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MAPPING GENOMES – genetic, physical & cytological maps
Genetic distance (in cM)1 centimorgan = 1 map unit, corresponding to recombination frequency of 1%
1 cM corresponds to ~ 1 Mbp DNA for human genome
Gibson Fig. 1.3
Physical distance (in bp)- determined by DNA sequencing (or restriction fragment sizing)
- no absolute relationship between genetic & physical distances
Fig. 3.25
Discrepancies (at times) between genetic & physical maps
Genetic mapping data may have limited resolution
or limited accuracy
(eg recombinational “hotspots”)
(eg if not enough progeny)
Yeast chr III
or “coldspots”
STRATEGIES USED IN GENOMIC ANALYSIS
Fig. 3.3
Markers used to anchor assembledsequences on map
- because of recent advances in rapid sequencing technologies & powerful bioinformatics tools, “shotgun” approach is now usually used
- except for genomes with high amounts of repetitive DNA
RFLPs – restriction fragment length polymorphisms
SSLPs – simple sequence length polymorphisms
SNPs – single nucleotide polymorphisms
DNA MARKERS USED IN GENETIC MAPPING
DNA marker must have (at least) two different alleles to be useful in monitoring inheritance patterns
Alleles – alternative forms of a gene (or DNA sequence) at a particular locus (chromosomal site)
Polymorphisms – occurrence of two or more variants (alleles, phenotypes, sequence variants) atsignificant frequencies in a population
if present < 2% in population, called “mutation” or “mutant allele”
Haplotype – set of alleles linked on a chromosomeusually inherited together as a block, “haploid genotype”
1. RFLPs (Restriction Fragment Length Polymorphisms)
Fig. 3.4
- inherited pattern of restriction sites follows Mendelian rules
How to detect RFLPs?
i. by Southern hybridization analysis
Fig. 3.5
- if “polymorphic” restriction site is present, detect two hybridizingfragments, vs. one if site is absent
Length of large fragment = sum of two small ones
Fig. 3.5
ii. by PCR analysis
Lane 1 = size markersLane 2 = DNA homozygous for allele 1Lane 3 = DNA homozygous for allele 2
Fig. 3.6
2. SSLPs (simple sequence length polymorphisms)
Microsatellites
- tandem repeats of short stretches ( < 5 bp or so) in clusters usually < 150 bp
Different number of GA copies in tandem
Fig.3.5- small fragments migrate more rapidly than large ones towards positive electrode
Electrophoretic mobility- detect length differences
Interpretation if lane A = DNA from person #1 and lane B = DNA from person #2 ?
How to detect SSLPs by PCR
Fig. 7.24
Differences in copy number in microsatellite array among individuals useful in genetic profiling
Blue, Green: fluorescent tagged PCR productsRed: DNA size markers
- high heterozygosity of microsatellites (multiple alleles in population)
homozygous vs.heterozygous state for a particularmicrosatellite locus?
Each lane = DNA profile (with microsatellite length variants) from different individual
- estimated that ~ 3 million differences between any two copiesof human genome (natural sequence variation)
DNA fingerprinting,forensic analysis
Fig. 3.7
3. SNPs (single nucleotide polymorphisms)
SNPs usually exist as just two variants in population, because for a third allele to arise, mutation must occur at exactly the same position (see p.69)
“...found population-specific allele frequency changes.... one SNP in EPAS1 (transcription factor gene involved in response to hypoxia) showed a 78% frequency difference between Tibetan [high altitude] and Han [sea level] samples.”
Yi et al. Science 329:75 (July 2 & August 13, 2010)
“Thus, a population genomic survey has revealed a functionally important locus in genetic adaptation to high altitude.”
Exome = “coding sequences of 92% of human genes”
Fig. 3.8A
- “stringency” of hybridization conditions (eg.temperature, salt…)influences stability of imperfect complementarity
- reduced stability of hybrid between oligomer probe & test DNA if any mis-matches
Oligomer-specific hybridization testsHow to detect SNPs
(i) Detecting hybridization by “dye-quenching”
- when oligomer probe forms stablehybrid with template DNA (vs. hairpin structure), generates fluorescent signal
Fig.3.8B
“molecular beacon”
FRET (fluorescence resonance energy transfer)
(ii) Detecting oligomer hybridization using DNA microarrays
“Oligo chips”
Tech.note 3.1
- for large scale analysis of SNPs (see later Topics for otherapplications)
- grid of different oligomers synthesized in situ on glass wafer by photolithographic process
- fluorescently labelled DNA (or cDNA…) allowed to hybridize
- hybrids monitored by laser scanning
- density ~ 10 6 oligos/cm2
“~150,000 polymorphisms can be typed in a single experiment...” p.71 Affymetrix's GeneChip
Griffiths p.404
Note: microarray Fig.T3.2 (p.71) shows RNA expression data not SNP data
Why might some signals be stronger than others in SNP analysis?
Advantages of DNA markers
- easy to generate large number & easy to detect
- codominant, can detect both alleles in heterozygote
- SNPs & microsatellites rather evenly spread in genome
Fig. 3.19 & 3.21Fig.7.24
Microsatellite analysis by PCR
Genetic markers DNA markers
PHYSICAL MAPPING APPROACHES
1. Restriction mapping
- determine relative positions of restriction sites in DNAmolecule by analyzing sizes of fragments generated byrestriction enzymes
Fig. 3.27
2. FISH (fluorescence in situ hybridization)
- intact metaphase chromosome (as ss DNA, denaturedwith formamide) hybridized with fluorescently labelled probe
Fig. 3.32
Drosophila centromere-specific tandem repetitive DNA probe
~ 1 Mbp resolution of markers
10 – 25 kb resolution
Fig. 3.31
Fibre-FISH
Probes: cosmid 1, cosmid 2, (overlap region in yellow)Nature Feb. 2001
- mechanically-stretched chromosomes or “molecular combing”of isolated DNA for higher resolution
3. STS (sequence tagged site) physical mapping
What are STSs?
- any short, known DNA sequence (100-500 bp)
which is unique in genome
- wish to have many STSs along chromosome
STSs A B C D E F G HI J K L MN O P Q R S T U V W
BACs
- if STS is present in two clones, can deduce that those clones are overlapping
… so can be used as physical mapping marker
1. ESTs – expressed sequence tags (from cDNA clones, ie. representing mRNAs for various genes see Fig.3.36)
2. SSLPs (Microsatellites)
3. Random genomic sequences
Examples of sequences that can serve as STSs
- tandem simple sequence repeats with unique flanking sequences
as long as single copy in genome
eg. only one hybridization signal in Southern experiment
Distance between 2 STSs will determine whether they are:
likely, sometimes or never located on same DNA fragment
In genomic analysis, want overlapping set of DNA fragments spanning genome (or chromosome separated by flow cytometry, Fig.3.38)
Fig. 3.35
(eg. clone library)
Fig. 3.39
1. Clone libraries
Fig. 3.1
Potential problems in mapping genomes with repetitive DNA
Fig.3.2A - omission of internal regions of tandem repeat array…
(DNA sequences shown as single stranded, eg. from computer file)
1. Tandem repeats
2. Dispersed repeats
Fig. 3.2B- omission of sequences located between two repeatsor incorrect linking (eg from different chromosomes)