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1、肿瘤分子病理诊断基础,闫庆国,分子病理与分子病理诊断 肿瘤的分子病理变化 肿瘤的分子病理诊断技术 肿瘤分子病理诊断的应用,肿瘤的分子病理诊断技术,Molecular pathology protocals,DNA extraction from paraffin-embedded tissues DNA extraction from fresh or frozen tissues RNA extraction from fresh or frozen tissues Single-strand conformation polymorphism analysis of mutations i
2、n exons 4-8 of the TP53 gene Cleavaes fragment length polymorphism analysis for genotyping and mutation detection Detection of telomerase by in situ hybridization and by the polymerase chain reaction based telomerase activity assay Detection of microsatellite instability Polymerase chain reaction cl
3、onality assays based on X-linked genes,Fluorescent in situ hybridization: evaluation for ploidy and gene amplification Her-2/neu oncogene amplification determined by fluorescence in situ hybridization A nested reversed transcription-polymerase chain reaction assay to detect BCR/ab/ Detection of t(15
4、;17)(q24;q21),inv(16)/t(16;16)(p13;q22), and t(8;21)(q22;q22) anomalies in acute myeloid leukemia Detection of t(14;18)(q32;q21)-Associated BCL-2/JH Gene Fusion in Non-Hodgkins Lymphoma Detection of Breast Cancer Cells Using Immunomagnetic Beads and Reverse Transcription-Polymerase Chain Reaction Mo
5、lecular Detection of Circulating Prostate Cancer Cells Methods to Detect Clonal Gene Rearrangements in Lymphomas and Leukemias,Monitoring of Bone Marrow Transplant Engraftment Direct Molecular Diagnosis of Multiple Endocrine Neoplasia Type 1 Molecular Detection of Multiple Endocrine Neoplasia Type 2
6、 Assay for Detecting the I1307K Susceptibility Allele within the Adenomatous Polyposis Coli Gene Detection of Human Papillomaviruses by Polymerase Chain Reaction and In Situ Hybridization Molecular Methods for Detecting Epstein-Barr Virus (Part I): Hybridization to Epstein-Barr VirusEncoded RNA (EBE
7、R) Transcripts Molecular Methods for Detecting Epstein-Barr Virus (Part II): Structural Analysis of Epstein-Barr Virus DNA as a Marker of Clonality Molecular Methods for Detecting Epstein-Barr Virus (Part III): EBV Viral Load by Competitive Polymerase Chain Reaction,Molecular Detection of Kaposis Sa
8、rcomaAssociated Herpesvirus/Human Herpesvirus-8 Diagnostic Applications of Quantitative Polymerase Chain Reaction for Cytomegalovirus, Polymerase Chain Reaction System That Detects Herpes Simplex Virus in Cerebrospinal Fluid and Discriminates Genotypes 1 and 2 Detection and Typing of Hepatitis C Vir
9、us Detection and Speciation of Mycobacteria in Formalin-Fixed, Paraffin-Embedded Tissue Sections Ultrasensitive Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma by the AMPLICOR and COBAS AMPLICOR HIV-1 MONITORTM Tests,Molecular Diagnosis of Hereditary Thrombotic Disorders Prenatal G
10、enotyping of the RhD Locus to Identify Fetuses at Risk for Hemolytic Disease of the Newborn Molecular Diagnosis of Hereditary Hemochromatosis Genotyping of Apolipoprotein E Genotyping for Functionally Important Human CYP2D6*4 (B) Mutation Using TaqMan Probes,常用的共性技术,核酸提取技术,电泳技术,凝胶成像,PCR 技术,分子病理诊断的代表
11、性技术,ISH In situ hybridizasion FISH Gene mutation analysis Clonality analysis,FISH: Method,Interphase FISH Performed on isolated nucleus or whole section. Advantage: Excellent correlation with histological and immunophenotypic features.Multiple probes on the same section. Disadvantage: Nucleus and si
12、gnal overlap truncation.,FISH: Protocols for interphase FISH,Slides preparation Prepare 2-4 m sections and dewax; Digest in 0.1% pepsin in 0.015N HCl for 20 min at 37C; Dehydrate and air dry slides. Hybridisation Apply probe to area of interest, place coverslip and seal with rubber cement; Denature
13、probe and target DNA at 80C for 25 min; Hybridise in a moisture chamber at 45C for 2 days. Post-hybridisation washing Remove rubber cement and cover slip; Wash 1- 0.4x SSC and 0.3% IGEPAL CA-630 buffer in a water bath set at 72 C; Wash 2- 2SSC and 0.1% IGEPAL CA-630 buffer at room temp for 1 min; Wa
14、sh 3 - 2SSC for 2-5 min at room temp; Apply anti-fade solution containing DAPI and coverslip. Read signals with a fluorescent microscope.,FISH: Probes Dual-colour break-apart probe independent of translocation partners,FISH: Probes Dual colour-dual fusion probe - translocation partners known,FISH: P
15、robes Single colour probe,Examples of specimen types applicable to FISH,Fresh/frozen tissue Cytology specimens Body fluids(urine) Intraoperative smears Cell culture preparations Fresh-frozen, parafin-embedded tissue Thin sections(46) Disaggregated nuclei Archived unstained sections Previously staine
16、d sections(e.g., negative immunohistochemistry controls),CISH,Detection of gene amplification, chromosome translocations and chromosome number Using conventional enzymatic reactions under the brightfield microscope on formalin-fixed, paraffin-embedded tissues Compared to FISH, CISHs advantages Detect under bright field The morphological details are readily apparent The probe signals are not subject to rapid fading,21,22,突变检测原理：根据野生型与突变型所表现出的差异,测序法：经典、荧光、焦
