分子诊断分子诊断 -1: Methods in -1: Methods in Molecular MedicineMolecular Medicine

张咸宁zhangxianning@zju.edu.cn

Tel ： 13105819271; 88208367 Office: C303, Teaching Building

2014/09

Trends of Medicine in 21th CenturyTrends of Medicine in 21th Century

• ““5P5P 医学”医学” : : PredictionPrediction 、 、 PreventionPrevention 、、 ParticipationParticipation 、 、 PersonalizationPersonalization 、、 PrecisionPrecision

– Treatment afterward Predictive & Preventive

– Molecular epidemiology: Sub-population

– Rare diseases Common diseases

– General Personalized

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

Infectious Disease 

Neoplastic Disease 

Genetic Disease 

Identity Testing 

HLA Typing

Pharmacogenetics

Areas of Application of Molecular Diagnosis

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

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

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 !

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)

分子杂交

Sanger (dideoxy) sequencing

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.

NGS

ASO

dHPLC ： WAVE 系统的组成

Interface Pump Degasser

Detector

Column Oven

Autosampler

Temperature Rack

dHPLC

高分辨率熔解曲线分析技术（ high resolution

melting ， HRM ）

MLPA

MLPA

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.

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。

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

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

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

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

Acknowledge （ PPT 特别鸣谢！）

• UCLA David Geffen School of Medicine

• www.medsch.ucla.edu/ANGEL/

• Prof. Prof. Gasson JC ((UCLA Jonsson Comprehensive Cancer Center ), et al.), et al.