Gene isolation

Introduction
Molecular manipulation of specific genes is one of
the very popular areas of research in
biotechnology.
These genes, therefore, should be either isolated
or artificially synthesized before they are
manipulated and used for transformation leading
to the production of transgenic plants and
animals.

Isolation of gene
During 1970s and 1980s, significant progress
was made in the techniques for isolation of a
variety of genes, including those for
1. Ribosomal RNA
2. Specific protein products
3. Phenotypic traits with unknown gene product
Different techniques have been used for the
isolation of these different types of genes.
Isolation of ribosomal RNA genes
Ribosomes consists of rRNA and proteins.
This rRNA makes 80% of cellular RNA and is
synthesized on ribosomal genes, which can be
isolated.
Isolation of ribosomal genes was considered
convenient due to their following characteristics
features:
1. Availability of homogenous rRNA
2. Differences between ribosomal genes and other
genes, due to relatively high G+C content (45-
60%) in rRNA.
3. Ribosomal genes are present in multiple copies.
Ribosomal RNA genes were isolated for the first
time by H. Wallace and M.L. Birnstiel in Xenopus.
Following steps were involved in this isolation:
i. Isolate rRNA from ribosomes of Xenopus
ii. make it radioactively labelled, due to replication
in a medium containing tritiated uridine.
iii. Isolate ribosomal DNA by density gradient
centrifugation followed by its denaturation (G+C
content of rDNA differs from that of bulk DNA
and helps in its separation by centrifugation.)
iv) Fix single stranded DNA on a filter paper
v) Add labelled rRNA on filter paper carrying single
stranded DNA
vi) Allow DNA-RNA hybridization to take place
vii) Wash excess labelled RNA.
viii) Measure radioactivity and isolate duplex
hybrids which on denaturation will give single
stranded DNA which can then be made double
stranded.
Isolation of genes coding for the
known specific proteins
Possible after the discovery of reverse
transcriptase enzyme in 1970.
RTase enzyme is used for the synthesis of cDNA.
This cDNA can then be used for the isolation of
corresponding gene from genomic DNA.
For the isolation of a specific gene, techniques
are available for the isolation of specific mRNA.
For this purpose, antibodies are produced against
a specific protein for which the gene is to be
isolated.
Steps involved in the isolation of a gene coding for specific
protein
Isolation of genes which are tissue
specific in expression
It is much easier to isolate genes which are
expressed in specific tissues because mRNA
extracted from specific tissues will belong to the
gene of interest.
Other mRNA molecules in minor quantities can be
eliminated, since these can be identified through
their isolation from tissues where this gene is
silent.
Genes for storage proteins are expressed only in
developing seeds and globin genes is expressed
in erythrocytes.


Steps involved in the isolation of a gene with tissue
specific expression using its mRNA.
Use of DNA or RNA probes in
isolation of genes
Specific molecular probes (DNA or RNA), can be
used for isolation of specific genes.
These probes may be available either from
another plant species for the same gene or may
be artificially using a part of the amino acid
sequence of their protein product of the gene in
question.
Use of heterologous probes
Probes obtained from one plant species and used
for another plant species are called heterologous
probes.
Heterologous probes should ordinarily be used
with cDNA library and not with the genomic library
because unrelated or pseudogenes may be
isolated and cloned.
Effective in identifying gene clones during colony
hybridization or Southern blots.
Gene for chalcone synthase (CSH) has been
isolated from Antirrhinum majus and Petunia
hybrida using heterologous cDNA probe from
parsley.
Similarly, heterologous Antirrhinum cDNA
probe was used for isolation of CSH gene from
barley, and heterologous probes from maize
were used for isolation of barley genes Wx
(waxy gene) and A1 (aleurone gene).
Use of cDNA or synthetic probes
If protein purified using the technique of two
dimensional gel electrophoresis, is used for
microsequencing of 5-15 consecutive amino acids, this
information can be used for the synthesis of
oligonucleotides (using automated DNA synthesizers).
These oligonucleotides may then be directly utilized for
screening of cDNA or genomic libraries for isolation of
specific genes.
These can also be used as primers in PCR for the
synthesis of cDNA using mRNA isolated from a tissue.
Steps involved in the isolation of a gene using the probe,
artificially synthesized on the basis of amino acid
sequence of a part of the protein.
Isolation of genes coding for an
unknown product
Isolation of a gene whose phenotypic effect is
known but the gene product has not been
identified.
Such genes include those for some
morphological traits, dormancy, photoperiodicity,
disease resistance, etc.
This area of research, in which genetics is
studied by isolating the gene first without knowing
the gene product is described as reverse
genetics.
Use of transposable elements
Transposable elements are effectively used for
the isolation of genes, when gene product is
unknown.
Transposon works as a gene tag.
Procedure
A gene with a scorable phenotypic effect is
cloned.
TE is transposed to this gene to get an unstable
allele.
Unstable allele is cloned and TE is isolated from
this unstable allele.
TE is transposed to a gene of interest with known
phenotypic effect, to produce unstable allele.
The DNA is extracted from this mutant.
TE sequence is used as a probe to isolate and
clone the mutant gene (carrying inserted TE), so
that we can isolate the gene of interest.
Steps involved in the use of transposon
mutagenesis (or tagging) for identification
and isolation of a gene.
In maize, TE like Ac/Ds, En/Spm and Mu1 have
been isolated using the genes Wx, C2 and Adh1.
These TEs can be used for gene tagging
experiments leading to isolation of genes.
In maize, several genes like Bz1, P, A1,
C1 and C2 have been isolated successfully using
gene tagging method.
For gene tagging, often transposable elements
endogenous to species like maize and
snapdragon have been used.
However, rarely transposable available from one
plant can be moved into the genome of another
plant, whose gene is to be isolated.
Ac element of maize has been transferred to
tobacco, where it can integrate into any locus
permitting gene tagging and gene isolation.
Mutant complementation
Clones from the wild strain are selected which should
be able to complement the mutants transforming them
into wild types.
Using these clones, the protoplasts derived from the
mutant plant may be transformed and the transgenic
plants are produced.
After this the gene of interest can be isolated from the
DNA extracted from the transformed plants using wild
type complementary clone as a probe.
Use of RFLP maps or chromosome
walking for gene isolation
RFLP maps are prepared for different crops.
If RFLPs are known which are very close to the
target gene, then by locating the RFLPs on either
side of the target gene, a long intervening DNA
segment may be isolated.
This fragment may be used for subcloning
leading to the isolation of the desired gene.

If RFLPs are examined in two isogenic lines
differing for a single specific gene, the difference
in RFLP maps of these two lines will help in
locating the position of this gene on molecular
map which can then be utilized for isolation of the
gene.
Use of chromosome jumping for
gene isolation
Chromosome jumping will help narrowing the gap
between the gene and available molecular
markers.
After several cycles of chromosome jumping
followed by cloning the regions that are closer to
the gene, it will be possible to approach very
close to the desired gene and clone it.
The gene for Cystic fibrosis was isolated using
this technique.