Molecular Genetics

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Map-based Cloning: Walking

The strategy of map-based cloning is to find molecular markers very closely linked
to the gene of interest. Those molecular markers can serve as the starting point for
chromosome walking or jumping to the gene.

In the diagram, the walk begins with a clone containing mkrB. The ends of the clone
(boxed) are used to probe a library. Clones from adjacent genome segments are
thus identified and isolated. The distal ends of those clones are used to reprobe the
library. These steps are continued until a clone contains either mkrA or mkrC
sequences.

Clones between mkr B and mkrC must then be evaluated for the presence of yfg
(see right).

Chromosome walking has been used in the isolation of centromere sequences,

among others.

If the target species is a species whose genome has been completely molecularly
mapped, an ordered set of YACs, PACs, BACs or cosmid clones will be available.
Knowing which molecular markers are adjacent to the target gene automatically
identifies the YACs and/or cosmids that need to be tested.

One difficulty of chromosome walking is recognizing where the gene is located

between the two markers. Zoo (or garden) blots where DNAs of a variety of species
have been restricted, electrophoresed and Southern blotted can be useful. Gene
sequences are more likely to be conserved during evolution than intergenic
sequences. The identification of GC islands or the use of exon trapping can also be
useful. There are other problems with walking.
The strategy of using a genetic map to hone in on the physical gene is a general
one for isolating disease-associated genes.
Map-based Cloning: Jumping-Linking
• Chromosome walks can be interrupted if a
segment of the region to be walked through is
non-clonable (for example if it is toxic to the
host cell).
• Chromosome walks can be detoured in many
directions if one of the clone end probes
(filled box) is a repeated sequence.
Phenotype-based Cloning
• The jumping-linking strategy was
invented to overcome the problems
of unclonable segments and
repeated sequences and to enable
larger regions of the genome to be
scanned.
• Use is made of a restriction enzyme
that recognizes few sites in the
DNA (large thick bars) and an
enzyme that cuts frequently (small
thin bars).
• The jumping library contains clones
in which the two double digestion
fragments at either end of a large
fragment are cloned in the same
plasmid in the library (segments
connected by dotted lines).
• The linking library consists of
plasmids containing the fragments
generated by the frequent cutter that
also contain a recognition site for
the rare cutter.
• The two libraries are used in
alternation to hop down the
molecular chromosome.
• Complementation of a mutant phenotype in an easily transformable organism
can be used to obtain, from a different organism, a gene equivalent to the
mutant gene. The mutant is transformed with a library of the target
organism. Transformants whose mutant phenotype is corrected contain the
target gene.

• When complementation is not feasible, tagging mutagenesis can be used.

• Seeds of Arabidopsis are transformed at high efficiency with T-DNA using

Agrobacteria. Resulting transformants are germinated and plantlets screened
for mutant phenotypes. Top

• DNAs such as transposable elements and T-DNA that integrate into the
chromosomal DNA can create mutations by interruption of the gene in which
they insert. Top

• Further genetic manipulation may be needed after the initial tagging event to
assure that the tag is indeed associated with the mutant phenotype. Top

• When a transposable element transposes, it often integrates itself in genes,

causing mutations that can be recognized by phenotype. Top

• A genomic library is made from DNA of the tagged mutant organism. Top

• The genomic library of the tagged mutant organism is screened by

hybridization with the tagging agent as probe. Top

• DNA of the tagged mutant organisms is digested with a restriction enzyme

that does not cleave within the tagging agent. The fragments are circularized
 by ligation at dilute concentrations and used to transform E. coli. Top

• Oligonucleotides complementary to the ends of the tagging

agent and with their 3'-ends pointing away from the agent are
used to amplify the sequences flanking the tagging agent.

• Positive clones obtained from a rescued plasmid, inverse PCR or

a tagged genomic library can be used as nucleic acid hybridization probes to
screen a library of genomic DNA to identify clones containing the wild type
gene. Top

• Positively reacting genomic library clones need to be further examined to :

o Identify where on the clone the gene is located.

o Prove that the clone actually contains the gene.

Further Information
• In a variant of tagging mutagenesis, a population of tags is used to generate
the mutants. Each tag differs from the others in a pair of 20mer
oligonucleotides that serve as bar codes. The population of mutants is
subjected to a selective pressure. The bar codes in the survivors are
compared with those in the initial population. Any missing bar codes must
derive from a mutation in a gene required to deal with the selective
pressure. The presence of the bar code allows its isolation from the initial
population (ref).
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