Mapping the Human Genome

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Mapping the Human Genome
Genetic Mapping
Physical Mapping
DNA Sequencing
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Physical Mapping Systems
Yeast Artificial Chromosomes (YACs) 200-2000 kb
Bacteriophage P1 90 kb
Cosmids 40 kb
Bacteriophage l 9-23 kb
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Large Fragment Cloning
--- Hybrid cells multiple chromosomes 100
-3000 Mbp
--- Mono-chromosomal hybrids 50-
300Mbp
--- Sub-chromosomal hybrids 1-50
Mbp
--- Double-minutes 0.3-2 Mbp
--- Yeast Artificial Chromosomes YACs 0.05-2
Mbp
--- BACs (F-plasmid derived) - 300 kb
--- PACs (large P1 clones) - 300 kb
Insert Size
--- T4-packaging system - 120 kb ( -
300 kb)
--- EBV derived vectors - 180 kb
--- P1 P1 packaging - 80- 90 kb
--- Cosmid vectors 35 - 45 kb
--- Lambda replacement vectors 8 -
22 kb
--- Micro-dissection clones - 1.5 kb
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Bacteriophage Lambda

• Insertion Vectors
• cDNA cloning and expression GT10, GT11, Zap
• Replacement Vectors
• Genomic cloning EMBL3, EMBL 4

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Lambda gt11

• Insertion vector
• Small inserts (cDNAs)
• Expression cloning

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Lambda Zap

• Insertion vector
• Small inserts (cDNAs)
• Expression cloning
• In vivo excision of inserts

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Lambda Fix

• Replacement vector
• Larger inserts
• Genomic DNA
• Spi selection

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Lambda EMBL3

• Replacement vector
• Larger inserts
• Genomic DNA
• Spi selection

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SORTED CHROMOSOME ISOLATION PROCEDURE
Use for purity 1 analysis
of DNA and quantitation
Proteinase K/SDS SUPERNATANT
Phenol / CHCl3 Extract
Clone into gt 75-85 Lawrist of
DNA Vector
Proteinase K/SDS PELLET
Phenol / CHCl3 Extract Dialyze
SORT TUBE spun _at_ 3K rpm
Clone into gt 14-24 Charon of
DNA Vector
Proteinase K/SDS SORT TUBE
Phenol / CHCl3 Extract Dialyze
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CONSTRUCTION OF LARGE INSERT LAMBDA LIBRARIES
Charon 40 Vector (9 - 23 kb capacity)
Polystuffer
R arm
L arm
cos
cos
BamHI
BamHI
(1) Nae 1 (2) PEG precipitation (3) Phenol,
chloroform extraction (4) Ethanol
precipitation (5) BamHI
BamHI
Polystuffer
R arm
L arm
cos
cos
Plate the Lambda library and screen with probes
to identify phage which contain sequences of
interest
BamHI
BamHI
Ligate
Ba
Ba
Package Amplify on E. Coli host strain
K802 (rec A-)
Characterize Before Distribution to (ATCC)
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Cosmids and Fosmids

• Cosmids
• High-copy number replicon
• Limited size based on packaging reactions
• Chromosome-specific libraries
• Fosmids
• Low-copy replicon (F factor)
• Limited size based on packaging reactions
• Chromosome-specific libraries

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pFOS1 F-replicon based cosmid vector
AatII
cos
HindIII
cos
cos
cos
BamHI
cos
HindIII
AatII
CM
1
pFOS1 9.5 kb
pUCcos1
BamHI
pBAC
CM
parB
pUCcos1
oriS
parB
parA
repE
oriS
2
parA
repE
AatII
BamHI
BamHI
AatII
Kim, U-J., Shizuya,H, de Jong,P.J., Birren,B.,
Simon,M., Nucleic Acids Research 1992, 20,
1083-1085
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LAMBDA ORIGIN COSMID VECTORS (DE JONG ET AL) WITH
TWO COS SITES DERIVED FROM LORISTX (P.F.R. LITTLE)
cos
amp
neo
ScaI
AatII
LAWRIST n 8.2 kb
cos
1 SfiI
2 SfiI
l ori
USE FOR CLONING OF
CLONING SITES
VECTOR
T7
SP6
H
LAWRIST5 LAWRIST6 LAWRIST7 LAWRIST8 LAWRIST16
H HindIII B BamHI Partial
HindIII or partial MboI digests
S
S SalI B BamHI Partial
MboI digest, using partial fill-in of
MboI and SalI
(vector) sites
Ss
N
N NotI Ss SstI
(Partial MboI complete NotI or SstII) digests
B
M
M Mlul B BamHI (Partial
MboI complete Mlul) digets
T7
T3
B
H
H HindIII B BamHI Partial
HindIII or partial MboI digests
For cosmid protocols Choice and use of cosmid
vectors, by Peter F.R. Little, in DNA cloning,
Volume 3 (Ed. D.M.Glover, IRL Press, 1987),
19-42 Please note that the neo marker only
contains a prokaryotic promoter. Hence, the
cosmids can not be selected with G418 upon
transfection into mammalian cells.
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SORTED CHROMOSOME ISOLATION PROCEDURE
Use for purity 1 analysis
of DNA and quantitation
Proteinase K/SDS SUPERNATANT
Phenol / CHCl3 Extract
Clone into gt 75-85 Lawrist of
DNA Vector
Proteinase K/SDS PELLET
Phenol / CHCl3 Extract Dialyze
SORT TUBE spun _at_ 3K rpm
Clone into gt 14-24 Charon of
DNA Vector
Proteinase K/SDS SORT TUBE
Phenol / CHCl3 Extract Dialyze
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CONSTRUCTION OF CHROMOSOME-ENRICHED COSMID LIBRARY
cos
amp
cos
neo
ScaI
LAWRIST 16 Vector
cos
l ori
BamHI
Prepare (1) Digest vector with excess ScaI
Vector (2) Dephosphorylate the ScaI ends
with excess CIAP Arms (3) Digest the
cloning site with BamHI
Pick clones into microtiter plates, grow,
and determine percentage of human clones
(hybridize to total human and total hamster DNA
probes)
ScaI
Infect host DH5aMCR
Kanamycin plate
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Bacteriophage P1

• Insertion Vectors
• Large Inserts
• Limited to HEADFULL
• Packaging reaction followed by plasmid
  propagation

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P1 vector for the construction of recombinants by
P1 packaging
loxP
P1 plasmid replicon
Ad2
Sca 1
pAD10SacBII (30kb)
kan
pBR322 ori
pac
sacB
loxP
P1 lytic replicon
Sp6
T7
E.coli promoter
sacB
c 1 repressor binding site
BamH 1
Sfi 1
Not 1
Pierce et al, Proc. Natl. Acad. Sci.U.S.A. (1992)
89, 2056-2060
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Constructing P1 recombinants using packaging
extracts
insert of 80-90 kb
pac
S
packaging
loxP
B
B
loxP
kan
site-specific recombination
loxP
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Implications of particle size for P1 recombinant
viability
HEADFULL YARD STICK (110-115 kb)
insert too large
maximal insert
min. insert
insert too small
first cut during packaging reaction
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P1 Derived Artificial Chromosomes (PACs)

• Electroporation Based System
• Large insert size
• Low copy number origin for propagation
• High copy origin for DNA production
• Negative selection against non-recombinants
• Very stable inserts

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Preparation of pUC19-link
XbaI
PmeI
BamHI
ScaI
GATCTAGAGTACTGGGTTTAAACCCGGATCCGGGTTTAAACCCAGTACTC
TA ATCTCATGACCCAAATTTGGGCCTAGGCCCAAATTTGGGTCAT
GAGATCTAG
ScaI
XbaI
PmeI
Oligonucleotide adapter LL171
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P1 vector for the construction of recombinants by
electroporation
loxP
loxP
plasmid replicon
pCYPAC1 (19.3 kb)
plasmid replicon
kan
1
2
P1 lytic replicon
pAD10SacBII (30kb)
S
S
kan
pac
sacB
pac
P1 lytic replicon
loxP
sacB
loxP
sacB
loxP
B
pUC19-link
P
B
B
S
B
B
S
pUC19-link
B
S
B
S
S
1. Insert BamHI-digested pUC19-link into BamHI
site of P1 vector
2. Deletion of Ad2-fragment by in vivo
site-specific recombination
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Preparation of vector for cloning
B
B
kan
1
S
S
S
S
B
S
B
S
2
B
B
kan
S
S
S
S
1
Digest with BamHI and ScaI
2
Dephosphorylate and remove linkers (Centricon)
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Sizing of MboI partial-digest fragments
CHEF gel electrophoresis
1
2
NOT stained
stained
stained
2
1
3
4
5
1
correct size fraction identified (150-250kb)
4
3
2
3
4
5
for analysis
for ligation
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Bacterial Artificial Chromosomes (BACs)

• Electroporation Based System
• Large insert size
• Low copy number origin for propagation
• Very stable inserts

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Yeast Artificial Chromosomes

• Large Inserts
• Not Stable
• Chimeric inserts

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Problems with YACs!
1) Approximately 40-60 of the YACs from most
libraries are chimeric.
2) Approximately 40 of the YACs from most
libraries are deleted.
3) Low transformation efficiencies.
4) YACs are very difficult to manipulate.
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Physical Mapping Systems
Yeast Artificial Chromosomes (YACs) 200-2000 kb
Bacteriophage P1 90 kb
Cosmids 40 kb
Bacteriophage l 9-23 kb
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Question of Size?
Vector
Human Genome
Chromosome 19
1X
5X
1X
5X
13,335
44,445
2,780
222,225
556
P1
Cosmid
1,251
6,255
100,002
500,010
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Storage Space?
Vector
Human Genome
Chromosome 19
1X
5X
1X
5X
YAC
1
2
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139
463
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2,315
6
P1
Cosmid
14
66
1,042
5,209
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Advantages of BACs PACs
1) Bacterial based systems that are easy to
manipulate.
2) Libraries are generated using bacterial hosts
with well defined properties.
3) Transformation efficiency is higher than that
obtained for YACs.
4) BACs and PACs are non-chimeric, very stable
and do not delete sequences.
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Comparison of BACs PACs
1) Both allow replication of clones at
one copy/cell.
2) Both systems replicate clones faithfully
across 60-100 generations.
3) PACs also have a negative selection against
non-recombinants.
4) PACs have an IPTG inducible high copy number
origin of replication.
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Closure of Chromosome 19
Type of Approach A) Bacterial Artificial
Chromosomes (BACs). B) P1 Artificial Chromosomes
(PACs). C) Yeast Artificial Chromosomes
(YACs). D) Cosmids. E) All of the above.
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Closure of Chromosome 19
Yeast Artificial Chromosomes
Bacterial Artificial Chromosomes
P1 Artificial Chromosomes
Cosmids
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Large Fragment Cloning Vectors
PROPERTY P1 pBAC pucBAC pCYPAC YAC Vector Size
(kb) 31 6.5 7.2 19.3 11.5 Vector Copy
single single multiple multiple multiple Inser
t Size (kb) 75-95 0-300 0-300 0-300 0-2000 Cloni
ng Strategy 2 arms single digest single
digest single digest double digest BamHI/ScaI
Bam or Hind Bam or Hind Bam/Sca
link Bam/EcoRI Cloning Method Packaging Electrop
orate Electroporate Electroporate Spheroplast Mai
ntenance (copy ) single single single single sin
gle Chimeric Clones () 0 2 2 0
(24/24) 20-60 Positive Selection yes no no yes y
es Copy Induction yes no no yes no