《分子生物学检验技术》第12章 肿瘤的分子生物学检验.ppt

* * * * * * * Point 3: (FP: BCR and ABL genes in close proximity in interphase cells can overlap creating a fusion signal. FN: varying breakpoint locations could leave too many bp between markers, resulting in loss of fusion signal & ~15% FN rate) Point 5: Variant translocation - ABL inserted within the BCR gene * * * * * * CML 诊断技术 Focus on two main techniques: Fluorescence in-situ hybridization (FISH) for detection of t(9;22) Reverse transcriptase polymerase chain reaction (RT-PCR) detection of t(9;22) * FISH for detection of t(9;22) short probes (~100-300 bp), constructed via PCR, consisting of a known DNA sequence labeled directly with fluorescently labeled nucleotides (in RT-PRC or via nick translation) indirectly using a ligand (i.e. biotin or digoxigenin) against which a fluorescently labeled Ab can be applied * FISH for detection of t(9;22) The probes can be used with either metaphase chromosomes (involved preparation, poor resolution ~ Mbp level) or with interphase chromosomes (easier to obtain and better resolution (i.e. ~10000 bp)) Probes and target DNA are denatured; probes are then hybridized to the patient sample. UV light is used for visualization * FISH in action /scitable/nated/content/35120/10.1038_nrg1692-f1_mid_1.jpg * FISHing for CML: Directly labeled probes spanning the breakpoint regions of the ABL and BCR genes are applied to metaphase or interphase chromosomes, Ph+ cells obtained from peripheral blood TRITC (red) for BCR on ch 22, FITC (green) for ABL on ch 9 If the BCR/ABL fusion gene is present, a YELLOW fusion signal will appear * FISH application in CML * * Advantages of FISH: Can use peripheral blood Can use BM smears for hybridization experiments -Good resolution obtainable from easily obtained interphase chromosomes Reduced false positives and false negatives with larger probes spanning the gene breakpoints (vs older method using probes upstream and downstream of the genes) Adds value and diagnostic power when combin