指x旁学命 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
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
Trends of Medicine in 21th Century “5P医学”: Prediction、 Prevention、 Participation Personalization Precision Treatment afterward Predictive Preventive General Personalized Molecular epidemiology: Sub-population Rare disease>Common diseases TME 能E
Trends of Medicine in 21th Century • “5P医学”: Prediction、 Prevention、 Participation、 Personalization、 Precision – Treatment afterward →Predictive & Preventive – Molecular epidemiology: Sub-population – Rare diseases → Common diseases – General →Personalized
In times of transformation we are all students O 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 o 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 O preempt disease progression, as a result of early detection; O 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
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
Areas of Application of Molecular Diagnosis Infectious Disease Neoplastic Disease Genetic Disease Identity Testing HLA Typing Pharmacogenetics
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,S印P,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→ 1900000 affected, 270000 visible at birth
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
Beiiing s birth defect rate. 2013 (北京市卫计委,Se.1,2014) 14.68% for household registered neonates 226.22% for non-household registered neonates
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%o in living birth - Carter CO JM Genet197;l4:316 The estimated affected neonates in china 160000 per vear l
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(5m)→WBC→ lysing buffer (sds) and protease K-y centrifugation. collect supernatant -> phenol -> centrifugation, collect supernant→ RNase a and t1→ phenol > centrifugation, collect supernatant otassium acetate and etoH (absolute)->genomic DNA appears (like cotton fiber)
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)
TABLE 4. 1 Commercially Available dNa lsolation Reagents Chemistry Kit Name Manufacturer Starting Yield Kit available Material 「or Magnetic beads ChargeSwitch gDNA Invitrogen 1, 7 up to 2ug (50ul whole blood) 10 EZI DNA Tissue kit Qiagen 9-28g(1040 mg tssue Genfind System Agencourt Beckmann Coulter 1,3 7-12ug (200ul whole blood) 40 kb averag MagAttract Blood DNA kit Qiagen 0.4-1.2ug (50ul whole blood MagneSil Blood Genomic Promega 4-6ug (200ul whole blood) FlexiGene dna kit Qiagen 1,2,.5 11-14ug (0.3 mL blood) Up to 150 kb MasterPure DNA kit EPICEntRE Biotechnologies 1-10 3-9ug (200ul whole blood) 10 PAXgene Blood DNA kit PreAnalytiX gmbH 150-500pg 18 5mL blood) 50-150kb Pure Gene DNA kit Gentra systems 1,2,5915-50gmt 100-200kb Wizard Genomic dnA Po 1,5,7, 8 5-15Hg 300ul whole blood) Solid-phase adsorption ChargeSwitch direct gDNA Invitrogen From only 10ul of whole blood GenElute Blood Genomic Sigma Aldrich 4-10ug (200pl whole blood) Up to 50 kb Generation DNA kit Gentra Systems 1-8 -Bug (200ul whole blood) >23kb QIAamp DNA kit Qiagen 1-4.9 4-12ug(200ul whole blood) Up to 50 kb Wizard SV Genomic DNA Promega 5,7 20-30ug(1.2cm tissue) se blood (1), buy coat (2), plasma 3), body fluids (4), cultured cells (), cell suspension (6), tissue homogenates uccal cells (1
TABLE 4.2 Commercially Available RNA lsolation Reagents Chemistry Kit Name Manufacturer Starting Material Yield Kits available agnetic beads ChargeSwitch total RNA Invitrogen Up to 25ug from 106 cells EZI RNA Tissue kit Qiagen 10-40g5-10 mg tissue MagAttract RNA kit Qiagen 5,7 5-20ug RNA per 1 x 10 cultured cells MagMAX Blood kit Ambion 1,4 300-500 ng from 50uL of whole blood MagNA Pure total rNa Roche diagnostic 1,3-5,7 MagneSil Total RNA Promega 1,5,7 2ug from 10 cells RNAdvance cell kit Agencourt Beckmann Coulter 5 Iug from 10 cells RNAgents Total rna isolation 1,2,5.7,8 1.3 mg from 10 WBC blood Tri reagent Sigma Aldrich 1,3-5,7 5-15ug from 10 cells TRIzol RNA purification Invitrogen 1,3,5,.7,8 Solid-phase adsorption Gen Elute Mammalian total RNA Sigma Aldrich Up to 150ug from 10/ cells Purelink Total RNA kit Invitrogen 1,5,7,8 I mg from 10 cells QIAamp RNA kit Qiagen 1,7 1-5ug rna per ml of whole blood Recover ALL Total Nucleic Acid Ambion RiboPure kit Ambion 1,5,7 2-4ug/0.5 mL whole blood SV Total RNA Isolation Promega 2,5,7 20-30ug(1.2 cm tissue) Sample Types: whole blood (1), buffy coat(2), plasma 3), body fluids(4), cultured cells 5), cell suspension (6), tissue homogenates () bacteria(8), bone marrow (9), buccal cells(10), paraffin-fixed