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Chapter 21
Genomics
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Cytogenetic map:
Three types of
maps associated
with the Genome
sc (1A8)
w (3B6)
A B C D E FA
1
Obtained from analysis
of polytene
chromosomes
1
B
C
D
2
EF
2
A B
C
3
3
Linkage map:
sc
w
1.5 mu
The results from
each type of
mapping technique
may be slightly
different
Physical map:
sc
D E F A B
w
~ 2.4 x 106 bp
Brooker, Figure 21.1
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4
C
Fluorescence in situ hybridization
Sister
chromatids
Treat cells
with agents
that make
them swell
and fixes
them onto
slide.
Hybridized
probe
Denature
chromosomal
DNA.
Add fluorescently
labeled avidin, which
binds to biotin.
Denatured
DNA (not
in a doublehelix
form)
Fluorescent
molecule
bound to
probe
Add single-stranded
DNA probes that have
biotin incorporated
into them.
View with a
fluorescence
microscope.
Figure 21.2
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© : From Ried, T., Baldini, A., Rand, T.C., and Ward, D.C. "Simultaneous visualization of seven different DNA probes by in situ hybridization using
combinatorial fluorescence and digital imaging microscopy. PNAS. 89: 4.1388-92. 1992. Courtesy Thomas Ried
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EcoRI sites PRESENT
on both
chromosomes
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Region of two homologous chromosomes from individual 1
EcoRI
2000 bp
EcoRI
EcoRIEcoRI
EcoRI
5000 bp
1500 bp
3000 bp
EcoRI EcoRI
EcoRI
2000 bp
EcoRI
5000 bp
1500 bp
3000 bp
2500 bp
EcoRI
2000 bp
EcoRI
2000 bp
5000 bp
4500 bp
EcoRI
EcoRI
5000 bp
EcoRI
2500 bp
EcoRI
4500 bp
EcoRI
2500 bp
Region of two homologous chromosomes from individual 2
EcoRI
EcoRI
EcoRI
EcoRI
EcoRI sites ABSENT
from both
chromosomes
EcoRI
EcoRI
2500 bp
Figure 21.4
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21 - 17
EcoRI site found
only on one
chromosome
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Region of two homologous chromosomes from individual 3
EcoRI
5000 bp
2000 bp
EcoRI
2000 bp
The three individuals share
many DNA fragments that
are identical in size.
Indeed, if these segments
are found in 99% of
individuals in the population,
they are termed
monomorphic
EcoRI EcoRI
EcoRI
1500 bp
5000 bp
2500 bp
3000 bp
EcoRI
EcoRI
EcoRI
EcoRI
EcoRI
EcoRI
4500 bp
2500 bp
Cut the DNA from
all 3 individuals
with EcoRI.
Separate the DNA
fragments by gel
electrophoresis.
1
2
3
5000 bp
4500 bp
3000 bp
Polymorphic
bands are
indicated at
the arrows.
2500 bp
2000 bp
Figure 21.4
1500 bp
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21 - 18
Southern Blotting of RFLP
Figure not in Brooker
RFLPs (and other markers) can be mapped
Figure not in Brooker
Short Tandem Repeats (STR)
TTTTC = (TTTTC)1
TTTTCTTTTCTTTTC = (TTTTC)3
TTTTCTTTTCTTTTCTTTTCTTTTCTTTTC = (TTTTC)5
Cataloging the world’s SNP variation
www.hapmap.org
PCR of
microsatellites
Set of
chromosomes
Add PCR
primers.
The PCR
primers specifically
recognize sequences
on chromosome 2.
The two STS copies in this case are
different in length.
Therefore, their microsatellites have
different numbers of CA repeats
Figure 21.5
2
2
Many cycles of PCR
produce a large amount
of the DNA fragment
contained between
the 2 primers.
Gel electrophoresis
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Parents
1
2
Offspring
3
4
5
3
4
5
(a) Pedigree
Fragment length
bp
1
2
154
150
146
140
(b) Electrophoretic gel of PCR products for a polymorphic
microsatellite found in the family in (a).
Figure 20.12
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In human genetics, computer algorithms can be used to
determine linkage
The likelihood of linkage between two RFLPs is
determined by the lod (logarithm of the odds) score
method
– Computer programs analyze pooled data from a large
number of pedigrees or crosses involving many RFLPs
– They determine probabilities that are used to calculate
the lod score
Probability of a certain degree of linkage
Probability of independent assortment
• lod score = log10
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Physical Mapping
A
B
C
D
E
F
GH
I
J
K
L
M
NO
P
Q
R
Clone individual pieces
into vectors.
A
B
C
1
D
GH
F
3
J
I
5
NO
M
K
7
P
9
Vector
B
C
2
D
E
4
F
I
GH
6
K
L
M
P
8
Q
R
A collection of overlapping
clones, known as a contig
10
The numbers denote the order of
the members of the contig
Figure 21.7
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21 - 31
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Region of chromosome 11
Gene A
Gene B
1.5 mu
Gene A
1
2
Gene B
3
4
5
6
7
• Genes A and B had been mapped previously to specific regions of
chromosome 11
– Gene A was found in the insert of clone #2
– Gene B was found in the insert of clone #7
• So Genes A and B can be used as genetic markers (i.e., reference points)
to align the members of the contig
Figure 21.8
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21 - 33
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ARS
(yeast origin
of replication)
Each arm has a
different
selectable
marker.
Therefore, it is
possible to
select for yeast
cells with YACs
that have both
arms
EcoRI site
CEN
(yeast
centromere)
Chromosomal DNA
EcoRI is used at
low
concentrations so
only some sites
are digested
ORI (E.coli
origin of
replication)
Selectable
marker
gene
TEL
TEL
(yeast telomere)
Left
arm
Selectable
Marker
gene
BamHI
site
Cut with
EcoRI and
BamHI.
BamHI
site
Cut
(occasionally)
with a low concentration
of EcoRI to yield very
large fragments.
+
Right
arm
Fragment not
needed in yeast
+
Mix and add DNA ligase.
Yeast
artificial
chromosome
(YAC)
TEL
Selectable
marker
gene
Figure 21.9
ORI
CEN
ARS
Large piece of chromosomal DNA
Note: This is not drawn to scale. The chromosomal
DNA is much larger than the YAC vector.
Selectable
marker
gene
TEL
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lacZ
cm R
HindIII BamHI SphI
parC
parB
BAC
vector
repE
parA
oriS
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Chromosome 16
95 million bp
Cytogenetic map
(resolution of in situ
hybridization 3–5
megabases)
Linkage map
(resolution 3–5 cM;
not all linkage
markers are shown)
D16S144
D16S40
D16S160
22.1
22.2
22.3
23.1
23.2
23.3
24.1
24.2
24.3
21.0
12.2
13.0
*
D16S150
D16S149
11.2
D16AC6.5 12.1
11.1
D16S48
11.2
13.2
13.13
13.12
13.11
D16S159 12.3
12.2
12.1
q
D16S60
D16S85
13.2
Low resolution
(3–5 million bp)
p
*
YAC N16Y1
150,000 bp
*
310C4
N16Y1-29
Physical map of
overlapping cosmid
clones (resolution
5–10 kilobases)
= (GT)n
N16Y1-18
N16Y1-13
N16Y1-14
N16Y1-12
N16Y1-16
N16Y1-30
High resolution
(1–100,000 bp)
Cosmid
contig 211
5F3
312F1
309G11
N16Y1-19
N16Y1-10
*
STS N16Y1-10
Primer
3′
5′
3′
AGTCAAACGTTTCCGGCCTA
GATCAAGGCGTTACATGA
TCAGTTTGCAAAGGCCGGAT
CTAGTTCCGCAATGTACT
AGTCAAACGTTTCCGGCCTA
5′
3′ Sequence-tagged site
5′ (resolution 1 base)
5′— GATCAAGGCGTTACATGA — 3′
Primer
219 bp
Figure 21.11
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Gene B
1
Gene B
Gene A
2
3
4
5
Numbers indicate
regions that are
subcloned.
(Starting clone)
The number of steps required
to reach the gene of interest
depends on the distance
between the start and end
points
Cosmid
vector
Subclone.
Screen a library.
1
2
....n
(Second clone)
Subclone.
2
Screen a library.
2
3
(Third clone)
Repeat subcloning and
screening until gene A
is reached.
n
Figure 20.18
Gene A
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Isolate
chromosomal
DNA
Isolate
chromosomal
DNA
Clone large chromosomal
DNA fragments into BACs and
create a contig for each
chromosome.
Shear DNA into small and
large pieces. Clone
chromosomal DNA pieces
into vectors.
BAC
vector
Vector
BAC contig
Chromosomal
DNA
For each BAC, shear into
smaller pieces and clone DNA
pieces into vectors.
Chromosomal
DNA
Vector
Clones from
one BAC insert:
Chromosomal
DNA
From the clones of each BAC,
determine the chromosomal
DNA sequence, usually at
one end, by shotgun
sequencing. The results
below show the sequences
from three chromosomal DNA
clones.
CCGA CCT T A CCGACCA
G A C C A C C C GA T T A A T
T T A A T C GC GA A T T G
Based on overlapping
regions, create one
contiguous sequence.
C C G A C C T T A C C GA C C A C C C G A T T A A T C G C GA A T T G
(a) Hierarchical genome shotgun sequencing
Figure 21.14
Determine the chromosomal
DNA sequence, usually at
both ends, by shotgun
sequencing. The results
below show sequences of
three chromosomal DNA
clones.
T T A C C G G T A GGC A C C T
C A C C T GT T A C GGGT C
GGGT C A A A C C T A GG
Based on overlapping
regions, create one
contiguous sequence.
T T A C C GGT A G G C A C C T G T T A C G GG T C A A A C C T A G G
(b) Whole-genome shotgun sequencing
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Isolate genomic DNA
and break into fragments.
Deposit the beads into a picotiter
plate. Only one bead can fit into
each well.
Fragment of
genomic DNA
Covalently attach oligonucleotide
adaptors to the 5′ and 3′ ends of
the DNA.
Adaptors
Denature the DNA into single
strands and attach to beads via
the adaptors. Note: Only one DNA
strand is attached to a bead.
Add sequencing reagents:
DNA polymerase, primers,
ATP sulfurylase, luciferase,
apyrase, adenosine 5′
phosphosulfate, and luciferin.
Sequentially flow solutions
containing A, T, G, or C into the
wells. In the example below, T
has been added to the wells.
PPi (pyrophosphate) is released
when T is incorporated into the
growing strand.
T
C
Emulsify the beads so there is only
one bead per droplet. The droplets
also contain PCR reagents that
amplify the DNA.
A
T
T
C
G A
Primer
PPi + Adenosine 5′
phosphosulfate
ATP sulfurylase
ATP + luciferin
Luciferase
Light
Light is detected by a camera
in the sequencing machine.
21 - 48