Download 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
– 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 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 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 Technology
Control or treatment
Fluorescently labeled DNA or RNA
mRNA
Reverse transcriptase to generate
Cy3/Cy5 cDNA probes
hybridization
Hybridization to the gene chip
Data analysis
Eur J Nucl Med 2002, 29, 115-32
cDNA Microarray Chip
From the point of view of bioinformatics.
DNA arrays are yet another profilic stream
of data creation
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
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
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
5.4
c
3.5
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
Banding Patterns of Chromosomes
• Chromosome
– Physical objects
p: petite (短)
q: queue (長)
centromere
8p1.2
17q2.2
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
第二類核酸限切酵素
鈍端(blunt end)
黏端(sticky end)
連接酵素(ligase)
載體(vector)
Restriction Map
Restriction Map
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
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
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
Somatic Cell Hybrids
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)
Southern Blotting
Multiple Cycles of PCR (I)
Multiple Cycles of PCR (II)
Multiple Cycles of PCR (III)
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)