Download How to obtain a clone of a specific gene

How to obtain a clone of a specific gene
There are two basic strategies:
-Direct selection for the desired
clone
- Identification of the clone from
a gene library
(several techniques can be
employed)
Direct selection for the
kanamycin resistance
gene
Mutant strains of
E. coli can be
used as hosts for
transformation
and cloning of the
missing gene
ex: cloning of tprA
genes from
different bacteria
using an E. coli
mutant for the
same gene
The scope and limitations of marker rescue
Limitations
-a mutant strain must be available for the gene in question
-a medium on which only the wild-type can survive is
needed
Applications
-most genes that code for biosynthetic enzymes
-is not restricted to E. coli since many auxotrophs from
other bacteria and yeast are available
Identification of a clone from a gene library:
preparation of a bacterial gene library
In eukaryotes different genes are expressed in
different types of cell
Preparation of a cDNA gene library from eukaryotes
(cont.)
mRNA can be
cloned as
complementary
DNA
Identification of a clone from a gene library:
Method: nucleic acid hybridization
Any two singlestranded nucleic acid
molecules have the
potential to form base
pairs with one another
Hybridization mechanism
(cont.)
Southern hybridization (DNA-DNA hybridization)
E.M.Southern, 1970
Transfer of the DNA to a membrane
(cont.)
After hybridization
Gel separation
A
probe
B
Southern hybridization
Colony hybridization (developed in the late 1970)
(cont.)
Colony hybridization
-The colonies are transferred to a nitrocellulose or nylon
membrane
-The membrane/immobilized cells is treated to remove all
contaminating material, leaving just DNA denatured
-The DNA is bound to the membrane by heat or UV light
-The labelled probe is denatured and hybridization taskes
place
-Washing steps to remove unbound probe
-Probe detection by autoradiography, colorimetry or
chemoluminescence
Probes
-Can be labelled with radioactive nucleotides or with
non-radioactive (biotin, digoxigenin, fluorescein, etc)
-The probes have to share at least a part of the
sequence of the molecule of interest to detect
-The size of the probes is usually between 100-1000
bp
Labelling DNA by random
priming
The mixture of hexameric
oligonucleotides of random
sequence is sufficiently
complex to include at least
a few molecules that can
base pair with the target
sequence so that the
polymerases synthesize
the complementary strand
with at least one labelled
dNTP
Non-radioactive labelling of DNA probes
Examples of the practical use of a hybridization probe
Heterologous probing makes use of hybridization between
related sequences for clone identification
Identification of pgmG from S. elodea
Box I
Active site
PgmG S. paucimobilis (AF167367)
ExoC Azospirillum brasilense (P45632)
PmmA Prochlorothrix hollandica (AAC44309)
ExoC Rickettsia prowazekii (CAA14961)
AlgC Pseudomonas aeruginosa (P26276)
Pgm Neisseria gonorrhoeae (P40390)
XanA Xanthomonas campestris (AAA27610)
ManB Escherichia coli (P24175)
RFK7 E. coli (P37742)
CelB A.xylinum (P38569)
Pgm E. coli (P36938)
Pgm1 Homo sapiens (P36871)
Pgm1 Saccharomyces cerevisiae (S41199)
PgmU Oryctolagus cuniculus (P00949)
Box II
Box III
Metal-ion-binding
Sugar binding
QITGSHNPGN-109
MITGSHNPPD-111
QITGSHNPPE-110
MVTGSHNPRD-116
MLTGSHNPPD-113
MITGSHNPPD-108
MVTASHNPMD-102
EVTASHNPMD-103
EVTASHNPMD-101
GADGDGDRID-249
GFDGDGDRIG-247
AFDGDGDRIG-251
AFDGDGDRIG-296
AFDGDGDRVG-249
AFDGDADRLG-246
AWDGDFDRCF-244
AFDGDFDRCF-252
AFDGDFDRCF-250
LAGEMSGH-326
LAGEMSGH-327
LAGEMSGH-331
LAGEMSGH-376
LAGEMSGH-329
VAGEMSGH-326
YGGEMSAH-324
YGGEMSAH-332
YGGEMSAH-329
VITPSHNPPE-153
VITPSHNPPE-151
ILTASHNPGG-122
ILTASHNPGG-125
ILTASHNPGG-122
ANDTDADRHG-313
ANDPDYDRHG-311
AFDGDGDRNM-295
ASDGDGDRNM-298
AFDGDGDRNM-295
FGGEESAG-397
FGGEESAG-394
LCGEESFG-380
ICGEESFG-383
LCGEESFG-380
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Degenerated primers based on known proteins with
phosphoglucomutase activity to amplify an internal fragment of
pgmG followed by screening of the Sphingomonas elodea
genomic library