MicroBiology

Have you ever wondered that how life continues in human beings? How do we all human beings share the same characteristic features? Yes, DNA is the answer to all your questions. The most significant mechanism for all the life cells that plays an important role in producing off springs is the DNA replication. DNA replication is the process that duplicates the DNA of a cell.

Each cell in our body consists of one or more than one DNA polymer molecules. These molecules need to be duplicated, so that the process of cell duplication takes place. This is what we call as DNA replication. In living organisms, the formation of DNA takes place in two strands. Each strand contains units of nucleotides. These two strands of DNA appear like two chains forming the DNA Double Helix. The process of DNA replication opens up the Double Helix and separates them into two strands. These two strands are then copied, and the result is that two new molecules of DNA are formed.

Steps involved in the replication of DNA

The first step in the DNA replication process is the breaking of the hydrogen bonds. This is the starting point where the two strands are unwounded. The enzyme that brings about the splitting between the two strands is Helicase and the structure thus created is referred as “Replication Fork”. The next step in the process is binding the RNA primase. The RNA nucleotides are attracted by the RNA Primase that gets bound to the DNA nucleotides because of the presence of hydrogen bonds between the bases. The RNA Primers are very vital for the DNA polymerase for binding the nucleotides, and the result is that the daughter strand is elongated because of this.

The DNA polymerase in the outer strand reads the fragments and thus, the RNA Primers are detached from there. The action of DNA polymerase closes the gaps formed by adding complimentary nucleotides, whereas the DNA Ligase closes the gaps by adding phosphates. Finally, the last step of the process is the termination. This process takes place only after the DNA polymerase reaches towards the end of the strands. DNA replication process is completed only after a repair mechanism fixes all the errors taken place during the replication process. There are enzymes such as DNA polymerase that fills up the gaps and nucleases that eliminates the improper nucleotides.

The speed of DNA replication in human beings is approximately 50 nucleotides/ second/ replication fork. It is relatively low when compared with the speed of DNA replication in bacteria’s. Nevertheless, human genome can be copied in just a few hours as a number of replication forks occur at the same time. This is also known as multiple initiation sites.

DNA replication is also performed artificially with the use of same enzymes that are used within the cell. Artificial DNA Primers and DNA Polymerases are used for initiating the synthesis of DNA at known sequences in a molecule of a template. Common laboratory method and polymerase chain reactions makes use of artificial synthesis rapidly in a cyclic form and particularly intensify the targeted fragment of DNA from the DNA pool.

RNA is one of the nucleic acid. RNA stands for Ribose nucleic acid. RNA consists of long chains of nucleotide units. The nucleotides consist of the nitrogenous base, phosphate and ribose sugar. DNA and RNA are almost the same only there are some structural differences between the both. The structure of the DNA is usually double stranded while on the other hand the structure of the RNA is single stranded.

The nucleotides of DNA contain deoxyribose while that of the RNA contains the ribose only. Both the DNA has thymine as a base while RNA has uracil as the base. The enzyme called the RNA polymerases transcribes from the DNA. Later it gets processed over by some other enzymes as well. RNA is also known as the “main center for the protein synthesis”.

Structure of RNA

Each nucleotide present in the RNA comprises of a ribose sugar with carbons (the carbons are number between 1’ to 5’). Adenine, Cytosine, Guanine, Uracil are attached to the base to the 1’ position. Cytosine and Uracil fall under pyrimidines while the adenine and guanine fall under the purines. At the 3’ position of the ribose and 5’ position a phosphate group is attached. Due to the negative charge carried by the phosphate group, the RNA also becomes charged molecule.

There is a possibility of hydrogen bond formation between the guanine and cytosine or between guanine and uracil by the bases. Many other interactions also take place, interactions which involve group of adenine base binding with each other.

RNA has a hydroxyl group present at the 2’ position of ribose sugar which is one of the most vital features for distinguishing RNA from DNA. A-form geometry is adopted by the helix rather than B form due to the presence of the functional group. Due to this the major deep and narrow groove and wide shallow groove is formed.

Though originally RNA is transcribed of only 4 bases some other sugars and bases also mature in the RNA. A C-N bond to a C-C bond is changed by the Pseudouridine. Also, at number of places ribothymidine is found. Other such type of the modified base is the hypoxanthine whose nucleoside is referred as inosine. Inosine is very important as it plays the main role in wooble hypothesis of genetic code.

The DNA molecule in any living organism is generally comprised of 2 strands; one is the sense strand and the other being the antisense strand. In a double DNA strand, one of the strand codes for RNA, which is later on translated in to proteins. This particular strand of DNA is known as Antisense DNA strand. On the other hand, the strand that does not take part in the coding of the RNA is referred to as the sense strand. The antisense DNA is also called as the non-coding DNA. The antisense DNA strand is solely responsible for carrying the information required to make the proteins. It performs this function by getting bound to the corresponding mRNA (messenger RNA). These strands are, no doubt the exact replica of one another, but even then, only the antisense DNA strand has the information to make the proteins. The sense strand does not feature this functionality.

Antisense DNA is a technology that down regulates or restrains the production of a targeted protein by either using the antisense DNA molecules or the antisense RNA molecules. The antisense sequence is complimentary to the targeted nucleotide sequence existing in the cells. The method that depends on the targeted mRNA is referred to as the antisense strategy. This antisense strategy uses the capability of the hundred percent complimentary RNA or DNA sequence for hybridizing or interlocking with the targeted mRNA. This thus inhibits the translation of the targeted protein.

A single strand of the DNA molecule gets bounded to the complimentary base sequence in a specific mRNA molecule and this thus prevents the synthesis of a protein that is encoded by the mRNA. The antisense DNA has the capacity of blocking the expression of a specific gene, hence it can be used a therapeutic weapon to fight certain diseases. These antisense molecules interrelate with the complimentary strands of the nucleic acids and thus, play an important role in altering the expression of the genes.

This antisense DNA method is being used successfully for blocking the expression of a particular gene in vivo in the central nervous system. Studies in the last few years have shown that when the antisense DNA is directly administered in the brain, it can modify different types of behaviors. Thus, it is said that the antisense DNA method can be used as an influential tool for the study of casual relationships among the molecular processes in the brain as well as its effect on the behavior of a person.

DNA strand, being double stranded, the strand that is complimentary to the antisense sequence is referred to as the non-transcribed strand and contains the similar sense sequence alike the mRNA transcript. Several forms of the antisense are developed and classified broadly in to the enzyme-based antisense. These antisense nucleic acid molecules are used in experiments to get them bound to the mRNA and thus, prevent the expression of particular genes. The antisense therapies are developing rapidly in the United States. The FDA (Food and DRUG Administration) has given approval to the Vitravene and the phosphorothioate antisense Oligo for remedial use in human beings. 

MiRNA stands for micro RNA and it is abbreviated as miRNA. The miRNA Labeling and detection kit is a unique and a new method developed to label as well as detect the mature miRNA and the small miRNA’s from the entire splinted ligation RNA. This kit uses a proprietary labeling technique that offers exceptional sensitivity to detect the endogenous miRNA. You can now label any sample of RNA such as amplified sense RNA, enriched low molecular weight RNA, degraded RNA and total RNA with the help of this method.

The miRNA Labeling Kit is designed especially in order to enable the labeling of the RNA’s and its molecules for the purpose of microarray analysis with the help of a very simple and a vigorous labeling technique. In this strategy, the RNA molecules are labeled directly, thus offering you a highly selective and a labeling technique for plants, human beings and animals that requires no enrichment at all.

The miRNA Labeling Kit contains the following components:

Clean up columns, positive control, OptiKinase, detection Oligo, clean up mix, 10X OptiKinase Reaction Buffer, Gel Loading Dye, RNase-Free water, a Protocol Card and a Long Protocol Booklet.

Using this technique, one can achieve specificity upto 95%. The labeling method is uniform and extracts reproducible outcome with CV of less than 15% between the demonstrations and less than three logs of active range on most of the scanners like Agilent, Axon, etc.

Advantages of using the miRNA Labeling Kits:

The process is very fast. The direct chemical labeling and capturing of the miRNA is complete in just two hours. Thus, it saves valuable time and the chances of experimental error are at its least. The add-and-incubate reactions take place very simply.The kit can be used flexibly to detect a number of small RNA’s. The linear detection range can be measured quantitatively. It is between the ranges of 0.2 to 20 femtomoles. The miRNA can be detected in as less as 50 ng or even less of the total RNA. Thus, we can say that using the miRNA labeling kit to measure the RNA is a very sensitive specific, fast, flexible and a convenient method.

The miRNA Labeling kit is a four-step process that helps you to label the miRNA directly from the nanogram to microgram quantities. The first step takes about 40 minutes wherein the detection of Oligo preparation takes place. Here the unincorporated nucleotide is taken out with the Clean up column. The second step is capturing the micro RNA that takes about 15 minutes.

MiRNA Ligation is the next step in the process that is completed within 75 minutes. The captured miRNA is ligated and the non-ligated detection Oligo is removed using the Clean up Mix. And the last step is detecting the RNA that required the maximum amount of time for its completion, is time period ranges from three hours to overnight. In this step, the ligated miRNA is separated on a polyacrylamide gel. You can observe this using a phosporimaging system or an X-ray film.

Mitochondria are a substance that is present in all our cells in the body. They perform one of the important functions of producing energy for our day-to day activities. Mitochondrial DNA testing is a test that determines your maternal ancestry with the help of the mitochondrial DNA. Mitochondrial DNA is abbreviated as mtDNA.

The mtDNA is present in the mitochondria that are the regarded as the powerhouse of the cells of the body. This DNA is passed to a child from the mother. Thus, it is very helpful to map out the maternal lineage of an individual. However, the mtDNA is inherited by both a girl and a boy from their mothers, but only girls can pass this mtDNA to their offspring’s.

In mitochondrial DNA testing, the mtDNA of the individual is sequenced. Then these sequences are compared in order to trace out that the individuals share the similar maternal line or not. There will be predictable similarities between the mtDNA sequences of the maternally linked individuals.

Now, you may come across a question that who can be tested for the mtDNA testing? Basically, this test demands the participation of more than one person who are interested to know whether they are related biologically to their mothers. The participants can be both, males and females. The fee for this teat is calculated on the participation of per person, so there is no bar regarding the number of persons participating in the test.

Mitochondrial DNA testing process:

The DNA samples of the participating individuals are collected using a pain free method called buccal swab method. These buccal swabs resemble the cotton wool buds. At the time of sample collection, four buccal swabs are wiped against the individuals cheeks internally. Tow buccal swabs are used on both the cheeks. The rubbing movement collects the loose cells of the cheeks and the DNA needed for genetic testing is present in these cells. In this way, the DNA sample is collected from the participants.

Results of mtDNA testing:

mitochondrial DNA testing will possibly yield two results: that the participants are related to one another maternally and that they are not related to one another maternally. The time required for the test results to come out is about three to four weeks.

The mitochondrial DNA testing is used to: Find out whether you are linked with others having the same surname. It is used to verify if there is some relation between two people. It determines whether the two persons descend from the similar ancestors.  It offers clues or hints regarding your ethnic origin. It can be used successively to prove to disprove the research of your family tree.

The mitochondrial DNA testing has been carried out from many years ago. However, it is only recently that its cost has come variably down to the realm of possibility of an average individual, as well. There are even home DNA testing kits available. You can order them, send your DNA sample cells from the inside of your mouth and you will receive your results within a month.

RNA
RNA stands for ribonucleic acid. It is a nucleic acid that comprises of a big chain of the nucleotide units. Each nucleotide has a ribose sugar, a phosphate and a nitrogenous base. Ribonucleic acid (RNA) is similar to the deoxyribonucleic acid (DNA) to a great extent. The only difference lies in some of the structural details. DNA is double stranded and RNA is single stranded; DNA nucleotides contain deoxyribose and RNA contains only ribose; DNA has the base thymine instead of uracil as present in RNA.

There are generally three types of RNA. These three types include Messenger RNA (mRNA), Ribosomal RNA (rRNA) and Transfer RNA (tRNA). Now let us have a brief overview on each of these types of RNA.

Messenger RNA- mRNA is the replica of the data that is being transmitted on DNA by the genes. The function of this mRNA is to carry the data that is present in the DNA to the translation mechanisms. The mRNA is usually synthesized from the DNA’s gene segment. This RNA has all the data of the amino acids in proteins that are to be synthesized on the primary sequence. It transmits the code in the cytoplasm where the synthesis of proteins takes place. The messenger RNA is heterogeneous in sequence as well as structure. Due to this diversity in size and sequence, there is not a specific structure of mRNA determined. It always has a five-inch cap containing the triphosphate linkages. This cap is an identification that the RNA molecule present in the structure is the mRNA.

Ribosomal RNA- the rRNA is a constituent of ribosome’s that are the regarded as the protein synthesis factories in the cells. The rRNA molecules are found profusely in the cell. 80% of the RNA molecules found in the cells are comprised of the rRNA. The rRNA plays different roles in the process of protein synthesis. Firstly, it plays a catalytic role by forming a part of peptidyl transferrase activity. Then, it plays the recognition role by getting occupied in the exact positioning of the tRNA and the mRNA. And finally, it plays the structural role by getting folded into 3-D shapes and forming a gibbet, wherein the assembly of the ribosomal protein takes place.

Transfer RNA- the tRNA is referred as the data adapter molecule. It bridges the gap directly between the DNA information and the sequence of amino acids in proteins. Thus, it can interpret the information contained in the DNA. There are more than twenty various types of tRNA molecules present in the cells all between the range of 75 - 95 NT. The tRNA’s in all the organisms features a similar structure and orientation. It has three loops and four arms and sometimes, it has an extra loop. The synthesis of the tRNA takes place in two parts. The tRNA body is copied from the tRNA gene. All the tRNA molecules share the same type of acceptor stems and this is added after the synthesis of the body. This is generally replaced at the lifetime of the tRNA molecule.

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MicroRNA is also abbreviated as miRNA. It is a single strand of RNA molecules that is upto 21-23 nucleotides in length. It is responsible for regulating the expression of genes. The genes transcribed from DNA encode the miRNA. The mature miRNA molecules are complimentary to some of the messenger RNA to some extent. They perform the important function of down regulating the expression of genes.

MiRNA is attached to a small piece of mRNA that is the master model to build the proteins in a non-coding pattern at one end of the molecules. These micro RNA’s are very small molecules that are coded in the genomes of animals and plants. They are highly conserved and play the role of regulating the gene expression by connecting the untranslated areas of particular mRNA’s.

Generally, the miRNA’s are transliterated as a part of the long RNA molecule. About 60% of this micro RNA’s are expressed independently; whereas the 15% of it in clusters and remaining 25% are expressed in introns. The mammalian genomes can encode about 200-500 miRNA’s that can together control the expression of 1/3^rd of all the genes.

The micro RNAs are produced and they function in the tissues, cells and organisms. They regulate multiple genes and are present in the higher Eukaryotes. Thus, the miRNA has adequate potential regulatory circuitry. They are the key regulators of the processes including cell death, cell proliferation, cell differentiation, fat metabolism, and apoptosis. The recent studies reveal that the expression of the miRNA is associated with the brain development, viral infection, colonic adenocarcinoma chronic lymphocytic leukemia and Burkitt’s lymphoma.

MiRNA and Cancer- it has been found that there are some miRNA’s that have a link to some type of caner. The patterns of gene activity distinguishing the kind of cancer can be detected by measuring the activity between the 217 genes that encode the miRNA. The signatures of miRNA make it possible to classify the types of cancer. This helps the doctor in determining the original type of tissue causing cancer and thus, the treatment course can be targeted based on the original type of tissue. The profiling of the micro RNA has enabled in verifying the patients having chronic leukemia with slow or aggressive forms of cancer.

MiRNA and Heart Disease- miRNA plays an important role in the functioning of the heart and its role during its development is quite significant. Studies show that the level of expression of a particular miRNA alters in diseased human hearts. It has distinct roles to play at the time of the development of heart, and pathological functions include regulating the key factors necessary for cardiogenesis, cardiac conductance and hypertrophic growth response.

The miRNA’s are significant for the development of the organisms and are expressed differentially in the tissues. They are involved in the processes of viral infection and are also associated with oncogenesis. The activity of these miRNA’s can be blocked experimentally using a Morpholino Oligo, locked nucleic acid or methyl RNA Oligo. The steps in its maturation process can also be blocked using steric blocking oligos.

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DNA Sequencing is a process to determine the precise order of the billions of chemical blocks known as bases. These bases are adenine, thymine, cytosine and guanine and they are abbreviated as A, T, C, and G respectively. They constitute the DNA of the twenty-four different chromosomes present in the human beings. DNA Sequencing contains the heritable genetic data in mitochondria, nuclei, chloroplasts and plasmids. These are responsible to form the base of the developmental programs in all the living organisms.

Thus, determination of the DNA Sequencing is of great help in studying the fundamental biological processes in basic research and in the applied fields like forensic and diagnostic research.  DNA Sequencing has initiated significant acceleration in biological discovery and research. The advent of modern technologies has attained rapid sequencing speed that is influential in the sequencing the large-scale human genome in the Human Genome Project.

For sequencing a piece of DNA, you will require the following: a DNA template containing the DNA that you want to sequence, DNA polymerase enzyme, DNA primer complimentary to the DNA that is being sequenced and four nucleotides. The most common method used for DNA Sequencing is the chain termination method. In this method, modified bases known as dideoxy bases are used. Replication of the DNA piece and incorporation of a dideoxy base in the new chain ceases the replication reaction.

The DNA that is to be sequenced is taken in a single strand form. This single strand acts as template on which the synthesis of a new DNA strand will take place. A nucleotide is included in each reaction, which cannot be extended. This nucleotide acts as a chain terminator. Four reactions, all containing the similar primer and template are set up. The process of incorporation of a new DNA strand takes place randomly as only a small amount of chain terminator is contained in the reaction. Thus, a collection of fragments is generated in each reaction, but all the DNA strands will end up with the same bases (A, C, T or G).

Most of the DNA Sequencing is done by the chain termination method. In the chain termination, method the synthesis of a new DNA strand takes place on a single stranded template. This method generates a set of the DNA molecules that differ in length from one another only by one nucleotide. You can easily recognize the last base in each molecule depending on the size that positions them in an exact order in order to read off the sequence easily.

DNA Sequencing technology is used widely in sequencing the genes and genomes precisely. The more number of times the template is being sequenced, the more accuracy will be achieved. This single-pass and low-fidelity sequencing is of great help in accumulating the sequence information rapidly as well as accurately. Another application where DNA Sequencing is of great use is in resequencing the same type of DNA molecule repeatedly. This is essential in the typing of the single nucleotide polymorphisms.

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Microbial ID refers to microbial identification. It meets all of the identification needs to recognize the yeast, bacteria and fungi with the help of the necessary tools. More than 2500 species can be identified with Microbial ID. It provides polyphasic investigation exclusively of the fatty acids and the DNA results. These are combined in, as a single report that enables for higher confirmation as well as clarification if any required, of the ID’s.

The Microbial ID process utilizes carbon source metabolic fingerprints. This helps in analyzing the microbial communities, identifying about more than 1900 species of yeast, bacteria and fungi, and characterizing the microbes. The types of samples tested in Microbial ID include environmental, clinical, pharmaceutical, personal, water and food. The types of tests performed on the samples to be tested are bacillus, E.coli, Shigella, streptococcus, clostridia, listeria, enterobacter, vibrio, campylobacter and yeast and mold.

The best genotypic technique for Microbial ID is the comparative DNA sequencing analysis. The commonly used approach is the sequencing and amplification of all the 500 bp portion of the 16S rRNA gene. A comparison is made between the sequence database and the sequenced information. This contains only the validated sequences of the microbes, ideally.

Microbial Identification is widely used in areas like clinical diagnosis, plant pathology, bioremediation, animal science, dental research, medical microbiology, epidemiology, biopharmaceutical, biodefense, microbial forensics, marine science, entomology, soil science and taxonomy studies.

The most widely used system for Microbial ID is the Sherlock Microbial Identification system. This system is used throughout the world in environmental as well as clinical labs for identifying the anaerobic bacteria, aerobic bacteria and the species of yeast. The Sherlock technique is carried out on a gas chromatographic analysis of the cellular fatty acids methyl ester, also known as FAME. This method enables the entire identification process in a time period of about fifteen minutes from the pure culture.

The Sherlock Microbial ID process can classify up to six bacterial agents. There is additional add-on software known as Sherlock DNA along with the Sherlock Microbial ID system. This additional software can recognize about 2500 species of microbes via 28S and 16S ribosomal RNA gene sequencing. The results of the DNA sequencing when combined with the FAME results can identify 2750 species of microbes. There is an extra feature included in this software that enables the user to compare the samples of HPLC method visually to a reference chromatogram.

Key features of the Microbial ID systems:

The Microbial ID system is in use form a very long time from the year 1985and it is one of the widely accepted techniques to identify the aerobic bacteria. There are widespread libraries of the clinical and the environmental organisms. The identification of the different species can be completed in only fifteen minutes. This automated naming and analysis system is very easy to use. It does not require any biochemical cards, gram stains, or upfront tests. It is an extensively comprehensive tool for data analysis and strain tracking. The process of microbial identification is quite reliable.

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RNA stands for ribonucleic acid. There are different types of RNA like microbial RNA, transfer RNA, ribosomal RNA, small hair pin RNA. In this article let us focus on small hair pin RNA.

A small hairpin RNA (shRNA) is a RNA sequence. It resembles a hair pin and hence the name small hairpin RNA is derived. This sequence of RNA creates a tight hair pin turn, which is used to silence the expressions of genes through the RNA interface. The small hairpin RNA utilizes a vector that is being introduced in a cells and it uses U6 promoter in order to ensure that the small hairpin RNA is expressed always. This vector is generally passed to the daughter cells that allow the inheritation of gene silencing.

The cellular machinery chops the structure of the short hairpin RNA into siRNA. The RISC (RNA induced silencing complex) then brings about the binding of this structure. This complex structure gets bound to the microbial RNAs and cleaves it that matches the siRNA bound to it.

RNA polymerase III brings about the transcription of the short hair pin RNA. The production of short hair pin RNA may sometimes lead to an interferon response in a mammalian cell. This happens because these cells try to defend themselves from what is perceived as viral attack. This does not happen in microbial RNA because the transcription of microbial RNA is carried out by RNA polymerase III.

The small hairpin RNAs are used in plants and other systems, basically those which are not drived by the U6 promoter. In most of the plant species, cauliflower mosaic virus35s promoter is the traditional promoter for a constitutive and strong expression. In such cases RNA polymerase II is utilized for the expression of the transcribed destined in order to initiate RNAi. The other applications of short hair pin RNA consist of developing the cell lines with the loss of function phenotypes.

The structural requirements of short hairpin RNA - 2 different systems were used by Paddison and his Colleagues for accessing the structural requirements of the short pin RNA. In the first system, sea pansy luciferase expression plasmids, firefly luciferase and chemically synthesized short hairpin RNAs were added to the embryo lysapes of Drosophila. The silencing of the short pin RNA is later measured based on the amount of the reduction activity in the firefly luciferase after the sea pansy luciferase levels become normal.

In the second system the similar type of dual luciferase technique was used for measuring the silencing in mamilian cell lines. But, in this approach the short hairpin RNAs were synthesized chemically in vitro transcription or expressed by polymerase III expression plasmids. The small hairpin RNAs are symmetrical bilaterally and their orientation of insertion in polymerase III expression vendor is very significant for their activity.

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RNAi Analysis refers to RNA interference analysis. It is the study as well as utilization of the small interfering RNAs that play a vital role in silencing the expression of genes, in the order of one gene at a time. The RNAi Analysis is very useful in describing the network of the gene expression pathways. It also aids in validation of the drug target and thus, results in the invention of new medicines.

The RNAi Analysis is functional to the genomic studies, as well. Researchers have now began applying the RNAi for scanning all the genomes. This helps in determining the network of phenotypic functions as well as the developmental pathways. The technology of the RNAi is a straightforward technology, on which you can rely completely for accurate and precise results. The primary aim of this technique is to selectively silence a particular target mRNAs.

RNAi analysis is sometimes also termed as Reverse Transfection. Reverse transfection is a process wherein the mammalian cells are transfected with the help of the spotted arrays of the DNA plasmid. Thus, this permits the analysis of transfections in parallel, in large numbers. The vectors to be expressed are displayed with a matrix containing the lipid transfection reagents on a microarray plate, slide or dish with the Genetix Array System. This array is later on developed in the adherent cell culture in such a way that the monolayer of the cells grows over the surface of the slide and the cell patches are transfected. The genes to be transfected are frequently cloned as fusions to the fluorescent reporter proteins. This can be used to observe the expression of genes after the transfection process is complete.

One of the applications where the reverse transfection process is specifically well adapted to the microarray formatting is the RNAi (RNA Interface). Here, the transcription of a particular gene is silenced by the reaction of the siRNA (short interfering RNA) molecules. You can measure the efficacy of the RNAi construction by scanning the slide through the confocal scanner along with the blue laser.

The RNA interface is considered as one of the best methods for effectively knocking down the expression of genes in order to study the functioning of proteins in all the types of cells precisely. The traditional methods for knocking down the genes in a mammalian cell comprises of using the synthetic RNA duplexes. This contains two unmodified twenty-one meroligonucleotides linked together for forming either the small interfering RNAs or short interfering RNAs (shRNA). Nowadays, these traditional duplexes are improved with the use of proprietary chemical alterations for ensuring better RNA interface results.

The RNAi analysis technology is used for functionally assess as well as identify number of genes present in the genome that participates actively in diseased phenotypes. Additionally, this technique offers you effective ways to block the expression of a particular gene and then evaluate its reaction to the chemical compounds or any alterations in the signal pathways. The RNAi is the significant technological advance in the modern age that enables one to visualize directly the effects of loss of functioning of a particular gene in the mammalian cell.

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Oomycetes is also referred as water molds. They are a group of unicellular and filamentous Heterokonts that physically resemble fungi. They are the absorptive and microscopic organisms composed of mycelia. Mycelia are tube like vegetative body and they are also known by the term thalamus. The reproduction of Oomycetes takes place either sexually or asexually.

Oomycetes are also referred as water molds due to the fact that they were earlier classified as fungi. And fungi exist from conditions like high humidity and running water surface. Water molds have a relation to the organisms like diatoms and brown algae. Water molds are very significant scientifically as well as economically as they are the aggressive plant pathogens.

There are about five hundred species of Oomycetes that includes water molds and downy mildews. The absorption of food of these filamentous protists takes place from the adjoining soil or water or they may attack another organisms’ body to feed themselves. These Oomycetes play a very crucial role in the process of recycling and decomposition of the decaying matter.

Oomycota refers to the egg fungi and it is a structure that contains the female gametes. The Oomycetes are oogamous that produces non-mobile and big gametes known as eggs and the smaller gametes are known as sperms. The fossil record of the Oomycota is very sparse. Oomycetes that comprises of a big group of aquatic and terrestrial organisms resemble superficially to the fungi in terms of growth and mode of nutrition. But, discrete morphological features and molecular studies position them under the Stramenopila kingdom having golden and brown algae along with diatoms.

The terrestrial Oomycetes are basically the scroungers of the vascular plants and they contain different types of very imperative plant pathogens. For instance, Aphanomyces results in a root rot of a number of hosts that includes snap bean, pea, and sugar beet.

Oomycetes refers to a large collection of some significant species, including both the parasites and the saprophytes of plants, insects and animals. However, they lack the taxonomic similarity with what we refer to as fungi. But, instead they are categorized along with the brown algae and diatoms in a group named as Stramenopiles. The largest group of these Stramenopiles comprises of the Oomycetes that are found throughout the world in salt and fresh water habitats.

Oomycetes are not only plant pathogens, but studies have revealed that they are animal pathogens, as well. There are instances that show that the Oomycetes are pathogenic on species other than the pants. These are the Pythium insidiosum; this is a species infecting animals and results in swamp cancer. Then there is Lagenidium giganteum that parasitizes the larval stage of a mosquito and are utilized as a probable control agent. And lastly, there is Saproilegnia that causes infection in different species of fish.

There are some terrestrial Oomycetes that are considered as significant plant pathogenic organisms. These organisms are found in stagnant water as well as well aerated streams and they get their energy through anaerobic respiration. These members are filamentous and they lack septa with the only exception where the production of the reproductive cells takes place.

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There are many types of viruses, you must have heard about. A virus is responsible to cause diseased conditions in living organisms. Among the different types of virus, is the RNA Virus. A RNA Virus can be defined as a virus that has the ribonucleic acid (RNA) as its genetic material. This nucleic acid is often single stranded RNA, but there are possibilities of it occurring in double strands, too. The notable diseases caused by the RNA Virus in human beings include influenza, SARS (Severe Acute Respiratory Syndrome) and hepatitis C.

There are in all six classes of viruses present. Out of them, the DNA virus comprises of the first two classes and the RNA virus makes up the remaining classes. The class III RNA virus features a double stranded RNA genome, class IV ha a single stranded RNA genome that acts as a messenger RNA. The class V has a single stranded RNA genome that is being utilized as a template for the synthesis of the messenger RNA. The class VI viruses are also single stranded genomes of RNA, along with the DNA intermediate in mRNA synthesis as well as replication.

The structure of a RNA Virus is usually similar to that of the other viruses that comprises of a nucleus of the genetic material, basically enclosed in a protective capsid of protein as well as a lipid envelope, in many cases. The life cycle of a RNA Virus depends upon reproduction of the genetic material, host cell, penetration, emergence from the cell, and creation of a protective capsid. The only difference lies in the fact that the genetic data of the RNA Viruses are stored. This has crucial significance and plays a vital role in the life cycle of the virus and gives it the ability to outwit your immune system in the body.

RNA is ideal to store the viral data as it acts as a temporary messenger molecule. Your immune system learns to identify the infecting viruses and thus, they create antibodies against it to destroy them rapidly. Thus, the viruses will no longer be able to utilize that host for reproduction. The RNA molecules are unstable in nature and they have mutagenic factor that enables the RNA virus to progress very rapidly as compared to the DNA virus. The RNA viruses can change their surface structures very often and thus, this mutation makes it very difficult for any organism to build up lasting immunity to that virus.

There are two types of RNA Viruses: one having a sense strand of RNA as their genetic material, they have coded data regarding how to build proteins. And the other type is the antisense strand, this is the paired opposite of the coded data. The only difference between the sense RNA and antisense RNA is the origin of the RNA transcriptase, meaning from where it comes from.

The RNA virus is responsible to cause many infections in human beings. This includes colds, AIDS, rabies, influenza bird flu and HIV.

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A Dermatophyte is defined as a parasitic fungus that is responsible for causing the infections in your skin. It is not a specific fungus, but only a short hand label for a class of three genera of fungi that often cause diseases of the skin in animals and human beings. These asexual genera are Microsporum, Epidermophyton and Trichophyton. There are approximately forty species in these three classes of genera.

Dermatophytes results in the hair infections, skin infections and nails infections as they are able to absorb the nutrients from the keratinized material. The keratin tissue is inhabited by the organisms and this result in inflammation by the host response to the metabolic by-products. Dermatophyte are often restricted to the cornified, non-living layer of the epidermis as they are unable to go through the viable tissues of the host. This invasion of the Dermatophytes educes a host response that ranges from mild to severe. Dermatophytes do not attack the living tissues usually, but they settle on the outer layer of the skin.

Classification of Dermatophyte- Dermatophyte is categorized into three classes based on their natural habitat: Anthrophilic- humans, Zoophilic-animals and Geophilic- soil. The Anthrophilic Dermatophytes limited to the human hosts only and they result in a mild and chronic infection in human beings. The Zoophilic organisms have their hosts, basically in animals. They are responsible in producing remarkable inflammatory reactions in the human beings having contact with the infected dogs, cats, birds, cattle’s, horses or any other animals. The Geophilic species are generally recovered from soil, but they cause infections in animals and human beings very often. They result in remarkable inflammatory reactions in humans and animals that limit spreading of the infection. It may result in impulsive cure, but scars of the reactions will be left behind.

Dermatophytes Transmission- The transmission of the Dermatophyte takes place either by the direct contact with the infected host that may be an animal or a human being, or by direct or indirect contact with infected caps, skin, hairs in clothing, hair brushes, comb, theatre seats, towels, rugs, furniture, locker room floorings and bed linens. It is workable in a particular environment for a period of about fifteen months, depending on the organism’s species. Susceptibility to infections is higher in cases of an already existing skin injury like burns, scars, humidity and excessive temperature.

The infections caused by the Dermatophytes can be diagnosed readily depending on the physical examination, history and KOH i.e. Potassium Hydroxide Microscopy. The diagnosis usually needs historologic examination, Wood’s Lamp examination and fungal culture. Dermatophytic infections are mostly treated by using the Topical Therapy. The courses of treatment are short; whereas the rates of cure are high with topical fungicidal allylamines rather than the fungistatic azoles.

Dermatophytes, a group of fungi are capable to cause infections and survive only on the dead keratin, which is found on the topmost layer of the hair, skin and nails. They are unable to live on the moist skin that is found inside the vagina or the mouth. Thus, they are liable in causing a number of hair, skin and nail infections in living organisms.

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Escherichia coli is abbreviated, as E. coli is a diverse and a large group of bacteria. It is generally found in the lower intestine of the warm-blooded animals. The E. coli strains are harmless normally, but there are some strains like the serotype O157 are responsible in causing food poisoning in human beings. The harmless strains are beneficial to their hosts as they produce vitamin K2 and also prevents the development of pathogenic bacteria inside the intestine of the organisms.

The E. coli bacteria is one of the leading causes of bloody diarrhea and severe cramps in the body. Symptoms of E. coli infections are worst in children and elderly people, and particularly in those people who are already suffering form some other illness. The E. coli infections are prevalent normally during the months of summer and the northern states.

Causes of E. coli infections- One can easily catch these E. coli infections. The E. coli infections generally come from drinking contaminated or impure water, unpasteurized or raw milk, working with cattle’s and consuming undercooked ground beef. The dairy cattle’s and healthy beef carry this E. coli bacterium in their intestines. The meat can get contaminated with the E. coli bacterium at the time of slaughtering process. The E. coli bacterium gets mixed in the entire meat when the beef is ground up. Thus, we can say that one of the common ways of getting this infection is by consuming contaminated food.

Symptoms of E. coli infection are visible after about seven days when you get infected with the germ. Its symptoms includes watery diarrhea and severe abdominal cramps. The body loses fluids and electrolytes in a large amount due to diarrhea. This diarrhea later on changes to red, bright bloody stools. You may also experience mild fever or no fever at all and vomiting. There are some complications that arise due to this infection. Hemolytic uremic syndrome is the most common complication. People with this problem suffer from renal failure- kidney damage, thrombocytopenia- low platelet count and hemolytic anemia- low red blood cell count.

Diagnosis of E. coli infections is done by finding the E. coli in a stool sample of the concerned person. It is recommended to see a doctor soon, if you have bloody diarrhea. There is no special treatment available as such, for the treatment of the E. coli infections. You just need to drink adequate water and observe the complications. Medicine to stop the diarrhea should not be taken, as it will keep the intestines away form getting rid of the E. coli bacterium. In case of serious dehydration, fluids are put in the veins through IVs. Generally, the E. coli infections get better in a period of five to ten days.

The E. coli infections are caused by consuming the foods containing bacteria. Hence, to prevent the arousal of these infections, it is suggested to handle the food safely. Wash the fruits and vegetables before eating or cooking them, cook the meat well and avoid raw juices and milk,

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It is studied by researchers that a genetic component is responsible for most of the diseases and conditions in human beings. Some disorders like the cystic fibrosis or the sickle cell anemia are a result of mutations in a single gene. However, the cause of many other diseased conditions is very complex. Some of the common disorders like diabetes, heart disease and obesity are not a result of a single genetic cause, but they are caused due to the result of multiple genes, in combination with the environmental factors as well as the lifestyle of the person. Multifactorial Disorder is caused by the interaction of genetic and non-genetic factors along with the environmental factors.

Multifactorial Disorders are frequently seen clustering in families, but they do no have a well-established pattern of inheritance. Thus, it becomes difficult to determine whether a person is at risk to inherit or pass on this disorder to the next generation. Multifactorial Disorders are very complex, hence difficult to treat as well as study as the particular factor responsible for most of these disorders are not yet identified.

There are many common diseases seen running in the family, but the simple inheritance pattern of a single gene disorder is seen lacking there. These Multifactorial Disorders includes diseases like epilepsy, manic depression, asthma, schizophrenia and hypertension. Some of the developmental abnormalities also come under this category like the cleft palate, neural tube defects like spina bifida and congenital heart defects.

The Multifactorial Disorders have a very low heritability in comparison with the single gene disorder. For instance: only 5-7% of your close relatives or family members of diabetics suffer from the same disease, and lower would be the case for single gene disorder such as cystic fibrosis. This clearly states that a single genetic factor is never responsible to cause diseased conditions in your body. There are a number of genes that combine along with the environmental factors like exposure to hazardous chemicals or poor diets, which cause disorders in a person.

The occurrence of the Multifactorial Disorders is thought to be dependent on the balance of risks. There exists a balance between the variants of genes with positive as well as negative effects, and between environmental factors again with positive as well as negative effects. Too many negative factors, both environmental and genetic may tilt the balance towards various disorders.

In a Multifactorial Disorder, sometimes the risk may depend on the sex of the affected person. For instance, pyloric stenosis is a Multifactorial Disorder occurring five times more often in males as compared to females. If a female suffers from pyloric stenosis, her as well as her parent’s risk of having other child affected with this condition will be higher than if a male child would be suffering with it. The occurrence of this condition in females has a higher genetic liability apparently, a number of abnormal genes are isolating in the family.

Thus, we can shortly state that there is a similar risk for the first-degree relatives such as parents, siblings or offspring’s to develop Multifactorial Disorder. The severity of the disorder and the sex of the person can play a role in modifying the risk. Some of the common chronic diseases are Multifactorial Disorders of the genes. The conditions with Multifactorial Disorder include cancer, birth defects, coronary artery diseases and mental disorders.

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Any permanent change in the structure of DNA sequence which makes up a gene is called as gene mutation. The size of the mutation varies, it may either as small as a DNA or even large as a segment of the chromosome.

The mutation of the Gene occurs in two ways: 1. from a parent 2. acquired during the person’s lifetime. Hereditary mutations or the germ line mutations are those which occur from the parents to the children. The mutation of this sort can be seen in the persons entire like in every cell in his/ her body.

The other are the new mutations which are occurred in the egg or the sperm cell or the mutation which occurs just after the fertilization process is complete. The genetic disorders can be best explained by the De novo mutations, however in this type of disorder there is no family history for this disorder involved.

The somatic (also known as acquired) mutations are the one which occur directly into the DNA of the individual cell. There is a possibility of DNA making copies of itself during cell division due to immense expose to the ultra violet radiations from the sun which results in number of changes in the individual’s life. The acquired mutations in the somatic cannot be passed to the next generations.

Mutations can also occur in the single cell in the early period of embryo. During the division of the cells the individual has some cells with the mutation and some other without any genetic changes. This situation is referred as mosaicism.

Some of the changes in the genetic makeup of the cells are very rare to find, while on the other hand some changes are easily spotted. The Genetic changes which can be found in more than 1% of the population are known as polymorphisms. Polymorphisms are responsible for the differences like the color of the hair, blood type and the color of the eye. Usually, polymorphisms don’t have any negative effect which affects the health of an individual; however they may also cause number of variations which can develop certain disorders in the individual.

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