A Review on Gene Cloning

Suppose you have generated important data and you do not want to lose it. What would you do to protect it? You may either file it or store it in a folder. Or you would generate multiple copies of the information using a Xerox. The Xerox machine reads the information in your paper and gives you a printout in the form of a photocopy. Thus, Xerox creates exact replicas of the document. Similarly, our body protects the genetic material by generating multiple copies. An example involves the replication process.
Scientists felt the need to generate multiple DNA copies. The process of cloning involves obtaining multiple copies of a gene or a DNA segment using technology. Each identical copy of a DNA segment or a gene segment is known as a clone. Human genome project utilizes maximum amounts of clones in various experiments. Many such cloned DNA collections make up a clone library. Cloning is not only applicable to DNA or a gene segment but also possible with the entire cell. This type of cloning exploits the natural process of cell division to make many copies of an entire cell. The genetic makeup of the cloned cells is known as cell lines. They are identical to the original cell. Gene cloning involves recombining a population of DNA molecules into the vector. The recombined DNA molecules contain the gene of interest. The DNA is inserted into a vector so that each vector consists of single DNA from the original population. The screening of the colonies revealed the presence or absence of the DNA of interest. Thus, understanding cloning requires knowledge of the clones and their production.

Image 1: Gene Cloning

The review article discusses various aspects of cloning:

1.     Restriction enzymes and cloning vectors

2.     Collection of clones in the form of a library

3.     Screening of specific clones

4.     Analyzing cloned DNA through a molecular approach

5.     Identifying adjacent clones

6.     Applications

Cloning involves producing multiple copies of the genes or DNA segment of interest. In short, the procedure involves digesting a DNA segment with restriction enzymes, introducing it in a vector, transforming the recombinant vector into the host and observing the expression of the gene of interest. The screened clones are later on analyzed using molecular techniques.

Restriction digestion:

The restriction enzymes are used to digest the DNA. These enzymes are endonucleases capable of cleaving specific sites in the DNA known as the restriction sites. The restriction sites have specific nucleotide base pairs. A restriction enzyme cleaves a specific base by breaking the phosphodiester bond. Cleaving of the DNA can be achieved using one or a combination of restriction enzymes. The process of cleaving the DNA to obtain fragments of different sizes is known as restriction digestion. Separation of the DNA fragments is possible with the electrophoretic technique. Smaller fragments travel faster than the larger fragments, thereby forming bands. After visualizing bands, the portion of the gel consisting of a DNA fragment is cut and used for cloning experiments. Here are the two options. One of the options is to make multiple copies of the gene using amplification techniques such as PCR. The second option is to incorporate the DNA into the cloning vector. PCR or polymerase chain reaction is a process of amplifying the gene or a DNA using a sophisticated thermal cycler. Cloning DNA using a vector involves inserting the desired DNA into a plasmid or another vector.

Cloning vectors:

A cloning vector replicates within the host organism. It consists of a restriction site which can be cleaved for inserting the desired DNA fragment. Various cloning vectors are available. A plasmid vector is a plasmid or an extrachromosomal material of the bacteria. It is isolated and used as a vector. It consists of three sites such as the origin of replication, a selectable marker gene, and one or more restriction sites. For example, pUC 19 vector has a polylinker site or multiple cloning sites. On treating the restriction site with a restriction enzyme, that particular site gets cleaved and the desired fragment of DNA gets inserted in that place. Other examples of vectors include shuttle vectors, expression vectors, cosmids, phasmids, and artificial chromosomes. Shuttle vectors are bifunctional. They are capable of insertion into two or more hosts. Expression vectors express the desired gene to get the protein product.

Clone Library:

It can be anything from a collection of clones including cDNAs, chromosomes, and genomes. A cDNA library is nothing but a collection of cloned cDNA sequences synthesized from mRNA. A collection of cloned DNA in which the sequence appears at least once is known as a genomic library. The clone libraries appear analogous to book library. Just as the books provide plenty of information, the clone libraries provide a lot of information about the genome. Genome libraries production involves inserting the required DNA digest into a vector and storing the product. However, there are other methods too. Gene splitting with a restriction enzyme sometimes gives the undesired product. Such cases involve other techniques like mechanical shearing or partial digestion. Large DNA insertion involves mechanical shearing. The process of screening is made easy using chromosome libraries. 24 different human chromosomes libraries are available. The preparation of the cDNA libraries involves mRNA molecules as raw materials. After making clone libraries, a specific gene is found using screening techniques.

Image 2: Transformation of the recombinant plasmid

Screening procedures:

The screening of cDNA libraries involves an antibody probe. The first step is to transform the recombinant plasmid consisting of the required cloned cDNA into a host such as E. coli bacteria. Next step involves plating the bacteria on a selective medium to observe the growth of the colonies. Transferring the colonies into a microtiter well enables the bacteria to grow. The transfer of the colonies to the membrane filter also enables them to grow to get the expression of the gene. Removal of the filter and cell lysis enables the protein product exposure. Radioactively labeled antibody treatment with the protein product is the final step. Autoradiography helps the observation of clones in the form of dark spots. Screening a genomic library involves a similar system. It involves plating the transformed bacteria on a medium and processing them through replica plating. Here, the probed DNA is used instead of a radioactively labeled antibody. Complementation test helps to identify the specific genes.

Analyzing cloned DNA:

Analysis of clones involves molecular biology techniques such as restriction mapping, southern and northern blotting. Restriction mapping includes the physical mapping of the genes. Restriction digestion gives rise to DNA fragments of different sizes. Electrophoresis separates the fragments in the form of bands. Southern blotting uses a nitrocellulose membrane filter placed on the gel immersed in the buffer solution. Capillary action of the blotting paper enables the DNA to come in contact with the buffer and transfer to the membrane filter. The filter gets exposed to probes which hybridize with the DNA. Autoradiography helps to detect the DNA. Southern blotting mainly involves blotting DNA. Northern blotting involves blotting RNA instead of DNA.

Adjacent clone identification:

Genes between the flanking markers are found through chromosome walking. Hence, adjacent clone identification requires chromosome walking. The overlapping clones help to study larger DNA segments.

Applications of cloning:

1.     Recombinant DNA technology or genetic engineering: Cloning techniques involve obtaining large amounts of pure DNA. Expression of the desired products including proteins, vitamins, enzymes, growth factors, and other biomolecules is possible with recombinant DNA technology. Cloned genes help to detect mutations. Plant vectors consisting of the desired gene improve the crop quality. Cloning enables development of transgenic plant varieties with stress tolerance, drought tolerance, and herbicide or pesticide resistance. Particular protein synthesis is achieved using expression vectors.

2.     Transgenics: These organisms, plants, animals or microbes, involve manipulated genes cloned into several copies. The genes of interest express phenotypically. Examples include knockout animals.

3.     Gene therapy: It helps in replacing the gene that has lost its function. The defective gene identification accompanies cloning the normal gene inserted into the affected individual through a vector. The first gene therapy was successful in children suffering from ADA deficiency. The gene therapy is a new way to treat genetic disease because the abnormal gene is corrected or replaced.

4.     In vitro fertilization: Microsurgery involves removal of the nucleus from the donor’s oocyte. The microinjection of the surrogate mother’s somatic cell nucleus into the donor’s oocyte makes the cell chimeric. The chimeric cell cultured in the laboratory develops into an embryo. The embryo gets implanted into the surrogate mother’s uterus.

     References:
[1] Gene cloning and DNA analysis, T.A. Brown
[2] Recombinant DNA Technology, Sardul Singh Sandhu
[3]Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh
[4] Gene Cloning and Manipulation, Christopher Howe

   

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What is a gene?

Introduction to genes:

The basic unit of the hereditary material is known as a gene or a cistron. It is an ordered sequence of nucleotides. These sequences encode polypeptide chain via mRNA molecule. Both DNA and RNA are known as nucleic acids. Genetics is the study of structure, function, and the regulation of genes. Studying genes helps us to know more about proteins, cellular functions, and disorders associated with them. In humans, gene targets help to study mutations and target them through advanced drug discovery and therapeutics. In bacteria, genes are manipulated to obtain the desired product. The genetic engineering or recombinant DNA technology helps to manipulate the genes. The desired gene can be integrated into a vector to obtain the desired product.

Modification of plant genes improves the quality of the crop and improves the yield. The genes play an important role in the growth and reproduction of an organism. Slight gene mutations, if harmful, lead to drastic changes in the cell and cause genetic disorders. Genetics plays an important role in preventing genetic disorders through prenatal testing, cytogenetic and molecular genetic techniques. Genes form the main basis of inheritance. We all have some traits obtained from our ancestors. The genes get passed on from generation to generation. We look similar to our parents, yet appear different. Helpful mutations in the genes lead to genetic diversity and variation. That is why we all look different from each other. The complete set of chromosomal and extrachromosomal genes of an organism is known as the genome. It consists of the complete genetic composition of an organism. 

The gene is a heritable determinant of a trait showing the property of segregation. With this reference, the way genes pass within the family are studied. The genes in a family may or may not have diverged from each other. Gene family involves a set of genes that descend from a common ancestor. Genes express themselves in several generations. They also interact with each other. Several genes can collaborate with each other and give rise to one phenotypic trait. The genes may be present in the cell in a particular dosage. It is important to know the frequency of a gene. The genes are present in a specific place on a chromosome, described as gene loci. Gene mapping through annotation of DNA sequences is possible based on gene loci information. DNA sequence annotation with regulatory element sites, coding regions, non-coding regions, and mutations accelerates the mapping. Sometimes many copies of a gene may be present in a chromosome, known as gene redundancy. Thus genes are vast and diversified.

Image 1: Basic genetic structure

HUGO gene nomenclature

It is a standard for gene nomenclature decided by the Human Genome Organisation (HUGO) committee. It is a meaningful naming of a gene. Names accompany useful symbols. A gene symbol is a unique abbreviation of the name of a gene. It consists of uppercase letters in italics, letters in Latin and numbers in Arabic. A putative gene name is locus based.

Here are the naming guidelines:

1.     The symbols must be unique and prohibited to use elsewhere.

2.     The gene symbols must have Latin letters and Arabic numbers

3.     Punctuation marks are not allowed in gene symbols.

4.     The gene symbols should not contain any references of species.

5.     The nomenclature of genes must evolve with the latest technology rather than follow age-old rules.

Structure of a gene:

A gene includes regions preceding and following the coding and non-coding regions. The preceding region is known as the leader sequence which is at 5’ position. It is the untranslated region. There is a coding region known as Exon. An exonic region specifies for an amino acid sequence. These exons are interrupted by non-coding regions known as introns. The untranslated trailer sequences follow them. The untranslated trailer sequences are at 3’ end. The spliced RNA molecule consists of only exons as the non-coding regions or introns for splicing out. Although DNA is a double-stranded molecule, only one strand encodes for RNA synthesis. The sense strand runs from 5’ to 3’ direction and encodes specific molecules. The gene has an open reading frame which is an indication of sense strand direction. The extremities of the gene consist of regulatory sequences. Plus the gene also consists of promoters, enhancers, silencers and other regions.

Image 2: Gene structure

Gene Expression- The Central Dogma

Expression of the genes involves the conversion of the genes coded information into the structures present and operating the cells. The genes are expressed to initiate the synthesis of the mRNA molecule and translated into a protein. Other examples of RNA include rRNA and tRNA. The tRNA and rRNA genes remain untranslated. The gene expression also involves a phenotypic manifestation by a process known as gene action. Differential gene expression studies involve gene expression at different levels. They express differently under different experimental conditions. The central dogma is a two-step process describing the gene expression. Francis Crick proposed the central dogma after the discovery of nucleic acids. DNA undergoes a process of transcription to synthesize RNA which further undergoes translation to form proteins. Not all genes express proteins. Thus, not all genes are transcribed to get RNA. Originally through central dogma, it was postulated that the genetic information is transferred only from nucleic acid to nucleic acid and from nucleic acid to protein. Thus, the genetic information gets transferred from DNA to DNA, DNA to RNA and from RNA to protein. The genetic information never transfers from protein to nucleic acid. The cis-trans test determines the functionality of genes. It determines whether the independent mutations occur in a single gene or several genes.

Cis-trans Complementation test

It helps to determine whether the two mutant sites are in the same functional unit or a gene. It is an allelism test and determines whether two different recessive mutations on the opposite chromosome of a diploid complement each other. The same two mutations in a diploid or a partial diploid show a wild-type phenotype. Cis mutations exhibit a wild-type phenotype. There is no genetic complementation when the mutations are in trans. The term cistron indicates gene.

The gene characterization is possible with sequence, transcription, and homology if it does not contribute to a phenotype. A gene is a functional and physical unit of heredity. The gene code is said to be a triplet. There are total 64 codons that code for 20 different amino acids. 

References:

[1] What is a gene?- Genetics Home Reference - NIH

[2] Gene – Wikipedia
[3] HUGO Gene Nomenclature Committee

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