1. 分子诊断 -1: Methods in Molecular Medicine 张咸宁 zhangxianning@zju.edu.cn Tel ： 13105819271; 88208367 Office: C303, Teaching Building 2014/09

2. Trends of Medicine in 21th Century “5P 医学 ”: Prediction 、 Prevention 、 Participation 、 Personalization 、 Precision“5P 医学 ”: Prediction 、 Prevention 、 Participation 、 Personalization 、 Precision –Treatment afterward  Predictive & Preventive –Molecular epidemiology: Sub-population –Rare diseases  Common diseases –General  Personalized

3. In times of transformation, we are all students ！ ● predict individual susceptibility to disease, based on genetic, genomic, and other factors; ● provide more useful tools and individualized programs for disease prevention, based on knowledge of one’s susceptibility; ● detect the onset of disease earlier and before it is clinically evident, based on newly discovered biological markers that arise from changes at the molecular level; ● preempt disease progression, as a result of early detection; ● target medicines and their dose more precisely and safely to each patient, on the basis of a deep understanding of disease mechanism and the role that genetic and genomic factors play in the individual response to drugs. ---- Ginsburg GS, Willard HF. Essentials of Genomic and Personalized Medicine. Elsevier, 2010

4. Infectious Disease Neoplastic Disease Genetic Disease Identity Testing HLA Typing Pharmacogenetics Areas of Application of Molecular Diagnosis

5. China‘s birth defect （出生缺陷） rate （卫计委公布， Sep., 2012 ） Birth defect: A congenital abnormality ( 先天性 畸形 ). Not all are detected at birth or even soon after. ~5.6% ~16 million neonates （新生儿） /yr → ~900 000 affected, ~ 270 000 visible at birth

6. Beijing‘s birth defect rate, 2013 ( 北京市卫计委, Sep. 11, 2014) ~14.68% for household registered neonates ~26.22% for non-household registered neonates

7. Prevalence of the common monogenetic disorders in China?--- No accurate data! ~1% in living birth --- Carter CO. J M Genet 1977;14:316 The estimated affected neonates in China: ~160 000 per year !

8. Human genome DNA Extraction Venous blood(5 ml) → WBC → lysing buffer (SDS) and protease K → centrifugation, collect supernatant → phenol → centrifugation, collect supernant → RNase A and T1 → phenol → centrifugation, collect supernatant → potassium acetate and EtOH (absolute) → genomic DNA appears (like cotton fiber)

15. 分子杂交

16. Sanger (dideoxy) sequencing

17. Mosaicism （嵌合体） (A) Sequence analysis of the proband revealed a hemizygous insertion mutation (LAMP2/c.808dupG; p.A270Gfx3). (B) Sequences of DNA from the proband’s mother were variable and weakly suggestive of a c.808dupG “G” peak. (C) Sequences of DNA from unrelated healthy controls were normal.

18. NGS

19. ASO

20. dHPLC ： WAVE 系统的组成 InterfacePumpDegasser Detector Column Oven Autosampler Temperature Rack

21. dHPLC

22. 高分辨率熔解曲线分析技术（ high resolution melting ， HRM ）

23. MLPA

24. Using multiplex ligation-dependent probe amplifi cation (MLPA) to identify an exon 13 deletion in the BRCA1 gene. (A) Results from a control sample. Numbered peaks represent products from each exon; peaks labeled c are control probes. (B) The same analysis on DNA from a patient with breast cancer. In comparison with the control sample, the exon 13 peak is only half the size.

25. qPCR 。 A ： TaqMan 技术。 B ： real-time PCR 分析结果。 随着 PCR 循环次数（ X 轴）的增加， PCR 产物（ Y 轴，以 荧光强度表示。 Rn 即校正后的报告基因染色强度）呈指 数性增长。

26. Mass Spectrometry-based Diagnostics MALDI-TOF MS ： matrix-assisted laser desorption/ionization-time of flight mass spectrometry SELDI -TOF MS ： surface-enhanced laser desorption/ionization- time of flight mass spectrometry

27. Rosenblatt KP, et al. Annu Rev Med, 2004, 55:97–112

28. Petricoin EF, et al. Trends Mol Med, 2004, 10:59–64

29. 荧光条形码标记的单分子检测技术 （ nCounter Analysis System ）：一种高通 量检测基因表达谱、 miRNA 等分子的技术 ● Gene Expression 800 个基因分析通量 具有弹性的样品需求，适用于血液样本与 FFPE 样本 只需要 15 分钟的手动操作时间 ● miRNA Analysis 人类，小鼠 及大鼠的 miRNA 分析皆适用 完整包含 miRBase 资料库的 miRNA （人类、小鼠、大鼠） ● Copy Number Variation 同时检视人类基因组中 800 个区域 准确的标记感兴趣的区域 ● 帮助二代测序进行后期验证 NGS Validation

30. Acknowledge （ PPT 特别鸣谢！） UCLA David Geffen School of Medicine www.medsch.ucla.edu/ANGEL/ Prof. ( ), et al.Prof. Gasson JC (UCLA Jonsson Comprehensive Cancer Center ), et al.