Chapter 21Genomics
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A
1
Cytogenetic map:
Linkage map:
Physical map:
sc (1A8) w (3B6)
wsc
1.5 mu
wsc~ 2.4 x 106 bp
1 2 2 3 3 4
B C D E FA A AB B BC CC D DE EF F
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Brooker, Figure 21.1
Obtained from analysis of polytene
chromosomes
The results from each type of mapping technique may be slightly different
Three types of maps associated with the Genome
Figure 21.2Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Sisterchromatids
Treat cellswith agentsthat makethem swelland fixesthem ontoslide.
DenaturedDNA (notin a double-helixform)
Add single-strandedDNA probes that havebiotin incorporatedinto them.
DenaturechromosomalDNA.
Hybridizedprobe
View with afluorescencemicroscope.
Fluorescentmoleculebound toprobe
Add fluorescentlylabeled avidin, whichbinds to biotin.
Fluorescence in situ hybridization
21 - 13
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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 usingcombinatorial fluorescence and digital imaging microscopy. PNAS. 89: 4.1388-92. 1992. Courtesy Thomas Ried
Figure 21.4
EcoRI sites PRESENT on both
chromosomes
EcoRI sites ABSENT from both
chromosomes
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Region of two homologous chromosomes from individual 1
EcoRI
Region of two homologous chromosomes from individual 2
EcoRI EcoRI EcoRI EcoRIEcoRI
EcoRIEcoRI EcoRI EcoRI EcoRIEcoRI
EcoRIEcoRI EcoRI EcoRIEcoRI
EcoRIEcoRI EcoRI EcoRIEcoRI
2000 bp 5000 bp 1500 bp 3000 bp 2500 bp
2000 bp 5000 bp 1500 bp 3000 bp 2500 bp
2000 bp 5000 bp 4500 bp 2500 bp
2000 bp 5000 bp 4500 bp 2500 bp
EcoRI site found only on one
chromosome
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
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Figure 21.421 - 18
1 2 3
5000 bp
4500 bp
3000 bp
2000 bp
2500 bp
1500 bp
Polymorphicbands areindicated atthe arrows.
Separate the DNAfragments by gelelectrophoresis.
Cut the DNA fromall 3 individualswith EcoRI.
Region of two homologous chromosomes from individual 3
EcoRIEcoRI EcoRI EcoRI EcoRIEcoRI
2000 bp 5000 bp 1500 bp 3000 bp 2500 bp
EcoRIEcoRI EcoRI EcoRIEcoRI
2000 bp 5000 bp 4500 bp 2500 bp
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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
Gel electrophoresis
Many cycles of PCRproduce a large amountof the DNA fragmentcontained betweenthe 2 primers.
Add PCRprimers.
The PCRprimers specificallyrecognize sequenceson chromosome 2.
Set ofchromosomes
22
Figure 21.5
The two STS copies in this case are different in length.
Therefore, their microsatellites have different numbers of CA repeats
PCR of microsatellites
Figure 20.12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
1 2 3 4 5bp
154
150
146
140
(b) Electrophoretic gel of PCR products for a polymorphicmicrosatellite found in the family in (a).
(a) Pedigree
Fra
gm
ent
len
gth
Parents
Offspring
1
3
2
4 5
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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
• lod score = log10Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Probability of a certain degree of linkage
Probability of independent assortment
In human genetics, computer algorithms can be used to determine linkage
Figure 21.7
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The numbers denote the order of the members of the contig
21 - 31
A B
A B F
F
GH
GH
C D
C
B C
D
D E
E F GH I
I
I
J
J
K
K
K L
L M
M
NO
NO
P
P
PQ R
Q R
Vector
Clone individual piecesinto vectors.
1 3 5
8642
7 9
10
A collection of overlappingclones, known as a contig
M
Physical Mapping
Figure 21.8Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
• 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
21 - 33
1.5 mu
Gene A
Region of chromosome 11
Gene A Gene B
Gene B
1 2 3 4 5 6 7
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SelectableMarkergeneTEL
ORI (E.coliorigin ofreplication)
ORI
CEN(yeastcentromere)
CEN
Selectablemarkergene
Selectablemarkergene
Selectablemarkergene
TEL(yeast telomere)
TEL TEL
Yeastartificialchromosome(YAC)
Cut withEcoRI andBamHI.
Leftarm
Mix and add DNA ligase.
Note: This is not drawn to scale. The chromosomalDNA is much larger than the YAC vector.
Chromosomal DNA
Rightarm
Fragment notneeded in yeast
Cut(occasionally)with a low concentrationof EcoRI to yield verylarge fragments.
EcoRI site
ARS(yeast originof replication)
ARS Large piece of chromosomal DNA
BamHIsite
BamHIsite
+
+
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Figure 21.9
EcoRI is used at low
concentrations so only some sites
are digested
Each arm has a different
selectable marker.
Therefore, it is possible to
select for yeast cells with YACs that have both
arms
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21 - 37
lacZ
HindIII BamHI SphI
BACvector
parC
parB
parA
oriS
repE
cm R
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Figure 21.1121 - 39
Chromosome 1695 million bp
Lo
w r
eso
lutio
n(3
–5
mill
ion
bp
)
Hig
h r
eso
lutio
n(1
–1
00
,00
0 b
p)
p q
13
.2
13
.21
3.1
31
3.1
21
3.1
11
2.3
12
.2
12
.2
12
.1
12
.1
11.2
11.2
11.1
13
.0
21
.0
22
.12
2.2
22
.32
3.1
23
.22
3.3
24
.12
4.2
24
.3
D1
6S
85
D1
6S
60
D1
6S
15
9
D1
6S
48
D1
6S
15
0D
16
S1
49
D1
6S
16
0
D1
6S
40
D1
6S
14
4
500 times expansion
219 bp
Cytogenetic map(resolution of in situhybridization 3–5megabases)
Physical map ofoverlapping cosmidclones (resolution5–10 kilobases)
Sequence-tagged site(resolution 1 base)
YAC N16Y1150,000 bp
STS N16Y1-10
Primer
Primer
3′
5′
5′
*
*
GATCAAGGCGTTACATGA
5′—GATCAAGGCGTTACATGA — 3′
CTAGTTCCGCAATGTACT
Cosmidcontig 211
310C4
N16Y1-29
N16Y1-18
N16Y1-10
N16Y1-19
309G11
312F1
5F3
N16Y1-30
N16Y1-16
N16Y1-12
N16Y1-14
N16Y1-13
= (GT)n
Linkage map(resolution 3–5 cM;not all linkagemarkers are shown)
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D1
6A
C6
.5
3′
5′AGTCAAACGTTTCCGGCCTA3′
TCAGTTTGCAAAGGCCGGAT
AGTCAAACGTTTCCGGCCTA
*
*
1 2 3 4 5
Numbers indicateregions that aresubcloned.
(Starting clone)
Subclone.
Cosmidvector
Subclone.
Screen a library.
Screen a library.
(Third clone)
Repeat subcloning andscreening until gene Ais reached.
(Second clone)
....n
Gene B
Gene B
Gene A
Gene A
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1 2
2
2 3
n
Figure 20.18
The number of steps required to reach the gene of interest
depends on the distance between the start and end
points
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Figure 21.14 21 - 46
ChromosomalDNA
ChromosomalDNA
BACvector
BAC contig
Clones fromone BAC insert:
Vector
Vector
Clone large chromosomalDNA fragments into BACs andcreate a contig for eachchromosome.
Shear DNA into small andlarge pieces. Clonechromosomal DNA piecesinto vectors.
For each BAC, shear intosmaller pieces and clone DNApieces into vectors.
From the clones of each BAC,determine the chromosomalDNA sequence, usually atone end, by shotgunsequencing. The resultsbelow show the sequencesfrom three chromosomal DNAclones.
Based on overlappingregions, create onecontiguous sequence.
ChromosomalDNA
(a) Hierarchical genome shotgun sequencing (b) Whole-genome shotgun sequencing
CCG A C C T T A CCG A C C A
C T T A CC C GA C C GA C CA C CC GA T T A A T C GC GA A T T G
GA CC A CCCGA T T A A TT T A A T CGCGA A T T G
Determine the chromosomalDNA sequence, usually atboth ends, by shotgunsequencing. The resultsbelow show sequences ofthree chromosomal DNAclones.
Based on overlappingregions, create onecontiguous sequence.
T T A CC GGT A GGC A C C T
GGT A GT T A CC GC A C C T G T T A CG GGT C A A A CC T A GG
C A CC T GT T A CGGGT CGGGT CA A A CC T A GG
IsolatechromosomalDNA
IsolatechromosomalDNA
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21 - 48
Emulsify the beads so there is onlyone bead per droplet. The dropletsalso contain PCR reagents thatamplify the DNA.
Deposit the beads into a picotiterplate. Only one bead can fit intoeach well.
Add sequencing reagents:DNA polymerase, primers,ATP sulfurylase, luciferase,apyrase, adenosine 5′phosphosulfate, and luciferin.Sequentially flow solutionscontaining A, T, G, or C into thewells. In the example below, Thas been added to the wells.
PPi (pyrophosphate) is releasedwhen T is incorporated into thegrowing strand.
Isolate genomic DNAand break into fragments.
Covalently attach oligonucleotideadaptors to the 5′ and 3′ ends ofthe DNA.
Denature the DNA into singlestrands and attach to beads viathe adaptors. Note: Only one DNAstrand is attached to a bead.
Fragment ofgenomic DNA
Adaptors
PPi + Adenosine 5′phosphosulfate
ATP + luciferin
ATP sulfurylase
Light
Light is detected by a camerain the sequencing machine.
Luciferase
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CA
TG
CA
T T
Primer