Download 1 Mbp DNA for human genome

MAPPING GENOMES – genetic, physical & cytological maps
Genetic distance (in cM)
1 centimorgan = 1 map unit, corresponding to recombination frequency of 1%
Physical distance (in bp)
- determined by DNA sequencing (or restriction fragment sizing)
1 cM corresponds to ~ 1 Mbp DNA for human genome
Gibson Fig. 1.3
Discrepancies (at times) between genetic & physical maps
Genetic mapping data may have
limited resolution
(eg if not enough progeny)
or limited accuracy
(eg recombinational “hotspots”)
or “coldspots”
- no absolute relationship between
genetic & physical distances
Yeast chr III
Fig. 3.25
Why don’t tomatoes taste as good as
they used to?
Sl-GLK2 gene mutation improves uniformity of
red colour… but less sugar & less tasty
Nature May 31, 2012
Ottawa Citizen September 15, 2012
Powell, Science 336:1711, 2012
STRATEGIES USED IN GENOMIC ANALYSIS
- 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
Markers used to
anchor assembled
sequences on map
Fig. 3.3
DNA MARKERS USED IN GENETIC MAPPING
RFLPs – restriction fragment length polymorphisms
SSLPs – simple sequence length polymorphisms
SNPs – single nucleotide polymorphisms
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) at
significant frequencies in a population
if present < 2% in population, called “mutation” or “mutant allele”
Haplotype – set of alleles linked on a chromosome
usually inherited together as a block,
“haploid genotype”
1. RFLPs (Restriction Fragment Length Polymorphisms)
- inherited pattern of restriction sites follows Mendelian rules
Fig. 3.4
How to detect RFLPs?
i. by Southern hybridization analysis
- if “polymorphic” restriction site is present, detect two hybridizing
fragments, 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 markers
Lane 2 = DNA homozygous for allele 1
Lane 3 = DNA homozygous for allele 2
Fig. 3.5
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. 3.6
How to detect SSLPs by PCR
Interpretation if lane A = DNA from
person #1 and lane B = DNA from
person #2 ?
Profile for lane A sample
in blue & size markers in
brown
The more copies in tandem, the larger
the PCR product ... and the slower it
migrates electrophoretically
Fig.3.5
Differences in copy number in microsatellite array
among individuals useful in genetic profiling
DNA fingerprinting,
forensic analysis
homozygous vs.
heterozygous state
for a particular
microsatellite locus?
Fig. 7.24
Blue, Green: fluorescent tagged PCR products
Red: DNA size markers
Each lane = DNA profile (with microsatellite length variants)
from different individual
- high heterozygosity of microsatellites (multiple alleles in population)
- estimated that ~ 3 million differences between any two copies
of human genome (natural sequence variation)
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)
Fig. 3.7
“...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.”
Exome = “coding sequences of
92% of human genes”
“Thus, a population genomic survey has revealed a functionally
important locus in genetic adaptation to high altitude.”
Yi et al. Science 329:75 (July 2 & August 13, 2010)
How to detect SNPs Oligomer-specific hybridization tests
- reduced stability of hybrid between oligomer probe & test DNA
if any mis-matches
- “stringency” of hybridization conditions (eg.temperature, salt…)
influences stability of imperfect complementarity
Fig. 3.8A
(i) Detecting hybridization by “dye-quenching”
“molecular beacon”
FRET (fluorescence
resonance energy transfer)
- when oligomer probe forms stable
hybrid with template DNA (vs. hairpin
structure), generates fluorescent signal
NB: your text just shows a figure for this type of hybridization probe, but others
include tagged PCR products (ss), end-labelled oligomers, etc. see Topic 3
Fig.3.8B
(ii) Detecting oligomer hybridization using DNA microarrays
- for large scale analysis of SNPs (see later Topics for other
applications)
Tech.note 3.1
“Oligo chips”
- density ~ 10 6 oligos/cm2
- 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
“~150,000 polymorphisms can be typed in a single experiment...” p.71 Affymetrix's GeneChip
Applications for drug discovery:
World Array 1: Axiom® Genome-Wide EUR 1 Array Plate – European ancestry
World Array 2: Axiom® Genome-Wide EAS 1 Array Plate – East Asian ancestry etc.
“SNPs selected from disease and drug response GWAS databases [from
Genome-Wide Association Studies] … saturated coverage in over 5,000 gene
regions previously identified as disease-associated…. ”
Griffiths p.404
Why might some
signals be
stronger than
others in SNP
analysis?
Note: microarray
Fig.T3.2 (p.71) shows
RNA expression data
not SNP data
Advantages of DNA markers
- easy to generate large number
& easy to detect
Genetic markers
- SNPs & microsatellites
rather evenly spread
in genome
- codominant, can detect
both alleles in heterozygote
Microsatellite analysis by PCR
Fig.7.24
Fig. 3.19 & 3.21
DNA markers
PHYSICAL MAPPING APPROACHES
1. Restriction mapping
- determine relative positions of restriction sites in DNA
molecule by analyzing sizes of fragments generated by
restriction enzymes
How many EcoRI sites are present? Where? ...
Fig. 3.27
2. FISH (fluorescence in situ hybridization)
- intact metaphase chromosome (as ss DNA, denatured
with formamide) hybridized with fluorescently labelled probe
~ 1 Mbp resolution of markers
Ottawa Citizen newspaper Sept. 6, 2012 (see Topic 1)
46 human chromosomes
telomere-specific repetitive
DNA probe
Fig. 3.32
Fibre-FISH
- mechanically-stretched chromosomes or “molecular combing”
of isolated DNA for higher resolution
10 – 25 kb resolution
Fig. 3.31
Probes: cosmid 1, cosmid 2,
(overlap region in yellow)
Nature Feb. 2001
3. STS (sequence tagged site) physical mapping
What are STSs?
- any short, known DNA sequence (100-500 bp)
which is unique in genome
… so can be used as physical mapping marker
A B C D E F G HI J
K
L MN O P Q R S
T U V W
STSs
BACs
- wish to have many STSs along chromosome
- if STS is present in two clones, can deduce that those clones are
overlapping
Examples of sequences that can serve as STSs
1. ESTs – expressed sequence tags (from cDNA clones,
ie. representing mRNAs for various genes see Fig.3.36)
2. SSLPs (Microsatellites)
- tandem simple sequence repeats with unique
flanking sequences
3. Random genomic 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)
(eg. clone library)
Fig. 3.35
1. Clone libraries
Fig. 3.39
Fig. 3.1
Potential problems in mapping genomes with repetitive DNA
1. Tandem repeats
(DNA sequences shown
as single stranded, eg.
from computer file)
- omission of internal regions of tandem repeat array…
Fig.3.2A
2. Dispersed repeats
- omission of sequences located between two repeats
or incorrect linking (eg from different chromosomes)
Fig. 3.2B