It's a universal and a scientifically proven fact that DNA carries the genetic material of an organism. DNA analysis is diagnostically proven as very useful and a sensitive tool. In this modern diagnostic DNA analyses can diagnose inherit genetic defect and also disease causing pathogen can be detected by identifying genes of that organism. Furthermore, each person’s genome is structurally unique which helps in the field of medical forensics. The DNA analysis in disease diagnosis and medical forensics involves procedures like- hybridization technique, DNA profiling, etc.

In the laboratory, to begin with, it is very essential to identify a specific DNA sequence. Nucleic acid hybridization is one such tool to be reliable on for DNA analysis. The DNA probes, are used in this technique are synthetic single-stranded DNA molecule which recognizes and binds to the specific target DNA to be analyzed. The technique basically involves the single stranded target DNA bound to membrane support and addition of DNA probes, which in presence of specific circumstances binds to the complementary target DNA. Now the sequence of the target DNA can be analyzed on the basis of the sequence of known DNA probes complementary to the target DNA. The hybridization technique involves two kinds: radioactive and non-radioactive i.e., DNA probe tagged with radioactive isotope and DNA probed untagged. Non-radioactive labeled system has an advantage over radioactive labeled system that they are quite stable (biotin labeled DNA) at room temperature even for an year. The use of probes for disease diagnosis has several advantages over conventional methods like, they are simple, rapid, highly specific, powerful when combined with PCR, and viral infections can also be detected.

Another technique is DNA chip-microarray which contains thousands of DNA probes on a glass slide on which thousands of target DNA molecules can be scanned at a time. In this method, by the use of restriction enzymes DNA molecules are cut into fragments and labeled by fluorescent markers. These reacts with probes on the DNA chip i.e. bind according to their complementary sequences. The target DNA is identified by fluorescence emission which is recorded by the computer and DNA is identified.

In the diagnosis of infectious diseases the DNA analysis is a novel approach to identify the very specific pathogen. This is incorporated by genetically engineered techniques or DNA probes or direct DNA analysis. A specific DNA diagnostic test for malaria for identification of P. falciparum is developed, where in even as little as 1ng of P. falciparum is detected; in this method the DNA probe is bound and hybridized with DNA of P. falciparum genome and not with any other species of Plasmodium. Now even radioisotope labeled DNA probes are available for diagnosis of HIV DNA. Diagnostic tool for tuberculosis is developed by genetic engineering in bacteriophage using luciferase enzyme, this involves flash of light which confirms tuberculosis. Also diseases like lyme, periodontals and chagas’ have been diagnosed using PCR amplification, DNA probes and genetically engineered DNA probes.

Genetic diseases have ailments that are manageable but there is no cure except for gene therapy. But if, the gene identification is done for that genetic disease there are chances of development of management therapy, development of precautions to reduce its risk and helpful in terminating the foetuses affected. Foetal cells are obtained by amniocentesis or biopsies of trophoblastic villi and are detected by methods like- karyotype analysis, enzyme assays, hybridization technique or RFLP analysis. Then comes identification of gene causing genetic diseases in which first step is to do pedigree analysis where in the inheritance of the disease showing high incidences in the family is examined, followed by analysis of the identified region using STRs (Short Tandem Repeats) which would result in genome mapping and identification of the most likely gene causing genetic disorder is confirmed using blotting techniques, RT-PCR or northern hybridization.

Few examples of DNA analysis in genetic disease diagnosis are: (i) Sickle cell anemia can be detected by digesting mutant and normal β-globin gene and performing hybridization with a cloned β-globin DNA probe. (ii) Huntington’s disease can be diagnosed by the analysis of RFLPs in blood related individuals. (iii) Alzheimer’s disease, researchers have found a gene on chromosome number 21 which is believed to be responsible for inheriting this disease and they developed a DNA probe that located the genetic marker for this disease. There are also diagnostic systems developed and are is underdevelopment to detect the disease by DNA analysis for diseases like- fragile X syndrome, Friedrich’s ataxia, cystic fibrosis, muscle dystrophy, diabetes, cancers, obesity, etc.

There are other human diseases which are detected by DNA analysis. These includes: deafness- where mutation of the gene on chromosome 5 causes defective protein synthesis and disassembly of actin molecules, which in turn results is deafness, baldness- alopecia universalis known as inherited form of baldness is associated with a gene located on chromosome 12. Other diseases include Glaucoma, Parkinson’s disease, etc.

In the medical forensics, DNA analysis has proven to be genetic detective in settling paternity disputes, identification of criminals, thieves, rapists, etc. This novel term and technique was developed by Alec Jaffrey’s in 1985 called as DNA Profiling also widely better known as DNA fingerprinting. This procedure does not require a large quantity of DNA but minute quantities of DNA from skin fragments, hair, semen, blood stains are enough which are amplified using PCR. DNA fingerprinting is analysis of the nitrogenous base sequence in DNA of an individual which is unique in each person. The markers used in DNA fingerprinting as well as in disease diagnosis are microsatellites or simple tandem repeats, minisatellites or variable number tandem repeats, single nucleotide polymorphisms and Restriction Fragment Length Polymorphism. Also, it is now possible to carry out DNA profiling by automated DNA detection system.

The Ribonucleic acid (RNA) exists as three forms in a cell. They are transfer RNA or tRNA, Ribosomal RNA or rRNA and Messenger RNA or mRNA. The messenger RNA as the name implies is carrier of information from DNA to the protein factory of the cell called as the ribosome. In ribosome, the information carried by the mRNA is read by rRNA and they participate in the conversion of the received information into proteins through a process called translation with the help of the tRNA.

Transfer RNA (tRNA): tRNAs are tiny in nature and acts as a tool in translation of mRNA into proteins by linking the base pairs of mRNA and amino acid sequence on a polypeptide. Transfer RNAs are amino acid specific and it scans and detects the parts of mRNA coding the type of aminoacid and enables the exact placement of the aminoacid in the polypeptide chain. The physique of the tRNA molecule resembles that of a clover leaf with several extended loops. They are acceptor arm, Dihydrouracil arm, anticodon arm and TⱷC arm each having a special function.

The acceptor arm as the name indicates acts as the site for aminoacid attachment and the anticodon arm detects the codons in mRNA and aids in their binding. RNA polymerase III is the active enzyme in the process of tRNA synthesis which involves transcription of genes corresponding to tRNA. The sequential array of the nucleotides in tRNA is susceptible to modification by chemical groups which contribute to methylation, saturation of double bond, deletion of amino group, replacement by sulfur group and so on.

Ribosomal RNA (rRNA): rRNAs are the native RNAs of the cell organelle Ribosome (protein factory) and hence the name Ribosomal RNA. They signify their presence by deriving the information from mRNA and participating in protein synthesis. The cell relies on ribosomes for all its protein requirement and the amount of protein synthesized in a cell is directly proportional to the number of ribosome molecules present in the cell. S value denotes the size of the ribosome and they exist as 70s in prokaryotes and 80s in eukaryotes. 70s ribosome is the combination of 50s subunit and 30s subunit. The 50s subunit of prokaryotes has 2 rRNAs and the 30s subunit has 1 rRNA. Whereas the 60s subunit in eukaryotes possess 3 rRNAs and 40s subunit has 1 rRNA.

The occurrence of inter RNA molecule base pairing and intra molecular base pairing stabilizes the structure of the rRNA molecule. The functional proteins are found attached to the rRNAs in ribosomes. Few RNAs possess the characteristics of an enzyme and are called as ribozymes.
The process of formation of rRNA is complex involving several steps before the final product of mature rRNA. In prokaryotes, the RNA polymerase mediated transcription of rRNA genes results in the formation of pre-rRNA. The pre-rRNA exists in folded form and base pairing occurs resulting in the formation of stem-loop structure. This is followed by binding of ribosomal proteins to the folded pre-rRNA and modification of bases by methylation and action of RNAse III on specific points on rRNA causing cleavage and finally trimming the 5′and 3′of the rRNA by the M5, M16 and M23 ribonucleases resulting in the formation of mature rRNA. In eukaryotes, the steps involved in the formation of mature rRNA are similar to prokaryotes except for the additional step of ribonuclease activated trimming in prokaryotes.

Messenger RNA (mRNA): The carriers of information from DNA to the ribosome and poses as the template for synthesis of proteins. RNA polymerase II activated transcription of genes addressing proteins in nucleus results in the formation of mRNA. The format of coding regions separated by the non coding region exists in eukaryotes. The coding regions are called as Exons and the non-coding regions are called as Introns. Like the other two RNAs, mRNA formation is also initiated by the formation of pre-mRNA by transcription of both the coding and non-coding regions present as such. This is followed by a process called as splicing which removes the introns allowing the continuity of the Exons, making it an exact template for protein synthesis. Capping and polyadenylation occurs post splicing. Capping process protects the 5′ end of mRNA from the action of exonucleases and polyadenylation protects the 3′ end of the mRNA. All this described processes are skipped by the prokaryotes as the information is translated much earlier even before the completion of the transcription itself.

The transfer RNA and ribosomal RNA are considered stable whereas the life span of the messenger RNA is short.

We propose a new mathematical model for cell cultivation in a chemostat. Our model is based on the structuring of the biomass into two main groups: dividing and nondividing cells. The model is applicable both to existing static characteristics such as the Monod model and to the deviations from it.

We determined the range of chemostat stability at the specified flow rate D and concentration of the input limiting substrate S0. We also proposed the methods for determining parameters of the chemostat structured model.

The value of the derivative of dividing cells to nondividing cells of zero age is constant for a given flow rate. That value is no less important than the equality of specific growth rate and nutrient flow rate, which determines the equilibrium of the chemostat.

We showed that the corresponding specific rate constants of limiting substrate use by dividing and nondividing cells determine the system equilibrium. We also demonstrated that the new proposed structured model of the chemostat is more general than any other existing model. In each specific case, the model provides comparable equations to the well-known models of Monod, Pirt, Moser, Andrews and Ierusalimsky.

http://www.bioprocessintl.com/journal/20...tat-335634

Bioplastics are a kind of plastic, produced from natural compounds, and can be degraded into their base compounds. It is also known as Biodegradable plastics.

Genetic variations in plants have been made possible through many conventional methods since many years. The biotechnological approach for the desired plant was achieved by genetic engineering. Transgenes refers to gene or genes which are not native of an organism and are introduced by many methods. Transgenic plants refer to plants having one or more transgenes. This technique of involving plants to produce various chemical commodities and pharmaceuticals is known as phytofermentation.

A variety of biochemicals such as proteins, lipids and carbohydrates are obtained from plants. Production of transgenic plants has resulted in generating modified biochemical production in plants.

Bioplastic
A reasonable amount of interest has been developed in the field of bioplastic as a result of increased concern about non degradability of plastics and environmental hazards. Bioplastic have been known to be produced by various means including vegetable fats, cornstarch etc

Biotechnological role:
The role of biotechnology in the field of bioplastic has been accounted mainly to the presence of the property of biodegradation by naturally occurring polyhydroxy- alkanoates(PHAs). These are naturally occurring polyesters produced by bacterial fermentation. Polyhydroxybutyrate(PHB) is a form of PHA. It is produced in the bacterial cell as a result of physiological stress in the form of energy storing molecules. These PHAs are metabolized and used up by such bacteria when none of the other forms of energy source is available under conditions of stress. The properties of PHB include non toxic, water insoluble, high oxygen permeability and ultra violet resistance, thermostable and biocompatible.

Bacterial fermentation of Alcaligenes eutrophus produces polyhydroxy alkanoates.

The organism is cultured under normal conditions to attain stable growth. The conditions of the medium are then altered so that the bacterium induces production of PHA. This is usually induced in deficiency conditions of macro elements, oxygen etc or due to the excess presence of carbon sources in the medium. Acetyl-coA serves as a precursor in the production of polyhydroxy alkanotaes. The microbial fermentation of Alcaligenes for the production of PHAs is not favourable as the yield is very low. The cost involved in such mechanisms is substantially higher.
Though plants do not produce PHB naturally, they can be programmed to do so. For the production of PHBs by genetic engineering, the two genes involved in the production of enzymes namely, aceto-acetyl-CoA reductase (phbB) and PHB synthase (phbC) are isolated. These two enzymes are pivotal in the PHB synthesis from its precursor. The genes responsible are transferred and expressed in Arabidopsis thaliana plant. These two genes are so targeted so that they are expressed in plastids of plant. The PHB gets accumulated in leaves and can be extracted easily.
Thus synthesis of polyhydroxyl butyrate is a classic example of application of biotechnology in the production of goods of commercial value. Transgenic trees producing phbB and phbC are also produced where the PHB are synthesised in the leaves which can be extracted. Studies shows that plant produce PHB 20 -40% of their dry mass.
This method of production involves many advantages like;
(i) Upstream production (the production of biomass from which the biochemical has to be isolated) costs are much lower than microbial fermentation.
(ii) The post translation modification system of proteins in a plant cell is as advanced as in the animal cell. As a result the PHB which is formed undergo appropriate modification so that when extracted they do not require any further modification.
(iii) The plant seeds of transgenic plants can be stored and transported easily. These seeds could be used to produce transgenic plants to synthesize bioplastic when required.
(iv) Since animal system is not used, it does not face any concerns regarding to ethical issues.

Disadvantages:
(i) The production levels do not rise to the expected levels.
(ii) The increased level of trangenes in a plant genome results in accumulation of transgenic product. This can effect the plant growth and stability.

Recent Developments:
Several studies involving production of PHB in corn, potato etc has been initiated. Transgenic tobacco plants have also been extensively studied for PHB production. It is also estimated that potatoes can be a good source of PHB production from which PHB can be easily obtained and the resulting yield is also proposed to be higher is such ‘plastic potatoes’. If successful, it will be he most cost effective method involved.

I am a biotech postgraduate working in a microbiology lab.I have tried a lot of techniques to grow the cancer cells.but was not sucessful.i have read many articles relating to this from my searches in google.
My query is that can i grow cancer cells on soft agar from the blood or wat other medium should i use to let them grow.
secondly wat conditions should i maintain for them to grow like temperature,etc.
lastly what is the incubation time.

Drug designing is a process of constructing ailments specific drugs adopting the known properties of diseases applying different technologies.

Small organic molecules which bind and modulate the properties of specific biological receptors or targets is known as drugs. These receptors are mostly protein molecules which perform several important functions and are vital for the proper functioning of the cell. In case of abnormalities, these receptors get affected and are reflected as minor or major physical symptoms. Drugs acts to alter the defective receptors to restore its actual functions helping in treating the symptoms.

Drug Designing :
The process of constructing ailments specific drugs adopting the known properties of diseases applying different technologies is known as drug designing. This aims at designing drugs which can specifically and selectively bind to the target sites thereby modifying the same. Drug designing techniques employing computer based techniques is known as computer-aided drug design and those based on the information on the three dimensional structure of targets is known as structure– based design.

Tasks involved in drug designing:
(i) Analyse medical condition to determine the target site.
(ii) In-depth knowledge of the critical sites of target molecules.
(iii) Designing of drugs which specifically target the receptor molecules.
(iv) Synthesis and administration of drugs.
(v) Assessment of the drug-target interaction and record of the same.
(vi) Execute any necessary modification required.

Successful examples of drug designing:
Propanolol: It is one of the successful examples in drug designing.This drug is used as a treatment against heart attacks and hypertension. The heart ailments are mostly caused by excess amount of epinephrine and norepinephrine hormones. Both these hormones consist of alpha and beta receptors. The propanolol is so designed so that it binds with the beta receptors inactivating the hormones.

Cimetidine: This drug is useful in the treatment of stomach ulcers. In stomach ulcers, there is excess release of Hcl into the stomach induced by the histamine. Cimetidine blocks the binding of histamine to its H2 receptor present in the stomach lining blocking the Hcl release and thus cures the ulcer.

Drug delivery:
After a drug has been designed the next major step involved is the delivery of the drugs into the system. The normal routes of drug delivery are oral or parenteral. While following these common methods, drug delivery is not specific as it is distributed over the whole body. This demand administration of higher doses and may not always evoke a positive response. Other complications are proteolytic degradation of orally administered protein drugs, less permeability of such drugs owing its larger structure. As a result various other mechanisms have been developed for more efficient delivery of drugs:

(i) Delivery by alternative routes- other routes such as nasal, vaginal, anal, ocular, etc can be used while using protein based drugs. In order to improve the efficiency of such delivery, permeability enhancers to improve the permeability of drugs can be used. Commonly used enhancers are sodium deoxycholate, sodium glycocholate etc

(ii) Liposomes- these are artificially composed lipid vesicles. Drugs can be encapsulated in liposome and administered. Tissue specificity of liposome can be accentuated by use of specific surface ligands.

(iii) Polymers: Polymers, which are biodegradable, have been used extensively for successful delivery of drug; the drug is released by cleavage of drug fro polymer. This procedure is used for slow release of drugs of larger size.

(iv) Drug targeting: the procedure involving site directed delivery of drugs is known as drug targeting

Drug targeting
It is one of the most effective mechanisms involved in delivery of drugs to specific location or specific targets. The principle of such a mechanism is based on monoclonal antibodies.

In monoclonal antibodies, all the antibodies present in one single preparation reacts specifically with only one target. This property aids in drug targeting. The most widespread application in the use of monoclonal antibodies in this context is as immunotoxins. Monoclonal antibodies are linked with a toxin polypeptide to form immunotoxins. The target specific antibodies ensure selective and specific binding and the toxin in the immunotoxins inactivates or kills the targets.

Ricin: an example of immunotoxins and drug targeting tumour cells
The natural toxin ricin is isolated from endosperm of castor. It consists of two polypeptide chains called as A and B where the polypeptide A accounts for its toxicity. The immunotoxin formed as a conjugate between an antibody and ricin A chain results in specific binding to tumour cells and irreversible enzymatic modification of ribosomes preventing protein synthesis is such cells.

Gene knockout is a genetic process by which an existing gene function can be blocked by destroying a specific gene and such organisms are called 'Knockouts'. There is a loss of function in transgenic animal in the gene knockout which is in contrast to gaining function in introducing a foreign gene. The knockout technique is carried out by incorporating a DNA sequence (usually a selectable marker) into coding region. The chromosome carrying the target gene (with four exons) with flanking sequences is subjected to homologous recombination with a vector carrying a selectable marker gene. This homologous recombination results in Gene Knockout.

Gene knockout has many applications, especially done for research purpose; it also provides information about what that gene normally does. It is also an efficient method to study about the gene that has been sequenced but hardly known for its function. The biochemical and pathological basis of human diseases can be understood by inactivating specific genes. For these purposes the researchers produced a knockout mouse which lack genes for a single organ or an organ system.

As humans share many genes with mice and mice being animal closely related to humans knockout experiments are carried out on mice. Knockout mice also offer great opportunity in development and testing of drugs and therapies. There are many examples in which knockout mice had been useful for studying different kinds of diseases like- arthritis, anxiety, cancer, diabetes, heart diseases, obesity and Parkinson disease. Knockout mouse is a genetically engineered altered mouse lacking the genes or inactivated gene for an entire organ or organ system. These mice exhibits changes in phenotypic characters as well as behavioral and biochemical characteristics. As an example of knockout mice is p53 knockout mouse, named after gene p53 coding for a protein that suppresses growth of tumors by arresting cell. The knockout mouse is usually named after the gene that has been inactivated.

The typical procedure of producing knockout mice involves several steps. First the gene intended to be knocked out is isolated from mouse gene library, followed by engineering a new DNA sequence resembling the original gene. DNA sequence should carry a marker gene which is usually not present in the normal mice’s gene. This marker gene helps in observing the changes. Simultaneously, stem cells are isolated from mouse blastocyst and grown invitro. Then by incorporation technique the DNA sequence is incorporated into the grown stem cells. By the process of homologous recombination takes place, where in altered cells will have new sequence. Now the stem cells from the unaltered cells are isolated using marker gene and incorporated into mouse blastocyst. This blastocyst now carries both original and knockout cell, and implanted into female mice uterus for development. The newborn mice is the recombinant mice exhibiting characters of both stem cells (original and knocked out stem cells) differently in various parts of body, i.e. a chimeric mouse showing patches of grey and white. When such mice is crossbred with the wild type some of the offspring would carry copy of knockout gene in all their cells, but these mice would be white in color even after being heterozygous. Further if these heterozygous offspring are interbred, the progeny would contain few offspring inheriting knockout genes from both parents carrying no functional copy of the original gene.

Limitations with knockout mice have been discussed by National Institutes of Health, which mentions that about 15% of knockout mice are developmentally lethal i.e. they do not grow to adulthood which hinders the study of genes’ function in the embryonic and adult stage. Then other limitation is that some genes are really difficult to knockout. Sometimes knocking out fails to produce an observable change in the mice or may exhibit drastically different characteristics from those observed in human despite of same gene’s inactivation. Also sometimes there are certain developmental defects and die whilst as embryos.

There are few knockout mice that have been useful in studying human health. Knockout mouse for transplantation in which liver cells are destroyed, using suicide gene, that lacks immune system. In this mouse sample human liver cells were transplanted which could develop because of lack of immune system. This way organ transplantation is made possible in animals. SCID mice were developed by eliminating a single gene and the resultant mice lost ability of producing B-lymphocytes and T-lymphocytes, from which human mouse was developed. Knockout mice with memory loss were developed by gene knockout technique, where the mice lack hippocampus – specialized area believed to memory processor in brain. Research on knockout mice for allergy is undergoing and it is expected in near future to benefit millions of sufferers of allergic reactions.

Every single gene possess a distinct and definite location in a particular chromosome. This is well established by the ability to reconstruct gene maps using various physical methods and gene techniques. Besides this, the suspicion on gene relocation triggered researches to find out that some of the genes can actually relocate (change its position) in a chromosome. The relocation of the genes were identified to be the occurrence of two phenomenon like mispairing or unequal recombination and the presence of commuting elements called as Transposons. Transposons are one among the other two mobile genetic elements like episomes and cassettes.

Transposons are the genes present as the segments of DNA, able to commute independently from one site to the other on a chromosome. The DNA with transposon genes are also called as the selfish DNA or mobilized DNA. Transposons are also classified as mutagens. They are called by different names like mobile genes, jumping genes, roving genes etc. The scientist Barbara Mc Clintock is the founder of Transposon, as she was the first person to discover the presence of such mobilizing elements in maize crop.

The Different Transposons: Transposons were classified into prokaryotic transposons and Eukaryote transposons based on their existence in the type of cell. Prokaryote transposons have sub classes like insertion transposon first identified in E. coli and Transposons. The latter possess the genes that not only encode for the enzyme transposase but also encode genes for antibiotic resistance and heavy metal resistance. The process of transposition was found to be not frequent. Also the insertion type of translocation occurring in bacteria can be transferred either vertically to bacteria of the same species or horizontally to the bacteria of the different species.

The Eukryotic transposons are divided into two major groups called as class I transposon and class II transposon. The class I transposons are retrotranposons which undergoes two phases before fixing themselves into a new location. Retrotranposon commutes to the new location on a chromosome by first transcribing into RNA and then reverse transcribing into DNA. The former step is induced by the action of the enzyme RNA polymerase II or RNA polymerase III and the reverse transcription is due to the action of the reverse transcriptase enzyme. The long terminal repeats (LTR), long interspersed elements (LINEs) and short interspersed elements (SINEs) forms the class I transposons.

The class II transposons are those elements which relocate as DNA itself from the origin site to the target site (not RNA mediated). Transposase is the enzyme involved in this activity which snaps the new location, creating glue ends enabling the cut DNA to paste into the new site. The family of transposon elements is composed of autonomous members and non autonomous members. The autonomous members are like earning members in a family who code for their own transposition and non autonomous members are like non earning members in a family who always depend on the autonomous member for their movement into new location. Few examples of the identified class II transposable elements are Ac – Ds element in maize, Tam element in Antirrhinum, p element in Drosophila, Ty element in yeast, Tc1 element in the worm species Caenhorabditis elegans and Alu in humans.

Transposable elements are known mutagens and considered as rich source of mutation. The type of mutation caused by the transposable elements is the insertion mutation. Some of the diseases associated with the mutagenic property of transposons are cancer, muscular dystrophy, hemophilia A & B, porphyria and immunodeficiency.

The definite frequency at which the transposons relocate to a new site is considered as the unique property of transposons. The mutation causing property of transposons has derived ways for multiple applications in the field of medicine, genetics, gene therapy and biotechnology. Characterization of various strains of the species Plasmodium falciparum, the major source responsible for malaria in humans is made possible by the use of transposons as markers in clinical studies. Identification of carriers of genes responsible for diseases like sickle cell trait and Down’s syndrome is made possible by using transposon as a genetic tool. Also transposons are identified as suitable vectors in Transformation mode of gene transfer. Transposons are applied in molecular genetics which involves gene isolation and they are also used to construct gene maps. The research on transposons as tools for genetics and gene therapy are underway in the labs of Zsuzsanna Izsvak and Zoltan Ivics at Max Delbruck center for molecular medicine.

Human haemoglobin is available in abundance. It is also one of the most sought after protein available. Haemoglobin has been studied so extensively that all the properties and expression mechanisms is well known. This makes it easier to develop haemoglobin artificially (blood substitute) to fulfil the rising demand of haemoglobin.

Mechanism involved:
Human haemoglobin basically consist of two parts mainly heme and globin. The heme portion of the protein is common among many organisms. This property can be exploited to produce haemoglobin artificially by using recombinant DNA technology. In the production of recombinant haemoglobin, only the globin part needs to be integrated and expressed. Since it naturally produces the heme part, this proves to be an easy method to produce active haemoglobins. As in such host animals the native haemoglobin serves the similar function, recombinant protein is successfully expressed.

Different organisms serve as hosts to haemoglobin production. This include

Microbial host: In microbes, the gene encoding the heme part, which produces heme identical to human haemoglobin, is first isolated and purified and then combined with globin structure isolated from human blood. Thus a proper functional haemoglobin gene is inserted in vitro into a microbial cell and the expression of the same releases haemoglobin into the cell. This haemoglobin, produced inside a microbial system, can be easily isolated and purified. The major disadvantages involved are the high cost of production involved in this technique.

Mammalian host:
The haemoglobin production in mammals involves production of transgenic animals. The genome of such animals is integrated with genes responsible for production of haemoglobin. In this technique, it is possible to determine the amount of haemoglobin genes to be expressed by controlling the genome of transgenic animals. Thus the expression levels can also be easily regulated to increase or decrease production. The main disadvantage of the process is time required to produce actual results.

Functions
The main function of blood with respect to natural haemoglobin is to act as a carrier for oxygen. Apart from this, blood also exhibits several other functions like immunity by white blood cells, blood clotting by platelets, electrolyte balance, and several other functions by blood proteins. The blood substitute is expected to fulfil some properties of blood if not all. Research corresponding to production of actual blood compiling various functions of blood is also initiated.

Advantages of blood substitute
(i) High production rate:
By producing blood substitutes, it is possible to supply blood as the demand arises.
(ii) Production of haemoglobin exhibiting specific characteristics:
As a result of rDNA technology, the recombinant haemoglobin produced exhibit several desired properties.
(iii) In depth analysis of blood related disease: since the blood substitute resembles haemoglobin closely, the study of mutations or disease affecting haemoglobin can be studied buy inducing such defects in the blood substitute.
(iv) Gene therapy:
The defective gene responsible for certain diseases like anaemia and thalassemia can be treated by gene therapy. It is also a proposed cure for inherited hemoglobinopathy
(v) Protein delivery: It can also acts as a carrier for certain biomolecules like proteins.
(vi) Safe blood transfusion: Blood substitute can be used in the place of blood for transfusion. Since it is produced artificially under sterile conditions, safe and infectious free blood can be transfused to affected people.

Disadvantages:
(i) Renal toxicity: Filtration of blood with blood substitute particles has found to affect kidneys adversely. As a result, cases which indicate renal toxicity in persons with blood substitutes is reported.
(ii) Other side affects like hypertension, fatigue is also known
(iii) This is also known to induce heart attacks.
(iv) Since animal and human haemoglobin share many properties, the separation of recombinant protein from animal ones after production proves to be difficult.
(v) It is not economical to produce on larger scale.
(vi) It is less stable and has to be transfused immediately rather than normal blood which has extended shelf life, comparatively.

Future prospects:
The stem cell research has been suggested as a very prospective area wherein the human haemoglobin can be isolated in its natural form and developed on larger scale. The haemoglobin protein can be isolated from stem cells collected and cultured to produce the same under invitro conditions. Since in this technology, the human haemoglobin is isolated in its original form, complexities due to a foreign substance in the body does not arise. All the vital functions of blood will be fulfilled by blood produced by stem cell technology.

A biological preparation, which evokes an immune response when administered into the body, is termed as vaccines. This usually consists of parts of pathogen in its weakened state or its products. This triggers an immune response from the body to the particular disease without actually causing the disease.

Catering to the needs of large number of diseases, numerous vaccines for a variety of diseases has been developed and still continues to do so.

Recombinant vaccines:
Biotechnology sector has also played its part in developing vaccines against certain diseases. Such vaccine which makes use of recombinant DNA technology is known as recombinant vaccines. It is also known as subunit vaccines.

Recombinant vaccines can be broadly grouped into two kinds:

(i) Recombinant protein vaccines: This is based on production of recombinant DNA which is expressed to release the specific protein used in vaccine preparation

(ii) DNA vaccines: Here the gene encoding for immunogenic protein is isolated and used to produce recombinant DNA which acts as vaccine to be injected into the individual.

Steps involved:
Production of recombinant vaccines involves the following steps:

(i) First and foremost, it is important that the protein which is crucial to the growth and development of the causative organism be identified.

(ii) The corresponding gene is then isolated applying various techniques. Further to this, an extensive study of the gene explains the gene expression pattern involved in the production of corresponding protein.

(iii) This gene is then integrated into a suitable expression vector to produce a recombinant DNA.

(iv) This rDNA is used as vaccines or is introduce into another host organism to produce immunogenic proteins which acts as vaccines.

Recombinant protein vaccines:
A pathogen upon infection produces proteins, vital for its functions, which elicit an immune response from the infected body. The gene encoding such a protein is isolated from the causative organism and used to develop a recombinant DNA. This DNA is expressed in another host organism, like genetically engineered microbes; animal cells; plant cells; insect larvae etc, resulting in the release of the appropriate proteins which are then isolated and purified. These when injected into the body, causes immunogenic response to be active against the corresponding disease providing immunity against future attack of the pathogen.
Based on the proteins involved in evoking immune response recombinant protein vaccines are of two types:

Whole protein vaccines: The whole immunogenic protein is produced in another host organism which is isolated and purified to act as vaccines.

Polypeptide vaccines: It is known that in the immunogenic protein produced, the actual immunogenic property is limited to one or two polypeptides forming the protein. The other parts of the protein may be successful in evoking an immune response but do not actually cause the disease. For eg: in the case of cholera caused by Vibrio cholerae, consists of three polypeptide chains like A1, A2, and B. The A polypeptides are toxic while B is non-toxic. Thus while producing vaccines, the polypeptide B is produced by rDNA technology and used for vaccination.

DNA vaccines:
It refers to the recombinant vaccines in which the DNA is used as a vaccine. The gene responsible for the immunogenic protein is identified, isolated and cloned with corresponding expression vector. Upon introduction into the individuals to be immunized, it produces a recombinant DNA. This DNA when expressed triggers an immune response and the person becomes successfully vaccinated. The mode of delivery of DNA vaccines include: direct injection into muscle; use of vectors like adenovirus, retrovirus etc; invitro transfer of the gene into autologous cells and reimplantation of the same and particle gun delivery of the DNA.
In certain cases, the responsible gene is integrated into live vectors which are introduced into individuals as vaccines. This is known as live recombinant vaccines. Eg: vaccinia virus. Live vaccinia virus vaccine (VV vaccine) with genes corresponding to several diseases, when introduced into the body elicit an immune response but does not actually cause the diseases.

Advantages:
(i) Since it does not involve actual pathogen, recombinant vaccines is considered to be safe than the conventional vaccines.
(ii) It induces both humoral and cellular immune response resulting in effective vaccination.

Risks involved:
(i) High cost of production.
(ii) Have to be stored at low temperature since heat destabilizes protein. Hence storage and transportation is tedious.
(iii) Individuals with immunodeficiency may elicit poor immune response.

Oral vaccines: a novel approach
The latest hot spot in the field of vaccine research is the development of vaccine which can be taken orally. Immunogenic protein of certain pathogens is found to be active when administered orally. The gene corresponding to such proteins is isolated and a gene construct is produced. This is introduced and expressed in a plant genome which results in production of such immunogenic proteins in the parts of the plant where it is expressed. These when fed into animals or mainly humans, the person becomes vaccinated against certain pathogen. Such vaccines are also known as edible vaccines. An exciting invention is production of ‘melt in the mouth’ vaccines that can be administered by placing them under your tongue which delivers it into the blood stream. The most important example is the production of flu vaccine by Bacillus which melts in the mouth. The tremendous benefit of such vaccines is the comfort of administration, low cost and ease of storage.

I am a master student of Erasmus University Rotterdam, Strategic management studies. Presently, I am working on my master thesis about strategic alliances between profit-seeking organizations and non-profit-seeking organizations (i.e. government agencies, universities, research institute.), particularly in the biotech & pharmaceutical industry.

Hereby, may I ask you a favor to help me by filling in the questionnaire?

Survey Link: https://qtrial.qualtrics.com/SE/?SID=SV_85GNxMwSEobotHT
(list of questions are also listed below in Jessica's post)

It’s about how organisations collaborate with each other in terms of trusting and controlling each other. Therefore, I'm looking for alliance managers, marketing, R&D and financial controllers to participate. The questionnaire is anonymous and completing it will only take you approximately 5 minutes.

I will really appreciate your participation, which helps me to graduate my Master study. Should you request, I would send you the results of this study, which may be interesting for you as well.

Please leave a message if you're interested. The questionnaire is an online link that I can forward to you.

Thanks

The gene expression is determined by two features called as penetrance and expressivity of the genes. Penetrance is the ratio of individuals exhibiting expected phenotype and expressivity is the extent of gene expression in an individual. The phenotype of an individual is determined by the genotype or the type of gene expressed. In general, phenotypic changes occur in individuals when exposed to various environmental factors. But the query, “Is the genotype of an individual is influenced by external environment?” lead to several researches throwing light on the effect of external or environmental factors like temperature, light, chemicals and nutrition in gene expression. Besides the effect of internal factors like hormones and metabolism on gene expression, external factors were also found to affect the gene expression and ultimately exhibiting phenotypic changes.

Temperature and Gene Expression: The study on the coat color of the Himalayan rabbit with relation to the temperature reveals the effect of temperature. The rabbit with normal phenotype of white fur showed differences in the skin color with exposure to temperature. The body of the rabbit which is generally exposed to high temperature (>34 degrees) expressed white color whereas the other parts like ears, nose , tail and paws which are little exposed to temperature expressed black color. Keeping the rabbit under cold climate resulted in the expression of fully black colored skin. This study proves the sensitivity of the genes responsible for the skin color to the temperature.

Another study on wing development in Drosophila flies with response to the temperature also provided results with the effect of temperature. Flies exposed to the temperature of 25 degree Celsius showed less penetrance whereas when exposed to higher temperature penetrance also increased which was observed by the increase in the development of wings in the selected population of flies.

The research by the scientist Voolstra CR and his team from KAUST, Saudi Arabia on studying the gene expression by exposing the embryos of Coral Montastraea faveolata to different temperatures like 27.5, 29 and 31.5 degree C resulted in the continuous expression of stress related genes in the embryos that were exposed to 31.5 degree C. Also the effect of temperature on genes encoding the enzyme for the biosynthesis of starch in the wheat plant Triticum aestivum was studied by William J Hurkman and his co-workers from USDA, USA. The effect of temperature on these genes was observed by analyzing the starch accumulation.
Also the research on the effect of temperature reduction on gene expression and oxidative stress in skeletal muscle from adult Zebra fish by Ranae L and team and the study of sea water acidification and elevated temperature’s effect on gene expression pattern of the Pearl Oyster Pinctada fucata by Wenguang Liu and team, China shows the role of temperature, an external environmental factor on gene expression.

Light and Gene Expression: The study of a gene responsible for the anthocyanin pigment formation in Maize plant with relation to the light by researchers is a good example showing the role of light in gene expression. The plant carrying the homozygous gene for pigmentation when exposed to sun light developed bright red color and when the light was retarded by covering the area of the plant prone to pigmentation, the bright red phenotype was not observed. Also the prevalence of skin cancer in humans on exposure to sunlight is a classic example.

Chemicals and gene expression: The research by Mankame T and team from Texas University on a fungicide called Enable which has potential effect on endocrine regulated gene showed down regulation of 8 genes and upregulation of 34 genes on exposure to the chemical. The effect of chemical mutagens and carcinogens on gene expression profiles in human TK6 cells by the researcher Lode Godderis and his team developed results. In their experiment, they observed a linear trend in Dose- response of gene expression for chemicals like Trichloroethylene, Benz(a)anthracene, epichlorohydrine, benzene and hydroquinone. The effect of the sedative drug Thalidomide on fetus causing birth defects can also be coined as an example for the effect of chemicals.

Nutrition and Gene Expression: It is a very interesting fact that dietary and nutritional supplement also plays an important role in gene expression. The deficient nutrient supplement alters the genetic expression. What a pregnant woman eats determines the health of her offspring. Babies born with deformities to mother ingested with Thalidomide drug during 6th week of pregnancy in 1960s should be cited. The folic acid supplement to the pregnant women takes care of the development of the fetus whereas the deficiency of the same causes some birth abnormalities.

Thus the fact that the environmental factors also play a vital role in gene expression is understood by various research studies.

DNA Microarrays consist of a number of DNA spots which are attached to solid substrates like glass, silicone, nylon membranes.

After a gene has been isolated, the next step involved is to study the different characteristics of the isolated gene. In the past, it was possible to analyse the nature and properties of only a single gene but with the advent of microarray technology it is possible to analyse thousands of gene with a single experiment resulting in a faster and more accurate results.

Microarray: It refers to a recent hybridization technique which provides an opportunity to match known and unknown DNA samples under specific conditions to reveal different properties of the unknown gene. Here, a series of probes is fixed onto a solid substrate and used to hybridize a series of test DNA. It is of two types DNA microarray and antibody microarrays.

DNA microarrays (DNA chips, bio chip, gene chip):
This consists of a number of DNA spots which are attached to solid substrates like glass, silicone, nylon membranes etc. In a more recent technique, an array of microscopic beads is used as a platform for such arrays.

Probe and Target:
A probe refers to a small nucleotide sequences with known bases. This is fixed onto a solid substrate to form a neat arrangement called an array. Purified mRNA, isolated DNA, cDNA produced from mRNA; all can form probes.

Target refers to test a DNA sequence which has to be studied for its position, composition, gene expression, mutations etc; these are labelled using fluorochrome or other labels which aid in quantifying the results after hybridization.

DNA microarrays are of two different types

cDNA arrays:
The probes consisting of complementary DNA is spotted onto the glass substrate with the help of fine needles and a robotic arm.

Oligonucleotide arrays:
The probes here are oligonucleotides formulated in situ or produced externally and then immobilized on the arrays by different techniques like photolithography.

Earlier the oligonucleotide array was termed as DNA chips. But recently the term DNA or gene chips are applicable to both the kind of arrays.

Principle and mechanism:
In order to conduct such a study, the complementary base pairing nature of the polynucleotide chain of the DNA and the resulting double helix plays an important role. In the process of hybridization, these polynucleotides are subjected to conditions so that the hydrogen bonds between the two stands weaken but phoshodiester bonds between the nucleotides remains intact leading to production of two separate polynucleotide strands. When treated with appropriate probes, it results in the pairing of the probe with its complementary bases in the target DNA.

The DNA sequences of each gene to be analysed of an organism are labelled with fluorochrome like Cys green or red. DNA micro array is set up by spotting or other available techniques. When such a probe is treated with the labelled target DNA and hybridization is initiated, the double helix open up and hybridize with complementary DNA resulting in producing fluorescence at hybridized sites. These are scanned and quantified. Different fluorochrome representing different properties can be used to be probed with a single DNA chip resulting in conducting multiple tests using a single microarray.

Multiple and single channel microarrays:
In a multiple channel or two colour microarray, it is possible to analyse genes from different sample in a single test. Each target gene is labelled with fluorochrome having different fluorescence emission. This mixed sample is allowed to hybridize in a single test with a single microarray probe, and when scanned with microarray scanner after excitement with different corresponding wavelengths, the ratio of different gene can be quantified.

In a single channel or one colour array, a probe is hybridized with target DNA labelled with one colour fluorochrome. Two colour microarrays is usually used to detect different genes present in one mixed sample whereas one colour microarray usually is employed to estimate the amount of gene expression of same sample or between samples. Single channel microarrays are found to give more accurate results but need to conduct different test to quantify different gene expression. Multi channel makes use of only one test to give multiple results but different samples sometimes interfere with each other leading to non unique results.

Applications:

(i)Diagnostic applications:
Micro array technology can be used to detect presence of genetic diseases, presence of mutations, polymorphism, cancer etc.

(ii)Gene expression profiling:
Using this technique, the number of genes present, the expression rate each gene can be determined. Monitoring the gene expression is known as gene expression profiling. Quantitative and qualitative measurements of such expressions is possible with the help of microarrays.

(iii)Genomics:
With the help of microarrays, SNPs or single nucleotide polymorphisms can be detected. It refers to different nucleotides present at the same base position in different alleles. Such minute differences between different individuals or different alleles of same gene can be identified successfully and uniquely with the help of micro arrays.

(iv)Sequencing:
A microarray is prepared with oligonucleotides of specific length. When hybridized with target sequences of unknown lengths, the results obtained indicate the length of the target sequence.

Advances in the field of biotechnology, research and medicine has made it possible to produce artificial skin in vitro.

A culture refers to culturing of any kind of cells or growth of cells on a suitable nutrient medium under in vitro conditions. A cell culture initiated to formation of tissues can be termed as tissue culture. It is basically of two types:

(i) Cell culture: where the tissues which are cultured are broken down into cells by enzymatic or mechanical means.

(ii) Organ culture: where the tissue culture are further developed into maintaining their structure and resulting in production of specific organs.
Isolation of animal cells and establishment of its successful cell culture leads to formation of primary culture. This when sub cultured produces cell lines. Cell lines which dies after several subcultures is known as finite and those which survive multiple subcultures and continue to grow indefinitely is known as continuous cell line.

Organ culture:
Culturing of animal cells under invitro conditions leading to production of organs or parts of organs such that, they retain or closely exhibits their structure and functions is known as organ culture.

Artificial skin:
Human skin is the largest organ of the human body giving structure to the body and serving as the first line of defense of the body against infections. It is basically formed of two layers: the outer or epidermal and inner or dermal layer.

Advances in the field of biotechnology, research and medicine has made it possible to produce artificial skin in vitro. The first successful result in this field was accomplished in around 1970s by two scientists named Burke and Yannas. The synthetic skin they produced was termed as Silastic. The Silastic so produced was successful in producing only the epidermal layer of skin and could not, however, develop the dermal layer.

Living skin equivalent (LSE):
A further development led to production of structure called living skin equivalent which essentially resembles complete skin (epidermis as well as dermis).This is also known as graftskin.

The corresponding procedure involves isolation of living skin explants either from the required patients or from the newborn infants; culture and growth of such explants in a collagen matrix.

Techniques involved:
The skin at large is made up of cells known as keratinocytes. These cells produce dead cells or corneocytes which make up the outermost layer of skin. The keratinocytes during their transition to corneocytes expel lipid molecules. These dead cells along with lipid molecules together form the living skin equivalent under invitro conditions. The explant isolated consists of keratinocytes extensively. This undergoes trypsin treatment in order to break down the tissue into cells. Studies have revealed that irradiated 3T3 fibroblasts, which is a continuous and non tumorigenic cell line, promotes keratinocyte growth and proliferation. Thus, following the trypsin treatment the cells are cultured in 3T3 lined vessels. The proliferation result in the formation of colonies of cells which is again subjected to dissociation into individual cells. After a number of cultures and subcultures, a pure multilayered sheet of epithelium called cultured epidermal sheets (CES) is achieved. These sheets produced are then detached from culture vessels, cleaned and then applied for grafting. For the complete success of the procedure, it is important that the living explants are isolated from the respective patient so as to defer the possibility of rejection by the patient’s immune system.

Applications:

(i) Treatment of burns: - The most important application in developing a living skin equivalent is in the treatment of people suffering severe burns. Often in such cases when grafted with the patient’s own skin taken from elsewhere in the body, it does not regenerate rapidly to cure the burns effectively. Similarly, xenografting can induce severe rejection reaction from the body. But the graftskin technology has proved successfully to aid in such treatment to larger extent so much so that, majority of the fundamental skin components were regenerated. This is also of great importance to people affected with skin cancer wherein such graft skin can be used for developing non cancerous cells and thus aid in cancer treatment.

(ii) Wound healing: - Another vital role of graftskin is in healing of wounds produced on skin by various diseased conditions. Thus, chronic skin ulcers (eg: foot ulcers developed as a result of diabetes) can be treated with living skin equivalent.

(iii) Providing model environment for research: - Since the graftskin produced resemble the living skin extensively, it can be employed as a medium to carry out various skin related research.Variations in the medium used for developing the related tissue has been introduced such as, introduction of fibroblasts for better differentiation and vitamin c for improved barrier functions of differentiating cells. Diseased skin condition can also be induced under invitro conditions in such living skin equivalents so that they can be studied closely into developing probable cure.

(iv) Testing dermatological products: - Artificial skin can also be used to test the effectiveness of various dermatological products rather than testing on the lab animals.

Thus it can be concluded that the living skin equivalent or artificial skin exhibits a wide range of future prospects having clinical, laboratorial and medical applications.

There are certain disorders like cancer, parasitic and viral infections, which cause excessive production of specific proteins in day to day life. An alternative treatment for these disorders is known as Antisense Technology. A single stranded RNA that is complementary to messenger RNA (mRNA) is referred to as Antisense RNA. The antisense therapy includes inhibition of translation by using single stranded nucleotide, any DNA or RNA sequences or even synthetic ones. From the practical point of view, most of the antisense therapies work efficiently and produce best results if used with RNA since RNA specifically binds to target mRNA and blocks protein synthesis.

Antisense technology was also referred as Gene Subtraction, but it is proven to be a misnomer as this technology does not remove gene, rather it just involves inactivation of the gene. Naturally occurring mRNA antisense mechanism is the hok/sok system in E.Coli R1 plasmid.

The antisense technology is carried out on the basis of the principle that the cloned gene is ligated into the vector in reverse orientation. Now, as the antisense technology obstructs the mechanism of translation it is stoichiometric in nature, and it can prevent synthesis of the product of the gene that it directs against. The antisense RNA mechanism involves hybridization of the antisense and sense copies of RNA. Now, as the ds-RNA molecule is formed, it rapidly degrades by ribonucleases and the expression is blocked. Another reason could also be the antisense RNA preventing ribosomes to bind to the sense strand. In simpler words, if an oligonucleotide is introduced into the cell, it binds to specific mRNA which forms an RNA dimer in the cytoplasm and halts the translation mechanism; this is because the mRNA no longer has access to ribosome and dimeric RNA is rapidly degraded by ribonuclease which in turn on introduction of oligonucleotide complementary to mRNA leads to blockage of translation by particular gene, turning off the gene.

RNAi (RNA interference) and Antisense RNA technology though has the same effect but their mechanisms are quite different. Firstly, RNAi technology involves degradation of mRNA by small interfering RNAs triggering catalytic gene silencing; whereas in Antisense RNA technology mRNA is degraded by RNase H. Secondly, in comparison to antisense RNA, RNAi are twice larger.

The application of Antisense RNA technology is in many sectors. This technology was used in Flavr Savr, for tomato ripening; ripening in tomato produces enzyme Polygalactourodase (PG) which softens tomatoes and finally rotten them quickly. Two biotechnology companies: Calgene, USA and ICI Seeds, UK introduced a gene in plant which synthesizes a complementary mRNA to PG gene and inhibites the synthesis of PG enzyme delaying over ripening and rotting. Antisense therapy is considered for treating certain genetic disorders and infections. This is also referred to as Gene Therapy. It includes: isolation of specific gene; it’s cloning and inserting it into target tissue cells to make desired protein. It has to be ensured that the gene is expressed correctly and sufficiently without causing harm to patient in context with the immune response. Antisense RNA technology is also used for cancer therapy. This therapy is used for treating brain cancer – malignant glioma and cancer of prostate gland, in malignant glioma IGF-I was over produced and in the prostate cancer IGF-IR was synthesized more. These two were used to block the translation. Research is carried on in regard with Antisense RNA drugs for treatment of CMV, HIV, cancer, etc. Antisense antiviral drug named Formivirsen is developed to treat CMV, which was licensed by FDA in August 1998. In 2010, scientist at NIPGR by using Antisense technology developed tomato which could last longer for more than 30 days by silencing two genes (alpha - man and beta – hex) which causes softening and wrinkling in tomatoes during ripening process.

Despite of achieving some success, Antisense technology has few challenges. Like: delivery into the patients body, then possibility of toxic effects due to it’s regulation on both the normal and mutant alleles. Antisense RNA technology is also used to study certain gene functions.

Looking at the advancements in the Antisense RNA technology, it has potentials for development of pharmacological agents, studying physiological and pathological processes as well as it’s use in effective treatment as gene therapy.

Gene cloning involves application of many enzymes having specific functions to result in a modified product. Of the different enzymes involved, endonucleases are a type of enzymes which have the ability to cut or cleave the DNA molecule. Restriction endonuclease refers to a group of endonucleases which cleaves the DNA at specific points known as recognition sequences or sites. Different types of restriction endonucleases have been identified like type I, II, and III. Among these, the most available and most extensively used enzyme is type II restriction endonuclease. Examples are: ECoR I, Hind III, etc.

The importance of restriction enzymes lies in the property that it cleaves the DNA sequence, in most cases, within their specific recognition sequences unlike other restriction enzymes which cuts some base pairs away from their recognition sites. Some type II restriction endonucleases are also known to cleave the DNA sequence in close proximity of their recognition sequence rather than within the recognition site. This efficient nature of type II restriction endonucleases, combined with their comparatively smaller structure, has led to the wide application of these enzymes in gene cloning.

Mechanism of type II restriction endonucleases.
Recognition sites are specific area or sequences in the genetic molecule which these enzymes recognize as sites for cleavage. Recognition sites are unique for different restriction endonucleases. For type II restriction endonucleases, recognition sites are mostly palindromic sequences with rotational symmetry. DNA has a double stranded helical structure where, the nucleotides of the two strands of DNA are complementary to each other. There are certain sequences in such a structure where, the first half of the sequence is a mirror image of the second half of the complementary strand and reads identical from same end. Such sequences are termed as palindromic sequence with rotational symmetry.
Eg:-
5’GAATTC3’
3’CTTAAG5’

The restriction endonuclease moves along the surface of the DNA until it recognises its target sites. After recognition, it initiates DNA binding in the presence of Mg2+ ions resulting in cleavage at specific sites.

Cleavage:
The cleavage patterns produced by different restriction endonucleases are specific and each holds a novel role in gene cloning.
The two main patterns of cleavage are creating staggered cuts and even cuts. In staggered cuts, the cleavage occurs in different locations resulting in producing protruding ends of one of the strands in the double helix. Such ends are known as cohesive or sticky ends. The main benefit of such ends is that the protruding ends created are usually complementary in nature and can be used to link with vectors consisting complementary sequences for isolating the DNA fragment. It forms the basics for recombinant DNA techniques such as southern blotting. The even cuts, on the other hand, produces blunt ends where the two strands are cleaved at similar points. The importance of blunt ends in gene cloning involves many techniques which are utilized to modify the blunt ends in a manner so as to meet the specific requirements.

These include:
Tailing: This is a procedure which results in a protruding end of a defined length being created which aids in the pairing of required DNA segment with appropriate vector.

Linker: Linkers are chemically synthesized oligonucleotides. This can be used to modify the blunt ends so as to create cohesive ends of required bases. Linkers are so designed as to have a recognition site of a specific endonuclease. This can be linked to a blunt end DNA fragment created by the restriction digest. Such a modified fragment when digested by the linker specific restriction endonuclease can create cohesive ends complementary to vectors which can later be isolated to create multiple copies or, can be used in creating a recombinant DNA.

Adapters: These are short artificially synthesized double stranded fragments which can be used to link two blunt ends with different end sequence.

As a result of all these techniques, it is possible to alter a specific gene at a nucleotide level by identifying the respective restriction endonuclease enzyme which can cleave at the specific site. This is the principle adapted in gene cloning given that, to create a specific clone it is required to isolate the target gene. This isolation can only be done with the help of a restriction endonuclease enzyme. The type II restriction enzymes accentuates the importance as they have the ability to cleave at exact points resulting in producing definite fragments rather than random fragments.

Other Applications:
(i) RFLP (restricted fragment length polymorphism), which involves production of DNA fragments of different lengths which can be separated and utilized for several purposes like DNA fingerprinting, identification of mutations, preparation of genomic library etc.

(ii) A technique called restriction mapping make use of the capability of restriction enzymes to create DNA fragments of specific length thus distinguish alleles of a single gene having altered restriction sites.

(iii) Gene therapy: This employs the property of restriction endonuclease to recognize and remove a specific DNA fragment responsible for many diseases.

The best outcome of the Recombinant DNA Technology is Transgene. The Transgenic Plants or Genetically Modified Crops are the plants produced by having certain genes transferred from another species into that particular plant through various natural or artificial insertion techniques. This conventional technique goes handy and very precise in comparison to time consuming classical methods of breeding. The noble causes for producing transgenic plants are many; like enhancing the agricultural values (quality), for higher yields, for manufacturing certain commercially important products like pharmaceutical products, proteins; and also importantly to carry out the detailed study about various physiological and biological processes during the plant’s development. Tobacco was the first transgenic crop, in 1983, expressing the Kanamycin resistance. Well known Flavr Savr tomato was the transgenic plant to be first commercially launched in the market. Then started the never ending research and production of various transgenic plants and crops’ line expressing various biotic and abiotic stresses resistances and more.

Transgenic plants have both the commercial and applied benefits, which includes introduction of herbicide resistant gene, virus resistant genes, genes for self-incompatibility, pigmentation in floral products and tolerance to biotic and abiotic stress. Also as vaccines for immunization against various pathogens. As tools for studying plant molecular biology, mutations, etc. these GM plants have been aiming to produce various immunoglobulin, interferon and some useful polymers as well. The applications and the methods goes hand in hand to understand the GM plants. We shall discuss it in the paragraphs below. The method plays a very significant role in producing the transgenic plants and the foremost crucial part for production of the transgenes are construction of the gene and the gene transfer method. Plant gene in general comprises of regions: promoter, enhancer, cap site, leader sequence, initiation codon and a stop codon, exons and introns, an untranslated region and poly A tail. Each of these regions have different role and these specification signifies the assembly of DNA sequence designing and its expression in the transgenes.

The methods for gene transfer are categorized into two types: Vector Mediated gene transfer and Direct Transfer i.e., vector-less gene transfer method.

First one, Vector Mediated, as the name suggests is carried out by plant viruses being the vector or by Agrobacterium mediated transformation. Agrobacterium tumefaciens are considered the natural genetic engineer which conveniently infects any plant tissue or organ ensuring the large fragments of DNA with reasonably good stability and effective regeneration capability. Plant viruses are natural vector for genetic engineering and they can efficiently introduce the desirable genes into almost all plant cells systematically. The plant viruses are majorly inserted into a plant chromosome.

Second one i.e., Direct (vector-less) DNA transfer allows the foreign DNA to directly insert into the plant genome. These methods are more simple and effective. These methods includes: DNA absorption by cells/ tissues, physical gene transfer method and chemical gene transfer method. DNA absorption has very little or no success rate, but it is believed that the DNA gets absorbed and the cells get transformed in the cells suspensions. The physical gene transfer method includes: electroporation technique, gene gun (particle bombardment), microinjection, liposome fusion and silicon carbide fibres. The chemical gene transfer method includes: polyethylene glycol mediated and diethylaminoethyl dextran mediated transfer method. In addition to methods described here, there is one more new transformation technique known as chloroplast transformation which is in the developing stages and it holds efficiently promising future in plant biotechnology.

After the transformation of plants is accomplished, they need to be confirmed for being transgene by various selecting tools. These tools are set of genes referred as marker genes: selectable marker genes (eg. Bleomycin resistance genes, β-glucoronidase, Acetolactase synthase) and reporter genes (eg. Greeen fluorescent protein, Luciferase). Further step is to study and ensure the expression of genes which is carried out by promoters and terminators. Next step involves confirmation of integration of transgenes with the targeted plant genome; this is confirmed by techniques- southern hybridization and polymerase chain reaction. Later comes ensuring of transgene being stable in terms to avoid gene silencing followed by regeneration of the transformed plants which are transgenic plants.

These all genetic manipulations are done with the aim of improving crops with desired traits such as biotic and abiotic stress resistant, quality and yield, enhancing the nutrition and use of genetically modified plants as bioreactors. Biotic stress resistant crops include:

(i) Pest resistance by the use of Bacillus thuringieneses (Bt) toxins producing wide range of cry proteins; protease, lectins, etc. (Bt crops includes Bt cotton, rice, maize, tobacco, tomato, potato, cowpea and soybeans),

(ii) Virus resistance by incorporating virus coat proteins, antisense RNA technology, ribosome, etc. and

(iii) Fungal and bacterial disease resistance by incorporating pathogenesis related proteins, phytoalexins. While, abiotic stress resistant plants includes: herbicide resistant, drought and soil salinity resistant, freeze resistant, etc. Genetic modification however has contributed tremendously in the crop yield and quality; such as extended shelf life, slow ripening, and preventing discoloration in flowers, fruits and vegetables. Transgenic plants with improved nutrition have been engineered for human health improvement, e.g. Golden rice which is enriched with provitamin A, potato with increased protein and methionine levels, canola with cis-Stearates-lowering the risk of heart diseases, sugar beet with fructans-low calorie alternatives to sucrose. Also, transgenic have been engineered for allergen absence. Most importantly, transgenic plants as bioreactors have created boom in commercial aspect for manufacturing hemoglobin, monoclonal antibodies, interferons, serum albumin, proinsulin, edible vaccines, etc.

Despite of all these advantages the GM crops/ plants have been much controversial from ethical point of view. Nevertheless, GM crops have been potentially engineered in an eco-friendly way causing improvement to human health and environment.

The basic principle of genetic engineering is gene transfer, achieved by various methods to produce recombinant proteins, genetically modified microorganisms, transgenic plants and transgenic animals for commercial application. Genetic engineering, thus ultimately influences the growth of biotech industry. The two significant feature of genetic engineering is production of beneficial proteins and enzymes in surplus quantities and creation of transgenic plants, transgenic animals and genetically modified microorganisms with new characters beneficial for themselves using recombinant DNA technology. The discovery of a new protein either with a therapeutic property or application in food industry by a researcher or scientist would not have reached humans, for the use by humans without the application of genetic engineering in mass producing such proteins.

Recombinant proteins production and uses: The industrial production of proteins is done by transferring the desired gene responsible for the particular protein to be manufactured from the source organism to the preferred host organism through recombinant DNA technology. The host organism can be a bacteria or a eukaryote. The most preferred bacterial host is Escherichia coli for industrial production of proteins. The well established gene structure, faster growth rate, easy to cultivate and handle are the salient features of the E. coli bacterium fascinated the bio technologists to use this in recombinant protein production. Besides all these commendable characters of E. coli, the final output product is found to be unstable and difficult to purify. As a result research encouraged the use of eukaryotic host like yeast, cells of insects and cells of mammals in protein production. The proteins produced in this way find its way into pharmaceutical industry and food industry.

The recombinant proteins produced in the industry using the techniques of genetic engineering acts as drugs for various human diseases. To name a few, insulin produced for diabetes, alpha 1- antitrypsin in treating emphysema, calcitonin to treat rickets, interferon to treat viral infections and cancer, Factor VIII for hemophilia, production of growth hormone to act against growth retardation and chorionic gonadotrophin in the treatment of infertility. Some of the industrial manufactured enzymes occupy a vital position in the food industry. For example, the recombinant enzymes like rennin and lipase are used in cheese making, the role of alpha- amylase in beer industry, the antioxidant property of the industrially produced enzyme catalase and the use of protease in detergents.

Uses of Transgenic plants: In order to improve the quality and quantity of plants, traditional method of plant breeding is replaced by the creation of transgenic plants. The transgenic plants are plants carrying foreign genes introduced deliberately into them to develop a new character useful for the plant. The infection of plants by microorganism mostly viruses, poor production and decline in quality of plants due to attack by insects and the plants inability to withstand the pesticide or the weedicide used in the agriculture process welcomed the genetic engineering technology to develop transgenic plants with new characters like resistance to infections, defensive against the attacking insects and resistance to pesticides or weedicide.

The transfer of gene responsible for the protein protoxin from Bacillus thuringiensis to plants to develop resistance against the attacking insects is a remarkable example. Also the digestive action of the insects on the plants is restricted or inhibited by transfer of gene responsible for a particular protein with the property to arrest protease activity. The pesticides and weedicides used to destroy the pests and weeds is also a threat to the cultivated plants. The effects of such chemicals are alleviated by developing a new character called resistance to chemicals in plants. Development of resistance in plants against the weedicide glyphosate states the role of genetic engineering in plant breeding.

Uses Transgenic animals: Transgenic animals are animals carrying foreign genes deliberately introduced into them and exhibiting the characteristics of the introduced gene. Animals are suitable for various research activities trying to help mankind. In that way transgenic animals are created to study human diseases to derive appropriate treatment methods and to develop and identify the drug useful to treat the disease. The presence of human proteins in milk of animals is made possible by genetic engineering. Gene transfer is done in animals to increase the milk production and to increase the growth.

Like a coin has two sides, the other face of genetic engineering like creation of genetically modified organisms to be used as biological weapons is not welcoming.

Genotoxicity, a branch of toxicology is developed to identify the elements or compounds present in the environment having the potential to cause mutation by damaging the DNA. These compounds are also classified under the group of carcinogens, because of their cancer causing property. The necessity to identify the toxic compounds causing mutation is important in various industries like pharmaceutical, agriculture and food as the end user of the products from these industries are humans. As a result various methods were developed to detect and assess the toxic elements. The conventional method of using laboratory animals like mice and rat as test subjects is replaced by newly developed in vitro methods using microorganisms (bacteria) and animal cells. Few of such mostly used testing methods include Ames test, cell line tests and cytogenetic or chromosomal test.

Ames Test: Ames test employs bacteria in detecting the mutagenecity of the test compound. The mostly used species is mutant of Salmonella typhimurium, the ability to produce histidine of this organism is altered by mutating histidine operon gene present in this bacterium. The mutant organism is plated on an agar plate prepared with small amount of added histidine and the test element is placed at the centre of the plate using a filter disc. The toxicity of the compound is assessed by the growth of the bacteria. Initially, the bacterium grows till the presence of added histidine. Later, only those organisms whose mutation is reversed by the test compound, grows by producing histidine. This is qualitative test. The amount of compound required to cause mutation is quantified with the help of dose-response curve.

Later two types of mutations like single base substitution and frame shift mutation were adopted to create mutant organisms with the scope of studying more number of suspected toxic compounds. The sensitivity of the test is enhanced by altering the permeable nature of the bacteria and their cell repair mechanism. The use of bacteria as the test organism to study the mutagenic elements or compounds that are threat to humans has its own limitations. For example, once the compound enters the body, its toxicity is established only after the action of certain enzymes produced in the human body. To overcome this problem, extracts of liver with active enzymes were added along with the test element.

Use of Cell lines: The fact that the testing of Genotoxicity of an element on mammals is more beneficial rather than the use of bacteria, led to the use of mammalian cell lines in vitro. The cell lines derived from the mouse lymphocyte is used and the thymidine kinase heterozygote test is considered as the popular method of toxicity testing. The mouse cell lines are selected based on the mutation occurred on the thymidine kinase gene locus. The mutants are derived by treating the cells with toxic copies of thymidine. The selected cells are grown in the cell culture media by exposing them to the compound under study.

Chromosome test or cytogenetic test: This method involves the use of cell lines. The mutagenic property of the test compound is detected by observing the cells for chromosomal damage. Earlier, the mutagenic property of the test element is assessed by calculating the chromosomal damages like chromosomal breakages, chromosomal exchange, formation of ring chromosome, chromosomal dicentric and chromosomal translocation. Later, due to the difficulty in assessing the toxicity by this method, enumeration of sister chromatid exchange is done to detect the element for its mutagenic property. The increase in the frequency of sister chromatid exchange on exposing to mutagens excited the researchers and they preferred this method than counting the chromosomal aberrations. The sister chromatid are stained to study the type of exchange taken place using the fluorescence Giemsa staining method. In this method the cells are labeled with Bromodeoxyuridine, a thymidine analogue by growing them in the solution containing Bromodeoxyuridine. The staining is done by initial exposure of the chromosomes to fluorochrome, irradiation using ultra violet light and then staining with the Giemsa stain.

The Ames method is less time consuming where the results are obtained within 48 hours whereas the test methods using cell lines to detect the Genotoxicity of a compound takes about 21 days to get the result. This is because of the slow growth rate of the cell lines compared to that of the bacteria. Also methods using cell lines should adopt sophisticated techniques to maintain the cell line.

Bacteria living on teeth convert sugar into lactic acid which erodes enamel & causes decay. Florida-based company on biopharma has engineered a new bacterial strain called smart that can’t produce lactic acid. But it releases ankills the natural decay causing strain.

In a bid to fight tooth decay, researchers have come up with new weapon in the form of a bacterium that produces an enzyme which inhibits the formation of plaque.

There are as many as 500 different species of bacteria that inhabit our mouth and can colonize on your teeth and gums. When we have a meal, these bacteria forms layers called biotims on the teeth which helps to convert sugars sugars like sucrose, fructose and glucose.left on your teeth and gums to acids. This process leads to production of lactic acid which breaks down tooth enamel and leads to cavities. Streptococcus mutans and Lactobacillus are found to be the most cariogenic (promotes tooth decay) of these bacteria.

There are other beneficial bacteria like Streptococcus salivarius that is found on the tongue and soft tissues of the mouth and it is known to fight the biofilms built up by Streptococcus mutans.

A researcher in this study, accomplished the task of turning cavity causing bacteria into cavity fighting bacteria by stripping the bacterium of its ability to produce lactic acid. It is this byproduct of the breakdown of sugar by Streptococcus mutans that causes tooth decay. If the bacteria are not able to produce lactic acid tooth decay is stopped.

The genetically altered strain of Streptococcus mutans appeared to thrive on sugar. Researchers found that the strain was able to stay on the surface of the teeth indefinitely and prevented the natural strain from colonizing on the teeth. The altered strain is genetically stable and no ill effects have been noted.

Dentists will only need to swab smart now in clinical trials onto tooth once to keep them healthy for a lifetime. The bacteria convert the glucose, fructose & sucrose into lactic acid through a glycolytic process.

Other researchers in Tokyo discovered that an off-the-shelf FruA acquired from fungus Aspergillus niger also has the similar qualities despite the fact that it has a different amino acid sequence to the one found in the mouth. They used chromatography technique to isolate proteins from S. salivarius and these are used in to find out what was responsible for its cavity fighting powers. Cultures were prepared and mixed with S. mutans cells. The culture with protein FruA had the smallest biofilm and this was evidence that it was the most powerful biofilm blocker.

According to a report in Applied and Environmental Microbiology the researchers found that when there is increased sucrose concentration in the mixtures containing S. salivarius FruA and S. mutans their ability to prevent biofilm formation is decreased.


Another research in UK suggests that using microbes to fight against microbes or more precisely an enzyme from bacteria found on the surface of seaweed. Lab tests have shown that the enzyme is effective in fighting plaque.

An enzyme isolated from Bacillus licheniformis was identified during a screening for compounds that could disperse microbes from the surfaces of ship hulls. When under threat, these bacteria create a slimy protective biofilm barrier of extracellular DNA that joins them together while also sticking to a solid surface. This sticky matrix offers the microbes some protection from brushing, chemical washes or even antibiotics.
These researchers discovered that the enzyme could break down the external DNA, weakening and breaking up the biofilm layer so that the bacteria could no longer find a foot-hold and so get evicted. Initial experiments in the lab have shown promise in demonstrating that the enzyme has the ability to cut through plaque but more tests are scheduled to prove the discovery is both effective and safe.

The components of these discoveries may be used as ingredients in a paste, mouthwash or denture cleaning product. Although it could take a few years before anything appears on the shelves of local pharmacies the scientists say that these could also be useful for keeping certain medical implants clean. But researchers warn that this does not mean that you can get rid of your tooth brush. Brushing and other forms of dental hygiene would still be recommended to prevent plaque build-up.