Recombinant DNA Technology: Top 8 Techniques

The following points highlight the top eight techniques in recombinant DNA technology. Some of the techniques are: 1. Construction of Gene Library 2. Specific Gene Screening from Libraries 3. Chromosome Walking and Gene Cloning 4. Investigation of DNA Polymorphism by Random Amplified Polymorphic DNA (RAPD) Technique 5. Southern Blotting Procedure and Others.  

Recombinant DNA Technology: Technique # 1.

Construction of Gene Library:

It is very difficult to isolate a particular gene from the enormous number of genes present inside the plant genome. It is very advantageous to make these genes available in the form of a gene library which is of two different kinds – the genomic DNA library and cDNA library.

A genomic DNA library represents the entire donor genome. A genomic library can be prepared by the cleavage of the total genome of the organism with the help of restriction endonucleases. The resultant fragments may be of about 15 to several hundred kbp, which frequently contain only parts of genes.

Each fragment is then inserted into a suitable plasmid or bacteriophage vectors and then each fragment is amplified by cloning, usually in bacteria, virus or yeast. A target DNA sequence is identified, sub-cultured and maintained as cell lines, known as gene bank or clone bank.

A cDNA library represents only transcribed genes, which is well suited for eukaryotic structural genes. It is prepared by the following way.

From a specific tissue, the mRNA molecules are isolated and then transcribed into corresponding cDNAs by reverse transcriptase. The cDNAs are then inserted into a suitable vector and amplified by cloning. The cDNA contains no introns. After transformation the cDNAs are expressed in prokaryotes.

A prokaryotic promoter must be added to the cDNA for its expression, as it does not contain any promoter. Eukaryotic mRNA contains a poly (A) tail at the 3′ end that allows mRNA to be separated from the other RNAs by affinity chromatography. The column material consists of polydeoxythymidine oligonucleotide (poly dT)-linked solid particles of cellulose or other materials.

When the total RNA fraction extracted from a tissue is charged to the column, the mRNA molecules bind to the column by hybridization of their poly (A) tail to the poly (dT) of the column material, and the other RNAs are washed out. With a suitable buffer, the bound mRNA is then eluted from the column.

On the isolated mRNA template, cDNA strands are produced by reverse transcriptase. The poly (dT) fragment attached to poly (A) tail is used as primer for the enzyme. The mRNA is then partially degraded by RNase H to form mRNA fragments that can serve as primers for the synthesis of second cDNA strand by DNA polymerase.

Using DNA polymerase I these mRNA fragments are successively replaced by DNA fragments and these are then linked to each other by DNA ligase.

A short RNA fragment remains at its 5′ end, which is of minor importance as the most mRNA at its 5′ end contains non-coding region. The double-stranded cDNA molecules thus formed from mRNA molecules are then amplified by cloning using plasmids or bacteriophages as cloning vectors.

A gene library can be preserved in phages. The phage DNA possesses a cleavage site for the restriction endonuclease EcoRI. The cDNA double helix is first methylated by an EcoRI methylase at EcoRI restriction sites to protect the restriction site within the cDNA. Chemically synthesized double- stranded oligonucleotides (called linkers) are then joined to both ends of cDNA double strand by T₄ DNA ligase.

The restriction endonuclease EcoRI causes staggered cuts of the two DNA strands leaving four nucleotides of one strand unpaired at each resulting end. These unpaired ends are called sticky ends as they can form a base pair with each other, or with complementary sticky ends of other DNA fragments.

So, EcoRI cleaves the linker as well as k phage DNA to form sticky ends resulting in the pairing of the complementary bases of the cDNA and phage DNA ends. Finally, the ligation of the DNA strands is done by T₄ DNA ligase. Thus, cDNA is inserted into the vector.

A gene library can also be preserved in plasmids. For this purpose a cDNA is inserted into plasmids in more or less the same way as in the insertion into phage DNA. The recombinant plasmids are then transferred into E. coli cells by treating cells with CaCl₂ to make their membrane more permeable to the plasmid and with a short heat shock.

The plasmid vectors contain an antibiotic resistance gene, which makes the bacteria resistant to ampicillin or tetracycline. When such an antibiotic is added to the culture medium only the transformed cells survive, after plating on agar culture medium bacterial colony spots develop.

Plasmid vectors are constructed in which restriction cleavage site remains inside the β-gaIactosidase gene. The galactosidase enzyme hydrolyses the colourless X-Gal (5-bromo-4-chloro- 3-indolyl -β-D-galactopyranoside) into an insoluble blue product.

The recombinant plasmids with DNA inserts do not possess intact β-galactosidasegene and cannot produce the functional enzyme. So, on the agar plate culture medium containing X-Gal, blue and white colonies are produced. The colourless-colonies are selected as the cells containing plasmids with DNA inserts.

Recombinant DNA Technology: Technique # 2.

Specific Gene Screening from Libraries:

For screening of bacterial colonies of phage plaques containing desired gene, specific probes are used. A nylon or nitrocellulose membrane is placed onto the bacterial colonies or phage plaques growing on the agar surface. Some of the bacteria or the phages bind to the blotting membrane.

To screen the bacterial or phage clones bound to the blotting membrane two kinds of probes are used, like:

(1) Specific antibodies to identify the protein produced as gene product of the desired clone 

(2) Specific radioactive DNA probes to label the cDNA of the desired clone by hybridization.

(a) Antibody Screening Technique:

In this technique a sufficient amount of protein encoded by the gene of interest is purified beforehand, so that polyclonal antibodies against the protein can be made by immunization in a suitable animal.

For antibody screening:

1. The cDNA library must be inserted next to a promoter that controls the initiation of transcription of the inserted gene. This is called expression vector. A colony of host cells are plated onto solid medium containing antibiotics.

2. The resultant mRNA is then translated into the corresponding protein, which is recognized by the antibody. When a discrete colony is formed on the plate, it is then transferred onto nitrocellulose membrane. The exact position of cell colony on the plate is maintained on the matrix.

The orientation of the blotting membrane is marked so that the visible spots on the membrane will correspond to the locations of specific bacterial colonies of phage plaque containing DNA insert that can later be picked off the plate.

3. The bacteria bound to the filter are ruptured and the released bacterial proteins are fixed to the membrane, whereas the phages themselves lyse and thus liberate the cell proteins, which are then fixed to the filter. The protein is then visualized on the membrane using Western blotting procedure.

4. In this procedure antibodies are added for specific binding with the corresponding protein but are washed off from all other parts of the membrane.

5. A second antibody either radio-labelled or fluorescent-labelled or enzyme-conjugated, is used to detect the first bound antibody. The blotting membrane is then washed, dried and exposed to X-ray film.

6. A positive colony can be recognized as a dark spot on the autoradiograph (Fig. 17.14). The positive colony is picked up diluted and plated on to another agar plate and the process is repeated through screening to obtain a single pure clone. Positive phage plaques are also re-grown in bacteria and plated again to obtain pure clones.

(b) Screening through Specific DNA Probe:

Principally this screening technique involves hybridization between labeled DNA probe and a target DNA sequence. In the method of bacteria or phages present on the membrane are lysed and the proteins are removed.

The tightly bound DNA is then dissolved into single strands. 32P-labelled deoxynucleotide sequence complementary to the membrane bound DNA strand are then used as probes. These bind by hybridization to the desired cDNA present on the blotting membrane. Identification of the positive clones is done by autoradiography.

Depending on the techniques screening through DNA probes is of the following types:

i. Oligonucleotide Screening:

In this screening technique chemically synthesized oligonucleotide probe is used to isolate cDNA clones. An oligonucleotide probe is a short segment of DNA usually 10- 50 nucleotides long and radioactively labeled at 5′ end by 32P-phosphate. The technique is employed only when the gene product is available in very low quantity, which is not sufficient to raise antibody.

By micro-sequencing, N-terminal amino acid sequence of this protein is determined. From such information the corresponding DNA sequence is deduced to produce oligonucleotide probes by chemical synthesis using automatic synthesizer.

Degenerate oligonucleotides containing all the possible sequences for encoding the given amino acid sequence is produced. These radiolabeled oligonucleotide probes hybridize with the desired cDNA present on the blotting membrane. Positive clones are identified by autoradiography.

ii. Heterologous Probe Screening:

Heterologous probes are cDNA clones previously obtained from another species. These are used to screen specific cDNA library made from the mRNA of another species. It becomes successful if there is sufficient sequence homology of the genes between the two species.

iii. Transposon Tagging:

Transposons are discrete DNA sequences that can jump within the genome of a plant, which sometimes results in the dis-functioning of a functional gene due to disruption of its correct sequence. The transposons, with the help of an enzyme transposes, are cut out of one position in the genome and re-joined at another position in the genome.

Labeling a gene with an inserted transposon is called transposon gene tagging. A radiolabeled DNA probe based on the known transposon sequence is used to identify by DNA hybridization the region of the genome in which the transposon has been tagged. Such an identified bacterial clone will contain the gene of interest.

iv. Differential Screening:

Differential or (±) screening procedure is used to trace a physiological process of interest. Differential expression of genes, before and after induction, is utilized in this procedure. For example, many auxin-induced genes are de-repressed to produce mRNA after auxin treatment. A cDNA library can be prepared from the mRNA produced after auxin treatment.

Radiolabeled cDNA probes are prepared from the mRNA from both induced and non-induced tissues. Screening procedure is the same as described for the use of oligonucleotides. Any bacterial colony having the insert representing an induced gene will hybridize only with the cDNA probes made from induced mRNA. It is confirmed in Northern blots.

v. Isolation of Genes by Complementation:

Genes encoding unknown proteins can be isolated by complementation of deficiency mutants of bacteria or yeast after transformation with plasmids from a cDNA library. The protein is known only by its function. The genes for sucrose trans-locator proteins involved in phloem loading have been identified by complementation. A yeast deficiency mutant was used for the purpose.

The mutant had lost the ability to take up sucrose due to defective sucrose trans-locator gene and, therefore, could no longer use sucrose as a carbon source. This mutant was transformed with plasmids from a plant cDNA library, which were expressed under the control of yeast promoter.

After plating, a yeast clone was found to grow on the sucrose-containing medium indicating the generation of plant sucrose trans-locator protein inside the transformed yeast cells. This positive yeast clones had been amplified, the plasmid cDNA was isolated and sequenced. From the cDNA sequencing amino acid sequence of the sucrose trans-locator protein was determined.

Recombinant DNA Technology: Technique # 3.

Chromosome Walking and Gene Cloning:

The characteristics of an organism depend on the DNA base sequences of its genome. This base sequence differs not only between different species but also between individuals of the same species. Genetic maps are prepared on the basis of the recombination frequencies of the phenotypic characters of an organism.

The map shows the relative distance between two genes on a chromosome. Restriction fragment length polymorphism (RFLP) of DNA can be used to detect differences in the genes within a species without a detailed sequence comparison. RFLP of DNA can be used as genetic markers.

This is based on the use of bacterial restriction endonucleases that cleave a DNA at a specific palindromic recognition sequence known as restriction site resulting in a polymorphism of the restriction fragment length.

For the chromosome walking purpose, a genomic library of the plant of interest is made first. The DNA is cut into fragments of a manageable size with the help of appropriate restriction enzyme. The fragments are introduced into bacteria using suitable vectors for multiplication to generate a population of chimeric vectors.

To select fragments of certain sections of the genome, labeled DNA probes are prepared. For this purpose a certain DNA section of the chromosome located near the gene of interest is found out.

This DNA section is introduced into E. coli through plasmid vector and then propagated. The plasmids are then isolated and the multiplied DNA sections are cut out again, isolated and radioactively labelled. These probes are called RFLP markers.

DNA restriction fragments are analysed with the labelled probes through the Southern blot method. As it is possible to clone and identify thousands of restriction fragments from a genome of a particular organism, genetic markers covering an entire genome can be obtained using RFLPs. Thus, RFLP marker based genetic maps can be used to clone genes that can be linked to a particular RFLP.

This is done by walking from the site of known RFLP towards the exact locus of the gene of interest. The genomic library is probed with a cloned restriction fragment recombined with the gene of interest. DNA inserts from these clones are digested with restriction enzymes, and a map of the order of the DNA segments is constructed.

DNA fragments representing the ends of the inserts are isolated and used as hybridizing probes to screen additional clones from the library.

Each new clone will contain DNA inserts that are sequences continued from the previous insert. This walking cycle is repeated several times until the desired position is reached. The chromosome walking is tedious and time-consuming process.

RFLP markers are inherited according to Mendelian principles. These are used to characterize a certain variety. Several probes are used in parallel measurements and in plant systematics to establish phylogenetic trees. Defined restriction fragments can be used as labelled probes in order to localize certain genes on the chromosome.

With this technique chromosome maps have been established for Arabidopsis, potato, tomato and maize.

Recombinant DNA Technology: Technique # 4.

Investigation of DNA Polymorphism by Random Amplified Polymorphic DNA (RAPD) Technique:

Through the amplification of randomly obtained DNA fragments it is possible to analyse the differences between DNA sequences of individual or varieties of a species. This random amplified polymorphic DNA (RAPD) technique is much easier to work and is applied widely in a very short time.

The principle of the technique is the polymerase chain reaction (PCR), invented by Kary Mullis in 1984. Selected DNA fragments of a length up to 2-3kb can be amplified by DNA polymerase in this method. Oligonucleotide primers are necessary for this amplification. Two oligonucleotides each complementary to a short sequence in one strand of the desired DNA segment are synthesized.

The primers are positioned just beyond the end of the sequence to be amplified. In the first step, the isolated DNA containing the segment to be cloned is heated briefly to about 95°C to denature it, in the presence of excess of the cooled synthetic oligonucleotide primers.

In the next step, a heat-stable DNA polymerase called Taq I isolated from Thermus aquaticus living in hot wells, and the precursor deoxynucleoside triphosphates are then added.

The primed DNA segment is then selectively replicated. The steps are then repeated through 25 or 30 such cycles within a few hours. The amplified DNA segments can be readily isolated and cloned. Since in the polymerase chain reaction the number of DNA molecules formed is multiplied exponentially by the number of cycles, very small DNA sample can be multiplied.

In the first step the newly formed DNA length is not restricted at one end. Whenever the primer binds to the complementary base sequence of the newly formed DNA strand, a product is formed in the next cycle, whose length is restricted by both primers.

The large amounts of amplified products of single DNA fragment can be separated by gel electrophoresis and stained with ethidium bromide. The fragments can be detected as fluorescent bands under UV chromatolight. Changing the primer sequence can produce different DNA fragments. A variety of synthetic oligonucleotide primers are commercially available.

The RAPD technique is sensitive enough to detect as little as one DNA molecule in almost any type of sample. The technique is very rapid and requires neither the preparation of probes nor the time consuming procedure of a Southern blot.

It has been used to clone DNA fragments from mummies and the remains of extinct animals. It is also a potent new tool in forensic medicine. It is also being used in prenatal diagnosis of genetic diseases. Since the RAPD technique allows differentiation between varieties of a species, it has become an important tool in breeding.

Recombinant DNA Technology: Technique # 5.

Southern Blotting Procedure:

Southern blotting is a technique of DNA hybridization in which one or more specific DNA fragments are detected in a large population by means of hybridization to a labelled complementary nucleic acid probe. The method was developed by Edward Southern in 1975. The restriction fragments are first fractionated lengthwise by electrophoresis in an agarose gel. The mobility of the smallest fragment is greatest.

After denaturing the segregated fragments in the gel are transferred to a nitrocellulose or nylon membrane by placing the membrane on the gel. A flow of an appropriate buffer through gel elutes DNA fragments and blots them onto the membrane. The membrane is covered with a stack of tissue paper.

The bound DNA fragments are dissociated by the buffer into single strands. After blotting, the membrane is heated for 1-2 hours at 80CC or is exposed to UV light to fix DNA molecules to the membrane. A labeled DNA probe representing specific sequence of a gene of interest when added, is hybridized to DNA strand that is bound to the membrane.

After removal of the non-bound probes by washing, the hybridized DNAs are identified by autoradiography. The position of the band on the blot is then related to its migration and hence its size (Fig. 17.19).

Recombinant DNA Technology: Technique # 6.

Western Blotting Procedure:

Western blotting is an immunological method used to quantify expression of a gene at the protein level. Using the nucleotide sequence information a peptide can be synthesized by chemically connecting the amino acids in a specific sequence. Such synthetic peptide may be used to raise antibodies.

The antibody can be used to detect the proteins encoded by the gene. Sensitive quantification of a specific protein is done by Western blotting. From the tissues total proteins are extracted in a suitable buffer and are separated through electrophoresis on polyacrylamide gels.

The separated proteins are then electrophoretically transferred to a nitrocellulose membrane, which are then probed with either radiolabeled or enzyme linked antibodies. The radioactivity is detected on the photographic film and the enzyme linked antibodies produce colour on the membrane, which can be quantified by densitometry scanning. The method was developed by Burnette in 1981.

Recombinant DNA Technology: Technique # 7.

Northern Blotting:

By Northern blotting procedures quantification of expression of a gene at the RNA level is done through RNA-DNA hybridization. The technique was developed by Thomas in 1980. In this method either total RNAs or poly (A) mRNAs are isolated from the tissues. Size-based separation of the RNAs is done through agarose gel electrophoresis in the presence of formaldehyde or methyl mercury as denaturating agent.

Like the Southern blotting a nitrocellulose membrane is laid on the gel and the RNA molecules are eluted from the gel and blotted onto the membrane as an appropriate buffer is allowed to flow through the gel. The separated RNAs may also be transferred to the membrane by electrophoresis.

The transferred RNAs are fixed on the membrane either by baking at 60°C for 1 -2 hours or by exposure to UV light. A radiolabeled DNA probe complementary to the gene of interest is then allowed to hybridize with the RNA on the filter to screen the complementary mRNA species, which are then quantified by any appropriate standard method.

Recombinant DNA Technology: Technique # 8.

Slot-Blot Analysis:

Slot-blot analysis is a modification of Northern blotting procedure. This is used for rapid determination of the levels of selected mRNA species. In this method total RNAs or poly (A) RNAs, instead of separating on agarose gel are directly blotted onto a nitrocellulose filter by a slot-blot device. The subsequent processing is same as described above.