Chapter 15Genomics and Medicine.ppt
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1、 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Chapter 15 Genomics and Medicine,The impact of genomics on the practice of medicine, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Contents,Medical promise of
2、genomics High-throughput methods for genotyping Cancer genomics Microbial genomics and medicine Finding new drug targets Developing vaccines DNA vaccines Gene therapy, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,What we hope to gain from genomics,Drug
3、, diagnostics, and prognostics development Genotyping to predict patient susceptibility to disease Personalized healthcare based on an individuals genomic features, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Long term returns,Personalized genotype da
4、tabases Used to assess health risks throughout life Adjustments to lifestyle and medical treatment Simulated cells Reduce the need for time consuming experiments Allow experiments that would otherwise be impossible New frameworks for clinical trials Pharmacogenomics, 2005 Prentice Hall Inc. / A Pear
5、son Education Company / Upper Saddle River, New Jersey 07458,Short term returns,Faster characterization of disease genes Better disease diagnosis / prognosis with microarrays Better methods for genotyping More efficient drug / vaccine development, 2005 Prentice Hall Inc. / A Pearson Education Compan
6、y / Upper Saddle River, New Jersey 07458,Advances in disease genetics,Detection of disease genes is most direct medical use of genomics information Over 1,000 disease genes were characterized by 2000 How to exploit this information?,250,0,1981,2000,Year of discovery,Disease genes, 2005 Prentice Hall
7、 Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Preimplantation diagnosis,Couples with at least one child suffering from cystic fibrosis underwent preimplantation diagnosis Biopsied cells from in vitro 3-day old embryos were genotyped Implanted embryos (NN and ND) in one c
8、ouple resulted in a healthy baby girl,DD,NN,DD,NN,BAD,ND,diagnosis,homoduplex,heteroduplex,biopsied cell,DNA added,1,2,3,4,5,6, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Single nucleotide polymorphisms (SNPs),Benefits of characterizing SNPs High den
9、sity SNP map will greatly facilitate finding disease genes Detection of SNPs can serve as a diagnostic for genetic diseases Millions of SNPs presently in public and private databases Fast, cheap, and accurate genotyping of SNPs still a challenge Smallest linkage disequilibrium studies still out of r
10、each Genotyping 30,000 SNPs in 1,000 individuals required 10-fold increase in technological capacity at end of 2001, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Biochemical basis for SNP genotyping,Hybridization with allele-specific oligonucleotides (
11、ASOs) Allele-specific primer extension Minisequencing Oligonucleotide ligation Restriction site cleavage Invasive cleavage, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Hybridization of allele-specific oligonucleotides,Uses hybridization to detect SNP
12、Heat or electric field used to denature hybrids Caveat Each SNP hybrid will denature with different parameters Microarrays overcome this to some degree,Allelle-specific probes,Stable,Unstable, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Allele-specifi
13、c primer extension,Use allele specific primers that include the SNP PCR will extend primers that match SNP, but not mismatches Detection Fluorescence assay that detects incorporation of primer into PCR product,Allele-specific primers,Extension,No extension,PCR, 2005 Prentice Hall Inc. / A Pearson Ed
14、ucation Company / Upper Saddle River, New Jersey 07458,Minisequencing,Use DNA polymerase to extend primer sequence by a single nucleotide Detection Colorimetric assay using antibodies to chemical group attached to nucleotide Luminometric detection of chemical released upon nucleotide addition Mass s
15、pectroscopy,One-nucleotide extension,Minisequencing primer,No extension,PCR, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Oligonucleotide ligation,Three probes used Increases cost Ligase ties together matched probes Detection methods Changes in mobilit
16、y Microarrays,Left probes,Match, ligation,Mismatch, no ligation,Right probe,Ligase, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Restriction site cleavage,Presence of SNP creates a site for cleavage by restriction enzyme Pattern of restriction fragment
17、s reveals presence (or absence) of SNP Method not sufficient for genome-wide SNP scan,Restriction enzyme,Cleavage,No cleavage, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Invasive cleavage,Highly specific probe binds to target sequence Probe causes ch
18、ange in conformation of double stranded oligonucleotide New conformation provides target for cleavage by FLAP endonuclease Cleaved signal sequence indicates SNP Requires a lot of DNA,Invader probe,Mismatch, cleavage,Match, no cleavage,Flap Endonuclease, 2005 Prentice Hall Inc. / A Pearson Education
19、Company / Upper Saddle River, New Jersey 07458,An ideal SNP genotyping method,PCR is rate limiting step of most SNP genotyping techniques The ideal genotyping method Single molecule genotyping (i.e. no PCR) Example: atomic force microscopy with nanotube probes,AFM,labels,DNA,probe,height,DNA molecul
20、e, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Cancer genome projects,Cancer Genome Anatomy Project (CGAP) Established 1997 by National Cancer Institute (USA) Specializes in EST sequencing Human Cancer Genome Project (HCGP) Established 1999 by Brazili
21、an research groups Specializes in SAGE analysis Cancer Genome Project (CGP) Established 2000 by Wellcome Trust and Sanger Institute (United Kingdom) Specializes in genomic mutations leading to cancer, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Cancer
22、 genomics using ESTs and SAGE,Use EST/SAGE tags from normal and tumorous tissue Tags stored in publicly accessible databases Bioinformatics tools used to reveal patterns of gene expression that define cancerous states,normal,tumor,SAGE/EST,sequencing,tags,database and analysis, 2005 Prentice Hall In
23、c. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Searching for cancer-causing mutations in genomic DNA,Use human genome sequence to make PCR primers for target genes Compare PCR products from normal tissue and tumors using automated heteroduplex analysis Sequence genes when he
24、teroduplex analysis suggests tumor/normal differences Find genomic mutants,PCR,heteroduplex analysis,sequence mutants,Make PCR primers of target genes from normal and tumor tissue,1,2,3, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458,Benefits of cancer g
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