This article throws light upon the top four tools of genetic engineering.
The top four tools of genetic engineering are: (1) Genomic Libraries (2) cDNA and cDNA Library (3) Analysis of Genes and Gene Transcripts and (4) Colony Hybridization.
Contents
Tool # 1. Genomic Libraries:
Creation of genomic library involves cloning of whole genomic DNA. Therefore, a genomic library is a collection of recombinant DNA molecule (plasmids, phages) so that the sum total of DNA inserts in this collection represents the entire genome of the organism. Depending upon the size of genome, prokaryotic or eukaryotic, vector can be selected. This way whole genome can be cut into pieces and cloned in vectors or by PCR technique. This can be referred as genomic cloning.
Making of genomic library includes the following steps:
1. Isolation of chromosomal DNA;
2. Fragmentation of DNA is carried out by mechanical shearing or sonication or by using a suitable endonuclease for partial digestion of DNA (Fig. 15.1). Mechanical shearing generates blunt ends while endonuclease produces cohesive ends. Complete digestion is avoided as it generates fragments too heterogenous in size to be used.
3. Ligation of DNA fragments- The partial digest of genomic DNA are subjected to agarose gel electrophoresis for separation of fragments of required size. The fragments of appropriate size are eluted from gel. These fragments are then inserted into a suitable vector. These vectors are cut with the same restriction enzymes. After that the DNA fragments are ligated into vector using DNA ligase (Fig. 15.2). By this technique about 25kb DNA fragments can be inserted into vectors.
4. Identification of the desired clone- Identification of the bacterial colony containing the desired DNA fragment by employing a suitable hybridization probe in colony hybridization. The probe may be mRNA of the gene, cDNA of its mRNA, homologous gene from another organism, or a synthetic oligonucleotide representing the sequence of a part of the desired gene/DNA fragment. In order to detect hybridization the probe must be labelled usually with a radiolabeled isotope.
Alternative Vector Systems for making Genomic Library:
For prokaryotes (smaller genomes) viable to make gene libraries in plasmids. Plasmid inserts normally -5-10 kb, so only need a few thousand recombinants for a representative library. Eukaryotes (larger genomes), really need vectors that can hold much larger insert DNA fragments (Table 15.1).
Bacteriophage lambda shows much more efficient infection of E. coli than that can be achieved by plasmid transformation. Phage lambda binds to receptors on the surface of E. coli and injects DNA. Infection by lambda is much more efficient than plasmid transformation – can get ~ 109 plaques per microgram of DNA, vs ~ 106 colonies per microgram of plasmid DNA.
As the bacterial cells lyse, a plaque forms, which spreads as more cells become infected and lyse. The plaque contains the infectious viral particles or virions and each one represents an individual lambda clone (similar to a bacterial colony representing an individual plasmid clone).
The appearance is of a clear circle in a ‘cloud’ of bacterial cells (the bacterial ‘lawn’) (Fig. 15.3). If incubated for long periods, these circles will continue to grow (containing more and more viral particles) and spread until all bacterial cells are wiped out. To pick a single lambda clone, the plaque is ‘punched’ out from the agar plate and stored in a holding solution. This can then be used to infect more bacterial cells and to replicate more of the recombinant lambda DNA.
Vectors for Making Libraries with Larger Inserts:
Cosmid libraries are used for cloning genes with large introns and for sequencing larger chunks of the genome. Cosmid vectors are hybrids of plasmid and bacteriophage lambda λ DNA (a small ~5 kb plasmid containing the plasmid origin of replication (ori), an antibiotic resistance gene such as amp and a suitable restriction site for cloning along with the COS sequence from phage λ DNA). Because of the COS sequence, cosmid recombinants can be packaged into viral particles (allowing high efficiency transformation).
Since most of Genomic libraries in cosmid vector, the λ DNA has been discarded; it can be replaced by the DNA of interest, so long as it doesn’t exceed the 50 kb limit for packaging into the viral head. The insert sizes are of the order of 35-45 kb. Since the recombinant DNA does not encode any lambda proteins, cosmids do not form viral particles (or plaques) but rather forms large circular plasmids and the colonies that arise can be selected on antibiotic plates, like other plasmid DNA trans-formants.
Cosmid clones can be manipulated similarly, allowing ease of plasmid isolation. Since many eukaryotic genes are on the order of 30 – 40 kb, the likelihood of obtaining a DNA clone containing the entire gene sequence is increased significantly when using a cosmid library.
YAC libraries are used for cloning very large DNA fragments (of more than 1 Mb), and are useful for cloning large genes (such as the 250 kb cystic fibrosis gene) and for creating libraries of large overlapping clones, such as for individual chromosomes isolated from organisms (chromosomal libraries). These have been used extensively for mapping genomes of complex organisms (e.g. Homo sapiens).
YACs are yeast artificial chromosomes and are hybrids of bacterial plasmid DNA and yeast DNA. The components required for replication/segregation of natural yeast chromosomes have been combined with E. coli plasmid DNA. YACs are grown in the yeast Saccharomomyces cerevesiae and so contain selectable markers which are suitable for the host system. Rather than antibiotic selection, yeast selectable markers enable growth of the transformant on selective media lacking specific nutrients. (Non-transformants are unable to grow). The yeast strains that are used are auxotrophic – that is, they are unable to make a specific compound.
For example, Trpl mutants can’t make tryptophan so can only grow on media supplemented with tryptophan. If the mutant strain is transformed with a YAC containing an intact Trpl gene, then this will compensate for the inactive gene (complement) and the transfected cell is able to grow on media lacking tryptophan.
YACs are not used as extensively anymore because of inherent problems. For instance, YAC clones can contain non-contiguous segments of the genome. This means that 2 or more DNA fragments from separate parts of the genome can be integrated into an individual YAC (because they are able to support rather large inserts). A second problem is that YACs are unstable and frequently lose parts of the DNA during propagation.
BAC libraries are also used for cloning very large DNA fragments and have been particularly useful for sequencing large genomes. BACs are bacterial artificial chromosomes, and are based on a naturally occurring large bacterial plasmid, the F factor. BAC vectors can accommodate DNA inserts up to 300 kb, still fairly respectable when needing to clone large genes or map and sequence complex genomes. BACs have several advantages over YACs, which means they are used more extensively now. Most of the human genome has been sequenced using BAC rather than YAC clones.
Tool # 2. cDNA and cDNA Library:
Complementary DNA (cDNA) is synthetic DNA made from mRNA with the use of a special enzyme called reverse transcriptase (RNA dependent DNA polymerase discovered by Temin and Baltimore in 1970). Originally this enzyme was isolated from reteroviruses. This enzyme performs similar reactions as DNA polymerase and requires a primer with a free 3′ OH terminal. With the use of a mRNA as a template, reverse transcriptase synthesizes a single stranded DNA molecule that can then be used as template for double-stranded DNA (Fig. 15.4). Because it is made from mRNA, cDNA is devoid of both upstream and downstream regulatory sequences and of introns.
This means that cDNA from eukaryotes can be translated into functional protein in bacteria, an important feature when expressing eukaryotic genes in bacterial host. A short oligo (dT) chain is hybridized to the poly (A) tail of mRNA strand. The oligo (dT) segment serves as a primer for the action of reverse transcriptase, which uses the mRNA as template for the synthesis of cDNA strand. The resulting cDNA ends in a hairpin loop. When the mRNA, from RNA-DNA hybrid, has been degraded by treatment with NaOH or RNase H, a short piece of RNA is left behind. This hair pin loop becomes a primer for DNA polymerase I, which completes the paired DNA strand.
The loop is then cleaved by SI nuclease (which acts only on the single stranded loop) to produce a double stranded cDNA molecule. This double stranded DNA can be inserted into plasmid, cosmid, phage lambda, cloning vectors by blunt-ended ligation.
A cDNA library is a population of bacterial transforraants or phage lysates in which each mRNA isolated from an organism or tissue is represented as its cDNA insertion in a plasmid or a phage vector. The frequency of specific cDNA in such a library would depend on the frequency of the concerned mRNA in that tissue.
Tool # 3. Analysis of Genes and Gene Transcripts:
Recombinant DNA technology involves localization of gene of interest. This gene can be either isolated or synthesized before they are manipulated and used for transformation leading to the production of transgenic plants and animals. Different techniques have been used for isolation of different varieties of genes like ribosomal RNA gene, gene for phenotypic traits with unknown gene product, for specific protein products, and genes involve in regulatory functions, e.g. promoter genes etc.
Isolation of Ribosomal RNA Genes:
The ribosomal RNA makes the 80% of total RNA and is synthesized on ribosomal gene which could be isolated. Isolation of this gene was easy due to some characteristics features of rRNA (a) ribomsomal genes are present in multiple copies (b) difference between ribosomal genes and other genes, due to their relatively high G + C content in rRNA. The ribosomal gene was isolated for the first time in 1965 by H. Wallace and M.L. Birnstiel in an amphibian named Xenopus.
The different steps involved in isolation of rRNA genes are the following:
1. rRNA is isolated from the ribosomes and are made radiolabelled by allowing them to replicate in tritiated uridine containing medium.
2. Ribosomal DNA is isolated by density gradient centrifugation (high G+C content of rDNA make it easy to separate it by centrifugation from rest of DNA) followed by its denaturation.
3. Single stranded DNA is then fixed on filter paper and labelled RNA is added to the paper.
4. Now DNA and RNA hybridization occurs and excess labelled RNA is washed off.
5. Radioactivity is measured and duplex hybrid which on denaturation will give single stranded DNA which can be made double stranded. This is how rRNA gene can be isolated.
Isolation of Gene of Specific Proteins:
This is possible by the use of enzyme reverse transcriptase. This enzyme can make copy/ complementary DNA (cDNA) from RNA. So it is necessary that techniques for isolation of mRNA should be available.
The different steps involved are the following:
1. Protein product of the gene is purified.
2. Antibodies are produced against these protein products by immunizing animals like rabbit and mouse.
3. Precipitation of polysomes is done which are engaged in synthesizing specific proteins. This is done with the help of antibodies produced.
4. mRNA is isolated and purified from the polysome fractions. This mRNA is used for synthesizing cDNA.
5. These cDNA are then inserted into cloning vectors for preparation of cDNA library. After it, immunological and electrophoretic analysis of the translation products of cDNA clones is done, to identify the specific cDNA clone, having gene for specific protein of interest.
6. Specific cDNA probes are then selected for identification and isolation of the gene from genomic DNA through screening a complete or partial genomic library.
Isolation of Gene of unknown Products (with Tissue Specific Expression):
It is easy to isolate the genes products which are tissue specific. For example, genes for storage proteins are expressed only in developing seeds, globin gene is expressed in erythrocytes. Such genes can be easily isolated because mRNA extracted from such tissue will be largely of gene of interest. Other mRNA can be eliminated, which can be identified by isolation and comparison of mRNA from the tissue where this gene is not expressed. Then this isolated mRNA is used for synthesis of cDNA using enzyme reverse transcriptase. Then the process is same as described in isolation of genes coding for known specific proteins.
Isolation of Genes using DNA and RNA Probes:
If the specific molecular probes (DNA and RNA probes) are available then they can be used for isolation of specific genes. These probes can be obtained either from another plant species or may be artificially synthesized using a part of the amino acid sequence of the protein product of the gene of interest. The probes obtained from one plant species and used for another plant species are called heterologous probes.
These heterologous probes have been found to be effective in identifying gene clones during colony hybridization or plaque hybridization or on southern blot. For example, the gene for chalcone synthase has been isolated from Antirrhinum majus and Petunia hybrida using heterologous probe from parsely. Heterologous probes are generally used with the cDNA library.
Now with our knowledge of synthesizing cDNA probes, these probes can be helpful in synthesis of synthetic probes. In this procedure, the purified protein using the two dimensional gel electrophoresis is used for micro-sequencing of 5-15 consecutive amino acids, this information can be used for the synthesis of corresponding oligonucleotides using automated DNA synthesizers. These oligonucleotides may then be directly utilized for screening of cDNA or genomic library for isolation of gene of interest.
Tool # 4. Colony Hybridization:
Colony hybridization is the screening of a library with a labeled probe (radioactive, bioluminescent, etc.) to identify a specific sequence of DNA, RNA, enzyme, protein, or antibody (Fig. 15.5).
Hybridization has two important features:
1. Hybridization reactions are specific; probes will bind only to sites that have complimentary sequences.
2. Hybridization reactions occur in the presence of large quantities of molecules that are similar but not identical to the site. This means a probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.
This specificity allows one to find a specific sequence in a complex mixture full of similar sequences. A hybridization technique also allows scientists to pick out the molecule of interest from very complex mixtures and study the molecule on its own.
Method of Colony Hybridization:
The following steps are required:
1. Isolate and grow cultures in a suitable medium (agar).
2. Transfer a sample of the colonies onto a solid matrix such as a nitrocellulose or nylon membrane.
3. The cells on the membrane are lysed and the DNA is then denatured.
4. A labelled probe is added to the matrix and hybridization takes place.
5. The matrix is rinsed to remove the non-hybridized probe molecules.
6. For radioactive probes, one uses autoradiography and the matrix is placed on X-ray film.
7. The film is observed for black spots that correspond to colonies that hybridized with the probe.
8. Compare the X-ray film with the master plate to see which colonies had probe hybridization. These are the colonies that contained the specific sequence that actually hybridized with the probe.
9. Colonies on the master plate that have the desired sequence can then be sub-cultured if desired.
Advantages:
1. Allows you to pick one molecule of interest out of millions.
2. Does not require the isolation of nucleic acids.
3. It is possible to culture and identify microorganisms containing the hybridized sequence.
Disadvantages:
1. Time consuming, takes time for colonies to incubate and for the hybridization to show on the X-ray film.
2. When identifying microorganisms, the procedure will only work if the organisms will grow and form a detectable colony.