MicroBiology

Deoxyribonucleic Acid (DNA) is present in the chromosomes of all the humans. DNA is

the vital biological material which helps to enable an individual’s identity. DNA plays a vital role in solving crime and also determines paternity. DNA is a type of nucleic acid which is made up of thousands and thousands of nucleotides and certain amino acids. The long strand of DNA can be built by the nucleotide.

Half of the DNA is received from our parents and the other half of it comes maternally. DNA can be extracted form number of sources like our hair, blood, saliva, razor clipping etc. DNA is tested in the laboratory by which we can understand our genetic relationships. The DNA contains the blueprints of life and it also helps in exercising control over the functions performed by the cells and helps to indicate the ancestry.

DNA is found in all the living organisms even in some of the viruses. It is found in the nucleus of the chromosomes. The repair and growth of the body can take place easily as DNA is present in every single cell of our body.

Importance of DNA

DNA is biologically essential for life. DNA is found in all the living organisms in every cell and it contains the storage of complex information about our nature, behavior etc. DNA helps to determine the factual circumstances so it is a very unique component of our body. For legal procedures also we can identify the matches between the DNA samples and understand link between the individuals. Most of the police and investigations agencies rely upon the DNA tests as a fact finding technique. DNA is one of the accurate techniques for positive identification of the living organisms.

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.

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. 

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.

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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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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Deoxyribonucleic acid, DNA, which makes up the genetic material in cells, is comprised of units called nucleotides.

Nucleotides can be simply described as the nutritional building blocks of new cells. There are five key nucleotides, which form the crucial building blocks of DNA and RNA, and are therefore essential for ongoing new cell production and system repair.

The body has an ongoing demand for new cell production, and must create cells at the same rate at which its cells die. To do this, a typical cell must double its mass and duplicate all of its contents in order to produce the two new ‘daughter’ cells.

This multiplication of a cell starts with the doubling of the information (inter-phase) - namely the DNA. Previously we discussed how the DNA is formed by the five key nucleotides. A normal DNA consists of 3 billion nucleotides.

It is only after this multiplication of the DNA that the M-phase can start. This is where the two cell nuclei are formed and the cells start to divide into two separate cells.

Cell proliferation is a lengthy and complicated process, dependent mainly on energy and supply of the specific building blocks, the five key nucleotides.

Research has shown that by providing extra dietary sources of nucleotides, the multiplication of these new cells can be speeded up. This applies to the following cell types:

Intestinal mucosa cells – the cells lining the gut

Bone marrow cells, namely leukocytes (especially Lymphocytes) – more commonly termed as the white blood immune cells

Erythrocytes – Red blood cells

Certain brain cells

For years, nucleic acids and nucleotides were considered essential nutrients. Now these nucleotides are increasingly considered to be limiting in certain diets and during periods of stress and illness.

It was thought that the body could synthesise sufficient nucleotides to meet its physiological demands via ‘de novo’ synthesis of nucleotides. However, research during the last several years indicates that this is not correct. There are certain conditions in which the body requires dietary nucleotides to meet its physiological requirements.

These conditions include:

Rapid growth

Limited food supply

Stress

Chronic disease

Bacterial and viral infections

In these situations, metabolic demand exceeds the capacity of the ‘de novo’ synthesis, and therefore dietary nucleotides become conditionally essential.

Importantly, dietary nucleotides may also spare the energetic costs of ‘de novo’ synthesis of nucleotides.

Critical point in nutrition: energy

Building nucleotides by the ‘de novo’ process requires lots of time and energy. Supplementing purified nucleotides to the diet reduces the proportions required to manufacture, while accelerating all active processes and saving energy also.

Supplementary nucleotides have been demonstrated to enhance the efficiency of a number of vital organs; these include:

Immune organs including the bone marrow

Liver

Intestines

Consequently, supplementary nucleotides have an effect on a number of vital functions:

Immunity

Production of stress hormones

Digestion and absorption of nutrients

Vitality and well-being

Focussing on Immunity

Nucleotides are an essential part of an healthy immune system, because they have been shown to support the following:

The reversal of malnutrition or starvation-induced immunosuppression

Enhancement of T-cell maturation and function

Enhancement of natural killer cell activity

Increase of delayed cutaneous hypersensitivity

Aiding resistance to infectious agents such as Staphylococcus aureus and Candida albicans

Modulation of T-Cell responses toward type 1 CD4 helper lymphocytes or TH1 cells.

In layman’s terms, nucleotides help to boost the immune system by speeding up the process of creating new defence cells, which are essential for the body to fight infections.

Reducing the effect of an infection

In 2003 a double-blind placebo controlled trial was carried out by Dr Isobel Davidson at Queen Margaret University College, Edinburgh. This clinical trial was carried out to test the effects of nucleotides on reducing the severity of specific symptoms, secondary infections and healing time after a natural infection by the cold or flu virus. The findings showed that the specific formula of nucleotides significantly reduced the symptoms of painful sinuses and earache. Over the first six days of taking the nucleotides or a placebo, the discomfort level was on average greatly reduced for the nucleotide patients:

Dry mouth: 30% less

Sore throat: 20% less

Muscle aches: 15% less

Headache: 40% less

Salivary IgA (antibody) concentrations were similar at recruitment, but the subsequent to this were higher in general for the nucleotide group. For the layman, higher antibody levels indicate a greater immune response.

Preserving a strong immunity

The positive effects of supplementary nucleotides on immunity was also backed up with another placebo controlled trial carried out by Professor Lars McNaughton, University of Bath, 2002. McNaughton tested the effects of nucleotides on athletes under both short-term and high intensity, and moderate endurance exercise. Again these results were significant and conclusive. Athletes on the nucleotide treatment showed a 25% higher IgA concentration after 90 minutes endurance exercise, compared with the placebo group.

The stress hormone, cortisol, was 10% reduced for the nucleotide supplemented group. High cortisol levels have been shown to impede the effectiveness of disease fighting cells. Hence, the placebo group showed a greater drop in antibodies. High decreases in IgA (antibodies) are thought to be a reason for increased levels of Upper Respiratory Tract Infection (e.g. colds and flu) for athletes and people suffering high levels of stress.

In conclusion, the body needs nucleotides to help support a healthy immune response. The body can normally take these from food, but in times of stress, illness, poor diet or rapid growth, dietary nucleotides become conditionally essential; supplementing all five nucleotides has been shown to preserve a strong immunity, and reduce the severity of infections.