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Linkage and
Mapping
For linked genes, recombinant frequencies are less than
50 percent
Figure 4-8
Map distances are generally additive
A map of the 12 tomato chromosomes
• Genetic distance is measured by
recombination frequency
• A relative map can be constructed based
on genetic distances
Genetic vs. Molecular Maps
• What is the relationship of genetic distance to
molecular distance?
• How can genetic and molecular relationships be
reconciled?
• How can one be used to locate the other?
Genetic markers
• Genetic mapping between positions on
chromosomes
– Positions can be genes
• Responsible for phenotype
– Examples: eye color or disease trait
– Positions can be physical markers
• DNA sequence variation
© 2005 Prentice Hall
Inc. / A Pearson
Physical markers
• Physical markers are DNA sequences that
vary between two related genomes
• Referred to as a DNA polymorphism
• Usually not in a gene
– Examples
• SSLP (microsatellite)
• SNP
–
–
–
–
RFLP
Intergenic SNP
Silent intragenic SNP
Causative point mutation
SSLP
•
Simple-sequence length polymorphism
•
•
•
•
Most genomes contain repeats of three or four nucleotides
Length of repeat varies
Use PCR with primers external to the repeat region
On gel, see difference in length of amplified fragment
1
1 ATCCTACGACGACGACGATTGATGCT
18
2 ATCCTACGACGACGACGACGACGATTGATGCT
12
2
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle
River, New Jersey 07458
RFLP
• Restriction-fragment length polymorphism
– Cut genomic DNA from two individuals with
restriction enzyme
– Run Southern blot
– Probe with different pieces of DNA
– Sequence difference creates different band
pattern
1
2
1
2
GGATCC
CCTAGG
GGATCC
CCTAGG
200
200
KpnI
GGTACC
CCATGG
*
GCTACC
CGATGG
*
400
400
GGATCC
CCTAGG
*
600
400
GGATCC
CCTAGG
200
*
SNP
• Single-nucleotide polymorphism
– One-nucleotide difference in sequence of two
organisms
– Discovered by sequencing
– Example: Between any two humans, on average
one SNP every 1,000 base pairs
1 ATCGATTGCCATGAC
2 ATCGATGGCCATGAC
SNP
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle
River, New Jersey 07458
Physical mapping
• Determination of physical distance
between two points on chromosome
– Distance in base pairs
• Example: between physical marker and a
gene
• Need overlapping fragments of DNA
– Requires vectors that accommodate large
inserts
• Examples: cosmids, YACs, and BACs
© 2005 Prentice Hall
Inc. / A Pearson
Large insert vectors
• Lambda phage
– Insert size: 20–30 kb
• Cosmids
– Insert size: 35–45 kb
• BACs and PACs (bacterial and P1 artificial
chromosomes respectively)
– Insert size: 100–300 kb
• YACs (yeast artificial chromosomes)
– Insert size: 200–1,000 kb
© 2005 Prentice Hall
Inc. / A Pearson
Pros and cons of large-insert
vectors
• Lambda phage and
cosmids
– Inserts stable
– But insert size too
small for large-scale
sequencing projects
• YACs
– Largest insert size
– But difficult to work
with
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle
River, New Jersey 07458
BACs and PACs
• BACs and PACs
– Most commonly used
vectors for large-scale
sequencing
– Good compromise
between insert size and
ease of use
– Growth and isolation
similar to that for
plasmids
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle
River, New Jersey 07458
Contigs
• Contigs are groups of overlapping pieces of chromosomal
DNA
– Make contiguous clones
• For sequencing one wants to create “minimum tiling path”
– Contig of smallest number of inserts that covers a region of
the chromosome
genomic DNA
contig
minimum
tiling path
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle
River, New Jersey 07458
Pros and cons whole genome
shotgun sequencing
• Pros
– Very rapid
– Becomes cheaper as sequencing technologies advance
• Cons
– Alignment is more challenging, especially in repeats
– Requires more computing power
Phenotypic and molecular markers mapped on human
chromosome 1
Figure 4-20
SNP genotyping methods
Single SNPs:
– SSCP
– TGCE
– Differential PCR amplification
Many SNPs simultaneously:
– SNP arrays
– Direct sequencing (high-throughput)
Temperature
gradient gel
electrophoresis
Alignment of physical and recombination maps
Phase I (2005)
1M SNPs from 269 individuals
Phase II (2007)
3M SNPs from 270 individuals
Phase III (2010)
1.6M SNPs genotyped from 1184 individuals from 11 populations
Sequenced 10x 100kb regions from 692
Using haplotypes to deduce gene position
Figure 4-16