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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 - PowerPoint PPT Presentation
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Ch2. Genome Organization Ch2. Genome Organization and Evolutionand Evolution
阮雪芬阮雪芬Nov21, 2002Nov21, 2002
NTUSTNTUST
Protein ArrayProtein Array
• Detection of specific antibody–antigen interactions on the hEx1 cDNA array.
DNA MicroarrayDNA Microarray
• 或稱 DNA chip
• For checking a sample of DNA simultaneously for 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.
DNA MicroarrayDNA 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.
Application of DNA MicroarrayApplication 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
Chip TechnologyChip Technology
Eur J Nucl Med 2002, 29, 115-32
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
cDNA Microarray Chip
From the point of view of bioinformatics. DFrom the point of view of bioinformatics. DNA arrays are yet another profilic stream oNA arrays are yet another profilic stream o
f data creationf data creation
Eavesdropping on the Transmission Eavesdropping on the Transmission of Genetic Informationof 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 hu
mans
– Banding patterns of chromosomes– DNA sequences
Linkage maps of genesLinkage 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
Recombination FrequencyRecombination 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%
b a c5.4 3.5
Eavesdropping on the Transmission Eavesdropping on the Transmission of Genetic Informationof 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 hu
mans
– Banding patterns of chromosomes– DNA sequences
Banding Patterns of ChromosomesBanding Patterns of Chromosomes
• Chromosome– Physical objects
p: petite (短 )q: queue (長 )
centromere8p1.2
17q2.2
Restriction EnzymesRestriction 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
第二類核酸限切酵素第二類核酸限切酵素
鈍端 (blunt end)黏端 (sticky end)
連接酵素連接酵素 (ligase)(ligase)
載體載體 (vector)(vector)
Restriction MapRestriction Map
Restriction MapRestriction Map
Cystic FibrosisCystic FibrosisKnowing the general region of the chromosome
Search the DNA of that region to identify candidate genes
Pinpoint the particular gene responsible and sequence it
Cystic FibrosisCystic Fibrosis
• In 1989 the gene was isolated and sequenced.• CFTR: cystic fibrosis transmembrane conductan
ce 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
Mappings Between The MapsMappings 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
Somatic Cell HybridsSomatic Cell Hybrids
High-resolution Maps (I)High-resolution Maps (I)
• Variable number tandem repeats (VNTRs)– Minisatellites– 10-100 bp long, repeated a var
iable number of times– Genetic fingerprints– RFLPs (restriction fragment le
ngth polymorphisms)– Southern blotting – PCR (polymerase chain reacti
on)
Southern BlottingSouthern Blotting
Multiple Cycles of PCR (I)Multiple Cycles of PCR (I)
Multiple Cycles of PCR (II)Multiple Cycles of PCR (II)
Multiple Cycles of PCR (III)Multiple Cycles of PCR (III)
High-resolution Maps (II)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 al
ong a chromosome from an organism of interest– Human-stored in yeast or bacterial cells as YAC or B
AC• 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)