From Wikipedia,
the free encyclopedia.
A genetic screen (or
simply screen) is a
procedure or test to identify and
select individuals which possess a
phenotype of interest. A
genetic screen for new genes is
often referred to as forward
genetics as opposed to
reverse genetics that is
the term used for identifying
mutant alleles in genes that are
already known. Mutant alleles that
are not tagged for rapid
cloning are
mapped and cloned by
positional cloning.
Creating a mutant population
Since unusual
alleles and phenotypes are
rare, geneticists expose the
individuals that are to be
screened to a
mutagen, such as a chemical or
radiation, which generates
mutations in their
chromosomes. The use of mutagens
enables "saturation screens," one
of the first of which was
performed by
Christiane Nüsslein-Volhard
and
Eric Wieschaus. A saturation
screen is performed to uncover
every
genes that is involved in a
particular phenotype in a given
species. This is done by
screening and
mapping genes until no new
genes are found. Mutagens such as
random DNA insertions by
transformation or active
transposons can also be used
to generate new mutants. These
techniques have an advantage since
a new allele is tagged with a
known molecular (DNA) marker that
can facilitate the rapid cloning
of the gene without mapping.
Types of screen
A basic screen involves
looking for a phenotype of
interest in the mutated
population. One might screen for
obvious phenotypes such a
fruit flies with no wings or
an
Arabidopsis flower with no
petals.
More subtle is a temperature
sensitive screen that involves
temperature shifts to enhance the
mutant phenotype. A population
grown at low temperature would
have a normal phenotype, however,
the mutation in the particular
gene would make it unstable at a
higher temperature. A screen for
temperature sensitivity in
fruit flies, for example, might
involve raising the
temperature in the cage until
some flies faint, then opening a
portal to let the others escape.
Individuals selected in a screen
are liable to carry an unusual
version of a
gene involved in the phenotype
of interest. An advantage of
alleles found in this type of
screen is that the mutant
phenotype is
conditional and can be
activated by simply raising the
temperature. A
null mutation in such a gene
may be
embryo lethal and such mutants
would be missed in a basic screen.
An enhancer/supressor screen
is the most sophisticated type of
genetic screen. In this case a
mutagenised population has an
allele of a gene that leads to a
weak mutant phenotype in the
biological process of interest.
For example, with regard to fruit
fly wing development, a weak
allele may have small abnormal
wings whereas a strong/null allele
would have no wings. In this
sensitised background it is
possible to discover new mutants
that either enhance the phenotype
(small wings to no wings) or
suppress the phenotype (small
wings to normal wings). Such a
screen has two advantages. First,
new genes identified in the screen
are often involved in the same
biological process as the weak
allele in the
genetic background, in this
case wing formation. Second, due
to
genetic redundancy, the mutant
genes discovered may not have a
visible phenotype of their own. In
a more basic screen these would
not be discovered, however, in the
sensitised genetic background a
visible phenotype is clear.
Mapping mutants
By the
classical genetics approach, a
researcher would then locate (map)
the gene on its
chromosome by
crossbreeding with individuals
that carry other unusual
traits and collecting
statistics on how frequently the
two traits are inherited together.
Classical geneticists would have
used phenotypic traits to map the
new mutant
alleles. With the advent of
genomic sequences for model
systems such as Dropsophila,
Arabidopsis and
C. elegans many
SNPs have now been identified
that can be used as traits for
mapping. SNPs are the prefered
traits for mapping since they are
very frequent, on the order of one
difference per 1000 base pairs,
between different varieties of
organism.
Positional cloning
For many genomes SNP's are not
known and therefore positional
cloning is required to identify
polymorphisms that flank the
mapped allele. This process
requires that DNA fragments from
the closest known
genetic marker are
progressively cloned getting
closer to the mutant allele with
each new clone. This process
produces a
contig map of the
locus and is known as
chromosome walking.
For each new
DNA clone a polymorphism is
identified and tested in the
mapping population for it's
recombination frequency
compared to the mutant phenotype.
When the DNA clone is at or close
to the mutant allele the
recombination frequency should be
equal to zero. If the chromosome
walk proceeds through the mutant
allele the new polymorphisms will
start to show increase in
recombination frequency compared
to the mutant phenotype. Depending
on the size of the mapping
population, and luck, the mutant
allele can be narrowed down to a
small region (<30 Kb). Sequence
comparison between
wild type and
mutant DNA in that region is
then required to locate the DNA
mutation that causes the
phenotypic difference.
