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Ch2. Genome Organization and Evolution

Ch2. Genome Organization and Evolution. 阮雪芬 Nov21, 2002 NTUST. Protein Array. Detection of specific antibody–antigen interactions on the hEx1 cDNA array. DNA Microarray. 或稱 DNA chip For checking a sample of DNA simultaneously for the presence of many sequences. Can be used

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Ch2. Genome Organization and Evolution

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  1. Ch2. Genome Organization and Evolution 阮雪芬 Nov21, 2002 NTUST

  2. Protein Array • Detection of specific antibody–antigen interactions on the hEx1 cDNA array.

  3. DNA Microarray • 或稱DNA chip • For checking a sample of DNA simultaneouslyfor the presence of many sequences. • Can be used • To determine expression patterns of different proteins by detection of mRNA. • For genotyping The correlation between the abundance of an mRNA and the corresponding protein is imperfect.

  4. DNA Microarray • A DNA array may contain 100000 probe oligomers. • The spot size ~150 u in diameter • Oligomers of length ~50-80 bp • For genotype, genomic DNA fragments of length 500-5000 bp.

  5. Application of DNA Microarray • Investigating cellular states and processes. • Diagnosis of disease: • Huntington disease: expanded repeats of CAG • In normal, 11-28 CAG repeats • >41 CAG repeats, huntington disease • Genetic warning signs • Drug selection • Classification of disease: • Different types of leukaemia can be identified by different patterns of gene expression • Target selection for drug design • Pathogen resistance

  6. Chip Technology Control or treatment mRNA Reverse transcriptase to generate Cy3/Cy5 cDNA probes Hybridization to the gene chip Data analysis Fluorescently labeled DNA or RNA hybridization Eur J Nucl Med 2002, 29, 115-32

  7. cDNA Microarray Chip

  8. From the point of view of bioinformatics. DNA arrays are yet another profilic stream of data creation

  9. Eavesdropping on the Transmission of Genetic Information • Three types of maps have been essential • Linkage maps of genes • Classically determined by observed patterns of heredity. • The unit of length in a gene map is the Morgan. • 1 cM~1% recombination frequency~1x106 bp in humans • Banding patterns of chromosomes • DNA sequences

  10. Linkage maps of genes • Example: Cross 1: a+b x ab+1773 a+b, 1747 ab+, 104 a+b+, 96 ab Cross 2: b+c x bc+ 1348 b+c., 1312 bc+, 124 b+c+, 108 bc Cross 3: a+c x ac+ 1443 a+c, 1483 ac+, 51a+c+, 55 ac

  11. Recombination Frequency • Ra-b = (104 + 96 )/3720 = 0.054 =5.4% • Rb-c = (124 + 108)/2892 = 0.080 =80% • Ra-c = (51+55)/ 3031 = 0.035 = 3.5% a b c 5.4 3.5

  12. Eavesdropping on the Transmission of Genetic Information • Three types of maps have been essential • Linkage maps of genes • Classically determined by observed patterns of heredity. • The unit of length in a gene map is the Morgan. • 1 cM~1% recombination frequency~1x106 bp in humans • Banding patterns of chromosomes • DNA sequences

  13. Banding Patterns of Chromosomes • Chromosome • Physical objects p: petite (短) q: queue (長) 8p1.2 centromere 17q2.2

  14. Restriction Enzymes • 1970, Smith發現第二類核酸限切酵素，可以很準確分割DNA • 1973, Boyer-Cohen-Chang完成第一基因選殖的工作

  15. 第二類核酸限切酵素 黏端(sticky end) 鈍端(blunt end)

  16. 連接酵素(ligase)

  17. 載體(vector)

  18. Restriction Map

  19. Restriction Map

  20. Cystic Fibrosis Knowing the general region of the chromosome Search the DNA of that region to identify candidate genes Pinpoint the particular gene responsible and sequence it

  21. Cystic Fibrosis • In 1989 the gene was isolated and sequenced. • CFTR: cystic fibrosis transmembrane conductance regulator • CFTR codes for a 1480 amino acids protein that normally forms a cyclicAMP-regulated epithelial Cl- channel. • The mutation is a three base pair deletion---deleting the residue 508Phe from the protein

  22. Mappings Between The Maps • Several approaches to coordinating chromosome banding patterns with individual DNA sequences of genes • In Fluorescent In Situ Hybridization (FISH) • Somatic Cell Hybrids FISH

  23. Somatic Cell Hybrids

  24. High-resolution Maps (I) • Variable number tandem repeats (VNTRs) • Minisatellites • 10-100 bp long, repeated a variable number of times • Genetic fingerprints • RFLPs (restriction fragment length polymorphisms) • Southern blotting • PCR (polymerase chain reaction)

  25. Southern Blotting

  26. Multiple Cycles of PCR (I)

  27. Multiple Cycles of PCR (II)

  28. Multiple Cycles of PCR (III)

  29. High-resolution Maps (II) • Short tandem repeat polymorphisms (STRPs) • Microsatellites • Regions of only 2-5 bp but repeated many times • A conting (contiguous clone map): • A series of overlapping DNA clones of known order along a chromosome from an organism of interest • Human-stored in yeast or bacterial cells as YAC or BAC • A sequence tagged site (STS) • A short, sequenced region of DNA, typically 200-60 bp long, that appears in a unique location in the genome. • EST (expressed sequence tag)

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