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 geneDuring 1970s and 1980s, significant

progress was made in the techniques for isolation of a variety of genes, including those for

1. Ribosomal RNA2. Specific protein products3. Phenotypic traits with unknown gene

productDifferent 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 rRNA2. 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 paperv) Add labelled rRNA on filter paper carrying

single stranded DNAvi) Allow DNA-RNA hybridization to take placevii) 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 probesProbes 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 productIsolation 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 complementationClones 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.