaattgatagttgttctagcagtgaag29
<210>55 <211>23 <212>DNA <213>人工引物 <400>55 cacggtttctgtagcccatactt23
<210>56 <211>29 <212>DNA <213>人工引物 <400>56 caggtaaccttaatgcattgtcttaacac29
<210>57 <211>32 <212>DNA <213>人工引物 <400>57 aaggaatgttcccaatagtagacataaaagtc32
<210>58 <211>23 <212>DNA <213>人工引物 <400>58 atcttctaccaggctcttagcca23
<210>59 <211>33 <212>DNA <213>人工引物 <400>59 ggactccttatgtccaaatttaattgataatgg33
<210>60 <211>26 <212>DNA <213>人工引物 <400>60 atttgcaaatgtaagtggtgcttcaa26
<210>61 <211>30 <212>DNA <213>人工引物 <400>61 agctgtgaaactgtttagtgatattgagaa30
<210>62 <211>32 <212>DNA <213>人工引物 <400>62 ggcagcagtatatttgttatcttcattttcag32
<210>63 <211>28 <212>DNA <213>人工引物 <400>63 accaaaatatgtctggattggagaaagt28
<210>64 <211>32 <212>DNA <213>人工引物 <400>64 acttgcttggtactatcttctatttcagaaaa32
<210>65 <211>23 <212>DNA <213>人工引物 <400>65 acagcagactgtggaatgtatgg23
<210>66 <211>29 <212>DNA <213>人工引物 <400>66 tgaaaagactctgcatttttgctgttaat29
<210>67 <211>27 <212>DNA <213>人工引物 <400>67 acttcttccattgcatctttctcatct27
<210>68 <211>33 <212>DNA <213>人工引物 <400>68 gattccataaactaacaagcacttatcaaaact33
<210>69 <211>28 <212>DNA <213>人工引物 <400>69 ccagaggaaacctcagaaaaagtagaaa28
<210>70 <211>33 <212>DNA <213>人工引物 <400>70 ctcctagaattaaacacacatcacatacataca33
<210>71 <211>28 <212>DNA <213>人工引物 <400>71 ccatcgtgggatcttgcttataatactc28
<210>72 <211>30 <212>DNA <213>人工引物 <400>72 aaagctcttcctttttgaaagtctgttttt30
<210>73 <211>21 <212>DNA <213>人工引物 <400>73 tgacgtcctagctgtgtgaag21
<210>74 <211>28 <212>DNA <213>人工引物 <400>74 gacaaggaattggtttcagatgatgaag28
<210>75 <211>26 <212>DNA <213>人工引物 <400>75 ctgactggcatttggttgtacttttt26
<210>76 <211>29 <212>DNA <213>人工引物 <400>76 tcactcgaaaaagaatctgctttcaaaac29
<210>77 <211>24 <212>DNA <213>人工引物 <400>77 acttcctgagttttcatggacagc24
<210>78 <211>24 <212>DNA <213>人工引物 <400>78 gaggttttctactgttgctgcatc24
<210>79 <211>32 <212>DNA <213>人工引物 <400>79 tacatatgtgttggcattttaaacatcacttg32
<210>80 <211>33 <212>DNA <213>人工引物 <400>80 taatcatacctgacttatctctttgtggtgtta33
<210>81 <211>28 <212>DNA <213>人工引物 <400>81 gaactaattaactgttcagcccagtttg28
<210>82 <211>32 <212>DNA <213>人工引物 <400>82 tgacctgattctaaacactggtaattaaagac32
<210>83 <211>30 <212>DNA <213>人工引物 <400>83 tggcacttttgttgaagaaattactgaaaa30
<210>84 <211>26 <212>DNA <213>人工引物 <400>84 ctgagtgtttccctccttcataaact26
<210>85 <211>28 <212>DNA <213>人工引物 <400>85 ccaggtatcagatgcttcattacaaaac28
<210>86 <211>25 <212>DNA <213>人工引物 <400>86 acttgctttccacttgctgtactaa25
<210>87 <211>25 <212>DNA <213>人工引物 <400>87 tgtagctgtatacgtatggcgtttc25
<210>88 <211>32 <212>DNA <213>人工引物 <400>88 tgagggaatacataaaagttaacacacaatct32
<210>89 <211>25 <212>DNA <213>人工引物 <400>89 aacaactaccggtacaaacctttca25
<210>90 <211>28 <212>DNA <213>人工引物 <400>90 acaacagaaacgacaaatcctattaggt28
<210>91 <211>21 <212>DNA <213>人工引物 <400>91 tccagagccatttccatcctg21
<210>92 <211>22 <212>DNA <213>人工引物 <400>92 cccaaactacggacattttcgc22
<210>93 <211>29 <212>DNA <213>人工引物 <400>93 acctagcaagaaagaaaatgttgaacatc29
<210>94 <211>22 <212>DNA <213>人工引物 <400>94 tctggatttgacggctcctcta22
<210>95 <211>21 <212>DNA <213>人工引物 <400>95 aaaatgaagcggcccatctct21
<210>96 <211>27 <212>DNA <213>人工引物 <400>96 ctgaatgccttaaatatgacgtgtctg27
<210>97 <211>28 <212>DNA <213>人工引物 <400>97 tgctcttcttgattattttcttccaagc28
<210>98 <211>26 <212>DNA <213>人工引物 <400>98 agtaaccaggtaatattggcaaaggc26
<210>99 <211>31 <212>DNA <213>人工引物 <400>99 gagttccatattgcttatactgctgcttata31
<210>100 <211>23 <212>DNA <213>人工引物 <400>100 caggccttcatcctgaggatttt23
<210>101 <211>30 <212>DNA <213>人工引物 <400>101 tctgatgaatggttttataggaacgctatg30
<210>102 <211>27 <212>DNA <213>人工引物 <400>102 tcttttcatggctatttgccttttgag27
<210>103 <211>30 <212>DNA <213>人工引物 <400>103 tctgatgaatggttttataggaacgctatg30
<210>104 <211>27 <212>DNA <213>人工引物 <400>104 tcttttcatggctatttgccttttgag27
<210>105 <211>32 <212>DNA <213>人工引物 <400>105 tgctttgttttattttagtcctgttgttctac32
<210>106 <211>30 <212>DNA <213>人工引物 <400>106 gcaaaggtataacgctattgtcaaattctc30
<210>107 <211>32 <212>DNA <213>人工引物 <400>107 tgctttgttttattttagtcctgttgttctac32
<210>108 <211>30 <212>DNA <213>人工引物 <400>108 gcaaaggtataacgctattgtcaaattctc30
<210>109 <211>33 <212>DNA <213>人工引物 <400>109 aaggcttttcatataatgtggtaaattcatctg33
<210>110 <211>24 <212>DNA <213>人工引物 <400>110 ttctgagtttacacagtgctctgg24
<210>111 <211>33 <212>DNA <213>人工引物 <400>111 attgatggtactttaattttgtcactttgtgtt33
<210>112 <211>22 <212>DNA <213>人工引物 <400>112 ctgcaggcatgacagagaatca22
<210>113 <211>32 <212>DNA <213>人工引物 <400>113 gctatttactgatcagcacaacatatgtctta32
<210>114 <211>28 <212>DNA <213>人工引物 <400>114 actaatatttttcccacttgcagtctga28
<210>115 <211>33 <212>DNA <213>人工引物 <400>115 atgatcttgaacaatgtagtttttgtacagaga33
<210>116 <211>20 <212>DNA <213>人工引物 <400>116 agcaccctttctgggcttag20
<210>117 <211>29 <212>DNA <213>人工引物 <400>117 atttttagatccagactttcagccatctt29
<210>118 <211>27 <212>DNA <213>人工引物 <400>118 aatcagaggttcaaagaggcttacttt27
<210>119 <211>28 <212>DNA <213>人工引物 <400>119 cattgaccaccttttattactccagcta28
<210>120 <211>33 <212>DNA <213>人工引物 <400>120 aaatcaaagcattcttaccttactacatcatca33
<210>121 <211>33 <212>DNA <213>人工引物 <400>121 ttaaattttctttctctaggtgaagctgtactt33
<210>122 <211>32 <212>DNA <213>人工引物 <400>122 caagtttattttcatggtgttttatccctctt32
<210>123 <211>29 <212>DNA <213>人工引物 <400>123 tggaaaatttgtgcattgttaaggaaagt29
<210>124 <211>22 <212>DNA <213>人工引物 <400>124 tcctctgtcattcttcctgtgc22
<210>125 <211>28 <212>DNA <213>人工引物 <400>125 agaacattttgtttcctcactaaggtga28
<210>126 <211>25 <212>DNA <213>人工引物 <400>126 ggccaagaaattagagtcctcagaa25
<210>127 <211>25 <212>DNA <213>人工引物 <400>127 caggagtcttttgaactgccaaatc25
<210>128 <211>28 <212>DNA <213>人工引物 <400>128 gtgaaagagttcactccaaatcagtaga28
<210>129 <211>33 <212>DNA <213>人工引物 <400>129 aaagtaaagcttctataaagttaggtgtttcct33
<210>130 <211>27 <212>DNA <213>人工引物 <400>130 actcagtcataacagctcaaagttgaa27
<210>131 <211>23 <212>DNA <213>人工引物 <400>131 tagcgttatacctttgccctgag23
<210>132 <211>22 <212>DNA <213>人工引物 <400>132 agtagaactaagggtgggtggt22
<210>133 <211>32 <212>DNA <213>人工引物 <400>133 gacaaaatgtatcaaaaatacttcctcgtgtt32
<210>134 <211>27 <212>DNA <213>人工引物 <400>134 tcccaaaacatgaatgttctcaacaag27
<210>135 <211>33 <212>DNA <213>人工引物 <400>135 aggaaaaactacagttatttattaccccagaag33
<210>136 <211>28 <212>DNA <213>人工引物 <400>136 aaataagagtgctggcattttcatgatc28
<210>137 <211>32 <212>DNA <213>人工引物 <400>137 actgctactaaaacggagcaaaatataaaaga32
<210>138 <211>28 <212>DNA <213>人工引物 <400>138 ctgggacactttctttcagtattttgtg28
<210>139 <211>28 <212>DNA <213>人工引物 <400>139 tatggaatgtgcctttcctaaggaattt28
<210>140 <211>22 <212>DNA <213>人工引物 <400>140 ctagggatgacaggagaacagc22
<210>141 <211>28 <212>DNA <213>人工引物 <400>141 ttgaaattttagcactgtaagcaacagg28
<210>142 <211>32 <212>DNA <213>人工引物 <400>142 ggtctatccaaaactttattgccagtaaattg32
<210>143 <211>25 <212>DNA <213>人工引物 <400>143 ctgcagaaagacttgaaggcgtata25
<210>144 <211>22 <212>DNA <213>人工引物 <400>144 actgtatccccctgaagtccat22
<210>145 <211>23 <212>DNA <213>人工引物 <400>145 tggtaactcagactcagcatcag23
<210>146 <211>26 <212>DNA <213>人工引物 <400>146 aaactgaggctctttagcttcttagg26
<210>147 <211>22 <212>DNA <213>人工引物 <400>147 cagggaagctcttcatcctcac22
<210>148 <211>24 <212>DNA <213>人工引物 <400>148 gtcatgcatctcaggtttgttctg24
<210>149 <211>30 <212>DNA <213>人工引物 <400>149 ttccgataggttttcccaaatattttgtct30
<210>150 <211>29 <212>DNA <213>人工引物 <400>150 aaagttaatgagtggttttccagaagtga29
<210>151 <211>31 <212>DNA <213>人工引物 <400>151 agcctaatcttactagacatgtcttttcttc31
<210>152 <211>33 <212>DNA <213>人工引物 <400>152 ttctaatgtgttaaagttcattggaacagaaag33
<210>153 <211>27 <212>DNA <213>人工引物 <400>153 ggatcctgatatgtcttggtcaagttc27
<210>154 <211>22 <212>DNA <213>人工引物 <400>154 cttgacaccactggactaccac22
<210>155 <211>29 <212>DNA <213>人工引物 <400>155 aacaagacaaacaacagttggtattagga29
<210>156 <211>29 <212>DNA <213>人工引物 <400>156 tctgtcagttcatcatcttccataaaagc29
<210>157 <211>28 <212>DNA <213>人工引物 <400>157 cagtgatactgactttcaatcccagaaa28
<210>158 <211>28 <212>DNA <213>人工引物 <400>158 tggcaacagctcaacgtttttataattt28
<210>159 <211>32 <212>DNA <213>人工引物 <400>159 acaaaatggacattctaagttatgaggaaaca32
<210>160 <211>27 <212>DNA <213>人工引物 <400>160 ggtgatttcactagtaccttgctcttt27
<210>161 <211>26 <212>DNA <213>人工引物 <400>161 acagtggaattctagagtcacacttc26
<210>162 <211>23 <212>DNA <213>人工引物 <400>162 acagcataccacccatctgtaag23
<210>163 <211>23 <212>DNA <213>人工引物 <400>163 acttgcccctttcgtctatttgt23
<210>164 <211>25 <212>DNA <213>人工引物 <400>164 atgtgtggtgatgctgaaaagtaac25
<210>165 <211>29 <212>DNA <213>人工引物 <400>165 ttagctcatttttgttaatggtggctttt29
<210>166 <211>29 <212>DNA <213>人工引物 <400>166 ggacttcttgacttaatcggtttaggaat29
<210>167 <211>22 <212>DNA <213>人工引物 <400>167 tcgcctcatgtggttttatgca22
<210>168 <211>20 <212>DNA <213>人工引物 <400>168 ccaggacacgtgtagaacgt20
<210>169 <211>30 <212>DNA <213>人工引物 <400>169 cttcctttatttcaccatcatctaacaggt30
<210>170 <211>23 <212>DNA <213>人工引物 <400>170 aacagagggccaaaattgaatgc23
<210>171 <211>23 <212>DNA <213>人工引物 <400>171 ccccatcatgtgagtcatcagaa23
<210>172 <211>23 <212>DNA <213>人工引物 <400>172 gcttagcaaggagccaacataac23
<210>173 <211>31 <212>DNA <213>人工引物 <400>173 agtttattcactgtgttgattgacctttcta31
<210>174 <211>31 <212>DNA <213>人工引物 <400>174 tgttagcaatttcaacagtctaatcaatgtc31
<210>175 <211>31 <212>DNA <213>人工引物 <400>175 ttcagaaaactactttgaaacagaagcagta31
<210>176 <211>23 <212>DNA <213>人工引物 <400>176 agtgattggcaacacgaaaggta23
<210>177 <211>29 <212>DNA <213>人工引物 <400>177 ttcccatggaaaagaatcaagatgtatgt29
<210>178 <211>30 <212>DNA <213>人工引物 <400>178 tcctttcattagctacttggaagacaaaat30
<210>179 <211>29 <212>DNA <213>人工引物 <400>179 caaaggaatctttggacaaagtgaaaaac29
<210>180 <211>26 <212>DNA <213>人工引物 <400>180 caccacagtctcaatagaaacaaggt26
<210>181 <211>31 <212>DNA <213>人工引物 <400>181 taaaactagtagtgcagatacccaaaaagtg31
<210>182 <211>29 <212>DNA <213>人工引物 <400>182 caatgactgatttttaccaagagtgcaaa29
<210>183 <211>25 <212>DNA <213>人工引物 <400>183 ggttttgtaattttgcatcggcatg25
<210>184 <211>25 <212>DNA <213>人工引物 <400>184 gccctgaagtacagtctttagttgg25
<210>185 <211>35 <212>DNA <213>人工引物 <400>185 cacagcataatatgtgtcacattataaagattcag35
<210>186 <211>30 <212>DNA <213>人工引物 <400>186 tatctcactcgataatctggatgactcatt30
<210>187 <211>33 <212>DNA <213>人工引物 <400>187 gacaatacctacataaaactctttccagaatgt33
<210>188 <211>24 <212>DNA <213>人工引物 <400>188 aagttccccaattgaaagttgcag24
<210>189 <211>25 <212>DNA <213>人工引物 <400>189 ttatagacctcaggttgcaaaaccc25
<210>190 <211>27 <212>DNA <213>人工引物 <400>190 acctgaaagagaaatgggaaatgagaa27
<210>191 <211>27 <212>DNA <213>人工引物 <400>191 ccgcctatcattacatgtttccttact27
<210>192 <211>23 <212>DNA <213>人工引物 <400>192 gatttgaacaccactgagaagcg23
<210>193 <211>27 <212>DNA <213>人工引物 <400>193 ccgcctatcattacatgtttccttact27
<210>194 <211>23 <212>DNA <213>人工引物 <400>194 gatttgaacaccactgagaagcg23
<210>195 <211>27 <212>DNA <213>人工引物 <400>195 gggaatcaggctttactagaagaacag27
<210>196 <211>26 <212>DNA <213>人工引物 <400>196 ccaatgtggtctttgcagctatttac26
<210>197 <211>28 <212>DNA <213>人工引物 <400>197 tcaatccagactctgaagaacttttctc28
<210>198 <211>27 <212>DNA <213>人工引物 <400>198 tcctctgcaagaacataaaccaaatct27
<210>199 <211>29 <212>DNA <213>人工引物 <400>199 cccaaagtgtaaagaaatgcagaattctc29
<210>200 <211>28 <212>DNA <213>人工引物 <400>200 aggctgaattttcaatgactgaataagg28
<210>201 <211>34 <212>DNA <213>人工引物 <400>201 aatggtctatagacttttgagaaataaaactgat34
<210>202 <211>33 <212>DNA <213>人工引物 <400>202 attatataccatacctatagagggagaacagat33
<210>203 <211>32 <212>DNA <213>人工引物 <400>203 tttaaggcagttctagaagaatgaaaactctt32
<210>204 <211>32 <212>DNA <213>人工引物 <400>204 atcatacctgtatagggtatgctctttgaata32
<210>205 <211>25 <212>DNA <213>人工引物 <400>205 gcatatactgcatgcaaatgatccc25
<210>206 <211>31 <212>DNA <213>人工引物 <400>206 agccacataacaaccacattttctaattaag31
<210>207 <211>33 <212>DNA <213>人工引物 <400>207 tacctgttaagtttgtatgcaacatttctaaag33
<210>208 <211>27 <212>DNA <213>人工引物 <400>208 atatcattacaccagttcgtccctttc27
<210>209 <211>22 <212>DNA <213>人工引物 <400>209 gtagtatgagcagcagctggac22
<210>210 <211>32 <212>DNA <213>人工引物 <400>210 aacattcatcgttgtgtaaattaaacttctcc32
<210>211 <211>29 <212>DNA <213>人工引物 <400>211 taatgttattacggctaattgtgctcact29
<210>212 <211>27 <212>DNA <213>人工引物 <400>212 ggctctaggttttgtctatcatctcag27
<210>213 <211>27 <212>DNA <213>人工引物 <400>213 gaactaccctgatacttttctggatgc27
<210>214 <211>29 <212>DNA <213>人工引物 <400>214 ctctaatatagccagttggttgatttcca29
<210>215 <211>27 <212>DNA <213>人工引物 <400>215 gaactaccctgatacttttctggatgc27
<210>216 <211>29 <212>DNA <213>人工引物 <400>216 ctctaatatagccagttggttgatttcca29
<210>217 <211>29 <212>DNA <213>人工引物 <400>217 actttaacaggatttggaaaaacatcagg29
<210>218 <211>24 <212>DNA <213>人工引物 <400>218 acattcatcagcgtttgcttcatg24
<210>219 <211>24 <212>DNA <213>人工引物 <400>219 caggtgataaacaagcaacccaag24
<210>220 <211>24 <212>DNA <213>人工引物 <400>220 ctgaagctacctccaaaactgtga24
<210>221 <211>25 <212>DNA <213>人工引物 <400>221 cagcaaaaagtcctgcaacttgtta25
<210>222 <211>28 <212>DNA <213>人工引物 <400>222 tcggagagatgatttttgtcattttcag28
<210>223 <211>34 <212>DNA <213>人工引物 <400>223 ttttgtgtatttacagtaacatggatattctctt34
<210>224 <211>29 <212>DNA <213>人工引物 <400>224 gggaagtgttaacttcttaacgttagtgt29
<210>225 <211>24 <212>DNA <213>人工引物 <400>225 cagtgatggaggaaatgttggttg24
<210>226 <211>21 <212>DNA <213>人工引物 <400>226 ggtggctcagctacttgagag21
<210>227 <211>26 <212>DNA <213>人工引物 <400>227 ggagggagactgtgtgtaatatttgc26
<210>228 <211>28 <212>DNA <213>人工引物 <400>228 ctctttctcccctttacaagactttgaa28
<210>229 <211>25 <212>DNA <213>人工引物 <400>229 gaccaatggctaagtgaagatgaca25
<210>230 <211>29 <212>DNA <213>人工引物 <400>230 ttccaataaattctcagatccaggaagag29
<210>231 <211>28 <212>DNA <213>人工引物 <400>231 aaatcaaagtgtttgttccaatacagca28
<210>232 <211>21 <212>DNA <213>人工引物 <400>232 ggcgatggttttctccttcca21
<210>233 <211>31 <212>DNA <213>人工引物 <400>233 catgtttatttggagtaatgagtccagtttc31
<210>234 <211>26 <212>DNA <213>人工引物 <400>234 tctgggtccttaaagaaacaaagtcc26
<210>235 <211>26 <212>DNA <213>人工引物 <400>235 ccaagagattttgtgggttgtaaagg26
<210>236 <211>29 <212>DNA <213>人工引物 <400>236 aatgtttgatcttggtcatttgacagttc29
<210>237 <211>29 <212>DNA <213>人工引物 <400>237 tacctcagtcacataataaggaatgcatc29
<210>238 <211>24 <212>DNA <213>人工引物 <400>238 ggttctaagcaacactgtgacgta24
<210>239 <211>32 <212>DNA <213>人工引物 <400>239 atctacaaaaagtaagaactagcaagactagg32
<210>240 <211>24 <212>DNA <213>人工引物 <400>240 agggttagttgagaccattcacag24
<210>241 <211>22 <212>DNA <213>人工引物 <400>241 acaaatgggcaggactcttagg22
<210>242 <211>30 <212>DNA <213>人工引物 <400>242 aactacactactctgtaaatgtgcagatac30
<210>243 <211>29 <212>DNA <213>人工引物 <400>243 ggtcaaccagaaagaataaatactgcaga29
<210>244 <211>31 <212>DNA <213>人工引物 <400>244 tactggctcaataccagaatcaagtttattt31
<210>245 <211>32 <212>DNA <213>人工引物 <400>245 atgtgttatgtgaggtagattgtaaagtcaaa32
<210>246 <211>24 <212>DNA <213>人工引物 <400>246 ccaggtgcggtaaaatttggattc24
<210>247 <211>35 <212>DNA <213>人工引物 <400>247 ctggcctgatacaattaacttgaatgttatatatg35
<210>248 <211>35 <212>DNA <213>人工引物 <400>248 gtctctaagactttgttctcatattagaaataaca35
<210>249 <211>22 <212>DNA <213>人工引物 <400>249 agtctgtttccacacctgtctc22
<210>250 <211>25 <212>DNA <213>人工引物 <400>250 tggagtcatctgaggagaattcagt25
<210>251 <211>21 <212>DNA <213>人工引物 <400>251 aatggctacgacccagttacc21
<210>252 <211>29 <212>DNA <213>人工引物 <400>252 tccaatacatggaaggatgagaatttcaa29
<210>253 <211>27 <212>DNA <213>人工引物 <400>253 tgtcagataccacagcatctttacatt27
<210>254 <211>33 <212>DNA <213>人工引物 <400>254 tgaattatcactatcagaacaaagcagtaaagt33
<210>255 <211>27 <212>DNA <213>人工引物 <400>255 tgtcagataccacagcatctttacatt27
<210>256 <211>33 <212>DNA <213>人工引物 <400>256 tgaattatcactatcagaacaaagcagtaaagt33
<210>257 <211>30 <212>DNA <213>人工引物 <400>257 ccttgattttcttccttttgttcacattca30
<210>258 <211>25 <212>DNA <213>人工引物 <400>258 tccattgggacatgaagttaaccac25
<210>259 <211>28 <212>DNA <213>人工引物 <400>259 aggaaaataccagcttcatagacaaagg28
<210>260 <211>33 <212>DNA <213>人工引物 <400>260 ctttaaccacttctctgtattacatactagctt33
<210>261 <211>31 <212>DNA <213>人工引物 <400>261 tttttccttatgatctttaactgttctgggt31
<210>262 <211>28 <212>DNA <213>人工引物 <400>262 gctgattataagatggtttcctttgtgg28
<210>263 <211>26 <212>DNA <213>人工引物 <400>263 agtgacaaaatctccaaggaagttgt26
<210>264 <211>26 <212>DNA <213>人工引物 <400>264 ccactgtttcctcatttaatggcttc26
<210>265 <211>27 <212>DNA <213>人工引物 <400>265 taatcaaaagaaactgagcaagcctca27
<210>266 <211>29 <212>DNA <213>人工引物 <400>266 gcacttcaaatgtactcttctgcaatatg29
<210>267 <211>28 <212>DNA <213>人工引物 <400>267 agctattcctaccattctgatgaggtat28
<210>268 <211>27 <212>DNA <213>人工引物 <400>268 tgcatttttatttttgcagggtgaaga27
<210>269 <211>22 <212>DNA <213>人工引物 <400>269 attgcagcacaactaaggaacg22
<210>270 <211>26 <212>DNA <213>人工引物 <400>270 acacactgttcaactctgtgaaaatg26
<210>271 <211>27 <212>DNA <213>人工引物 <400>271 tgggtgttttatgcttggttctttagt27
<210>272 <211>26 <212>DNA <213>人工引物 <400>272 gagagtctaaaacagcttctcacctt26
<210>273 <211>22 <212>DNA <213>人工引物 <400>273 gtggcaccaaatacgaaacacc22
<210>274 <211>26 <212>DNA <213>人工引物 <400>274 acaacgtcgtttcagtctgagataat26
<210>275 <211>29 <212>DNA <213>人工引物 <400>275 tgatttgcttgagatcaagattgcagata29
<210>276 <211>27 <212>DNA <213>人工引物 <400>276 gagggaactcaaagtacatgaacttgt27
Claims (10)
1.一种用于遗传性肿瘤胚系突变的多重PCR引物组，所述多重PCR引物组至少包括SEQID NO.：1～276所示的引物序列。
2.根据权利要求1所述的多重PCR引物序列组，其特征在于：所述多重PCR引物组还包括检测如下遗传病相关的引物组：
ADH1B、AIP、ALDH2、ALK、APC、ARID1A、ATM、AXIN2、BAP1、BARD1、BLM、BMPR1A、BRCA1、BRCA2、BRIP1、BUB1、BUB1B、BUB3、CDC73、CDH1、CDK4、CDKN1C、CDKN2A、CEBPA、CEP57、CHEK2、CTNNB1、CYLD、DDB2、DICER1、DIS3L2、EGFR、EPCAM、EPHB2、ERBB4、ERCC1、ERCC2、ERCC3、ERCC4、ERCC5、EXT1、EXT2、EZH2、FANCA、FANCB、FANCC、FANCD2、FANCE、FANCF、FANCG、FANCI、FANCL、FANCM、FH、FLCN、FOXP1、GATA2、GPC3、HDAC9、HNF1A、HOXB13、HRAS、KIT、KLLN、KRAS、LZTR1、MAX、MEN1、MET、MITF、MLH1、MLH3、MPL、MRE11、MRE11A、MSH2、MSH3、MSH6、MUTYH、NAT1、NBN、NF1、NF2、NSD1、PALB2、PBRM1、PDE11A、PHOX2B、PIK3CA、PMS1、PMS2、POLE、POLH、PRF1、PRKAR1A、PRSS1、PTCH1、PTEN、PTPN11、RAD50、RAD51、RAD51C、RAD51D、RB1、RECQL4、RET、RHBDF2、RUNX1、SBDS、SDHA、SDHAF2、SDHB、SDHC、SDHD、SETD2、SLX4、SMAD4、SMARCB1、SPINK1、STK11、SUFU、TGFBR2、TMEM127、TP53、TSC1、TSC2、TSHR、VHL、WRN、WT1、XPA、XPC。
3.一种用于遗传性肿瘤胚系突变的多重PCR试剂盒，其特征在于：其使用的多重PCR引物组如权利要求1或2所述。
4.一种多重PCR的方法，其特征在于：其使用的多重PCR引物组如权利要求1或2所述。
5.根据权利要求4所述的方法，其特征在于：PCR反应体系中，dNTP的初始浓度为0.3mM，Mg2+的浓度为2.5mM，每50μL反应体系中的聚合酶为5U。
6.根据权利要求4或5所述的方法，其特征在于：PCR反应体系中，引物的浓度为0.15μM。
7.根据权利要求4或5所述的方法，其特征在于：PCR扩增体系如下：
。
8.根据权利要求7所述的方法，其特征在于：PCR扩增的反应条件为：
9.根据权利要求4～8任一项所述的方法，其特征在于：还包括PCR产物的纯化，所述纯化包括：
1)在PCR反应完成后的体系内，每一管加入9μL AMPure XP磁珠，混匀，静置；
2)吸附磁珠至溶液澄清，吸取上清至新的0.2ml PCR管中；
3)在所述新的PCR管中加入15μL磁珠，混匀，静置，吸附磁珠至溶液澄清，弃上清，留磁珠；
4)在PCR管中加入100μL 70％乙醇，充分悬浮磁珠后，吸附磁珠至溶液澄清，弃上清，留磁珠，挥干乙醇；
5)加入15μL洗脱液，充分悬浮磁珠，洗脱DNA，其中，所述洗脱液为10mM Tris-HCl，pH7.5-8.0，温度为55～65℃。
10.根据权利要求9所述的方法，其特征在于：所述洗脱液的温度为60℃。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911021454.6A CN110684848A (zh) | 2019-10-25 | 2019-10-25 | 用于遗传性肿瘤胚系突变检测的多重pcr引物组及试剂盒 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911021454.6A CN110684848A (zh) | 2019-10-25 | 2019-10-25 | 用于遗传性肿瘤胚系突变检测的多重pcr引物组及试剂盒 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110684848A true CN110684848A (zh) | 2020-01-14 |
Family
ID=69114452
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911021454.6A Pending CN110684848A (zh) | 2019-10-25 | 2019-10-25 | 用于遗传性肿瘤胚系突变检测的多重pcr引物组及试剂盒 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110684848A (zh) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130123120A1 (en) * | 2010-05-18 | 2013-05-16 | Natera, Inc. | Highly Multiplex PCR Methods and Compositions |
| CN106119405A (zh) * | 2016-08-31 | 2016-11-16 | 广州万德基因医学科技有限公司 | 一种肺癌的预后标记物、使用该标记预测肺癌预后的方法及应用 |
| CN106283199A (zh) * | 2016-08-27 | 2017-01-04 | 大连晶泰生物技术有限公司 | 检测肿瘤相关的50个热点突变基因的捕获文库和试剂盒 |
| CN106555226A (zh) * | 2016-04-14 | 2017-04-05 | 北京京诺玛特科技有限公司 | 一种构建高通量测序文库的方法和试剂盒 |
| CN107338308A (zh) * | 2017-07-28 | 2017-11-10 | 广州永诺健康科技有限公司 | 遗传性结直肠癌易感基因检测用的多重pcr引物***、检测方法和应用 |
| CN107723354A (zh) * | 2017-08-23 | 2018-02-23 | 广州永诺健康科技有限公司 | 一种基于高通量测序检测非小细胞肺癌致癌基因突变的多重pcr引物、试剂盒和方法 |
| US20180119214A1 (en) * | 2015-03-31 | 2018-05-03 | Fred Hutchinson Cancer Research Center | Compositions and methods for target nucleic acid molecule enrichment |
| CN108315416A (zh) * | 2018-03-02 | 2018-07-24 | 中国科学院合肥物质科学研究院 | 基于高通量测序技术确定肺癌基因突变位点的引物、试剂盒及方法 |
| CN108998507A (zh) * | 2018-07-24 | 2018-12-14 | 陈梦麟 | 一种应用于人群复杂亲缘关系鉴定的无创高通量检测方法 |
| CN109554443A (zh) * | 2018-12-29 | 2019-04-02 | 杭州迪安医学检验中心有限公司 | 一种基于高通量测序技术检测dmd基因变异的引物及其应用 |
| CN110241212A (zh) * | 2019-06-25 | 2019-09-17 | 苏州泓迅生物科技股份有限公司 | 一种用于brca1和brca2基因扩增子测序检测的引物组及其应用 |
-
2019
- 2019-10-25 CN CN201911021454.6A patent/CN110684848A/zh active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130123120A1 (en) * | 2010-05-18 | 2013-05-16 | Natera, Inc. | Highly Multiplex PCR Methods and Compositions |
| US20180119214A1 (en) * | 2015-03-31 | 2018-05-03 | Fred Hutchinson Cancer Research Center | Compositions and methods for target nucleic acid molecule enrichment |
| CN106555226A (zh) * | 2016-04-14 | 2017-04-05 | 北京京诺玛特科技有限公司 | 一种构建高通量测序文库的方法和试剂盒 |
| CN106283199A (zh) * | 2016-08-27 | 2017-01-04 | 大连晶泰生物技术有限公司 | 检测肿瘤相关的50个热点突变基因的捕获文库和试剂盒 |
| CN106119405A (zh) * | 2016-08-31 | 2016-11-16 | 广州万德基因医学科技有限公司 | 一种肺癌的预后标记物、使用该标记预测肺癌预后的方法及应用 |
| CN107338308A (zh) * | 2017-07-28 | 2017-11-10 | 广州永诺健康科技有限公司 | 遗传性结直肠癌易感基因检测用的多重pcr引物***、检测方法和应用 |
| CN107723354A (zh) * | 2017-08-23 | 2018-02-23 | 广州永诺健康科技有限公司 | 一种基于高通量测序检测非小细胞肺癌致癌基因突变的多重pcr引物、试剂盒和方法 |
| CN108315416A (zh) * | 2018-03-02 | 2018-07-24 | 中国科学院合肥物质科学研究院 | 基于高通量测序技术确定肺癌基因突变位点的引物、试剂盒及方法 |
| CN108998507A (zh) * | 2018-07-24 | 2018-12-14 | 陈梦麟 | 一种应用于人群复杂亲缘关系鉴定的无创高通量检测方法 |
| CN109554443A (zh) * | 2018-12-29 | 2019-04-02 | 杭州迪安医学检验中心有限公司 | 一种基于高通量测序技术检测dmd基因变异的引物及其应用 |
| CN110241212A (zh) * | 2019-06-25 | 2019-09-17 | 苏州泓迅生物科技股份有限公司 | 一种用于brca1和brca2基因扩增子测序检测的引物组及其应用 |
Non-Patent Citations (3)
| Title |
|---|
| FALK ZAKRZEWSKI 等: "Targeted capture-based NGS is superior to multiplex PCR-based NGS for hereditary BRCA1 and BRCA2 gene analysis in FFPE tumor samples", 《BMC CANCER》 * |
| 孙吉 等: "高通量测序技术在肿瘤临床上的应用", 《科技风》 * |
| 曾溢滔 编, 上海科学技术出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220010385A1 (en) | Methods for detecting inactivation of the homologous recombination pathway (brca1/2) in human tumors | |
| US20200399713A1 (en) | Methods and materials for assessing loss of heterozygosity | |
| EP2794907B2 (en) | Methods and materials for assessing loss of heterozygosity | |
| JP2022519159A (ja) | 循環細胞の分析方法 | |
| Du et al. | MicroRNA expression distinguishes SCLC from NSCLC lung tumor cells and suggests a possible pathological relationship between SCLCs and NSCLCs | |
| EP3541934B1 (en) | Methods for preparing dna reference material and controls | |
| Busse et al. | Copy number alterations determined by single nucleotide polymorphism array testing in the clinical laboratory are indicative of gene fusions in pediatric cancer patients | |
| Kim et al. | Germline pathogenic variants in neuroblastoma patients are enriched in BARD1 and predict worse survival | |
| CN106337058B (zh) | Cryl1-ift88融合基因及其在原发性肝细胞癌诊断和治疗中的应用 | |
| CN110684848A (zh) | 用于遗传性肿瘤胚系突变检测的多重pcr引物组及试剂盒 | |
| US20220127601A1 (en) | Method of determining the origin of nucleic acids in a mixed sample | |
| CN110564851B (zh) | 一组用于非超突变型直肠癌分子分型的基因及其应用 | |
| Jóźwik et al. | Breast cancer in an 18-year-old female: A fatal case report and literature review | |
| Shimoda et al. | Development of two 410-cancer-gene panel tests for solid tumors and liquid biopsy based on genome data of 5,143 Japanese cancer patients | |
| WO2024112643A1 (en) | Fragmentomics based identification of tumor-specific copy number alteration states in liquid biopsy | |
| WO2024077041A2 (en) | Methods and systems for identifying copy number signatures | |
| EP4206335A1 (en) | Method for identifying gastric cancer-associated mutations in blood using a gene panel | |
| Micheloni | Circulating cell-free DNA: a powerful biomarker for tumor management and a possible monitor tool in other pathological conditions. | |
| JP2016198027A (ja) | 卵巣癌の診断方法。 | |
| WO2024118594A1 (en) | Methods and systems for mutation signature attribution | |
| WO2024112752A1 (en) | Methods to identify false-positive disease therapy associations and improve clinical reporting for patients | |
| CN116829736A (zh) | 用于将样品分拣为临床相关类别的方法 | |
| WO2024020343A1 (en) | Methods and systems for determining a diagnostic gene status | |
| WO2024112893A1 (en) | Systems and methods for tracking personalized methylation biomarkers for the detection of disease | |
| Pinyol et al. | Genomics and Epigenomics |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200114 |
|
| RJ01 | Rejection of invention patent application after publication |