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Deoxyribonucleic acid (DNA) is a molecule that carries genetic information from generation to generation. It is responsible to preserve the identity of the species over millions of years. DNA may be regarded as a reserve bank of genetic information or memory bank1.
The important function of DNA molecules are the storage of information for long-term. DNAs are present in all living things, like bacteria, plants, and animals. They determine a person's facial features, hair, skin and eye color, height, blood type, complexion and everything to make an individual unique. DNA replication is a process in which DNA copies itself to produce identical daughter molecules of DNA2.
STRUCTURE OF DNA
DNA is a double helix; with bases to the center (like rungs on a ladder) and sugar-phosphate units along the sides of the helix (like the sides of a twisted ladder). The structure of DNA is explained by James D. Watson and Francis H.C. Crick. DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription. Within cells, DNA is organized into long structures called chromosomes. These chromosomes are duplicated before cells divide, in a process called DNA replication3.
Figure 1(1): Structure of DNA
Each nucleotide consists of a deoxyribose sugar, a phosphate and a nitrogenous base. In DNA the four bases include Adenine (A), Thymine (T), Guanine (G) and Cytosine (C) and in RNA they are Adenine, Uracil (U), Guanine and Cytosine. Adenine and Guanine are double-ring molecules known as Purines; cytosine, thymine and uracil are single-ring molecules called Pyrimidines. The strands are complementary as deduced by Watson and Crick from Chargaff's data, (A) pairs with (T) and C pairs with G, the pairs held together by hydrogen bonds. Double ringed purine is always bonded to a single ring pyrimidine. Purines are Adenine (A) and Guanine (G). Pyrimidines are Cytosine (C) and Thymine (T). In DNA the sugar is deoxyribose. The bases are complementary, with adenine (A) on one side of the molecule the other side is T and similarly with G and C. The DNA regions which encode proteins are called genes4.
1.1.1 LOCATION OF DNA
location of DNA
Figure 1(2): Location of DNA
Eukaryotic organisms (animals, plants, fungi, and protists) store most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts. In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm5.
DNA is a long polymer made from repeating units called nucleotides. Within cells of an organism, DNA molecules are assembled into chromosomes, organelles that package and manage the storage, duplication, expression and evolution of DNA. In the chromosomes of a cell, DNA occurs as fine spirally coiled threads that in turn coil around another, like a ladder. The total length of all DNA in the Cell's nucleus would be 3km. The entire collection of chromosomes in each cell of an organism is its genome6. Human cells contain 23 distinct kinds of chromosomes carrying approximately 3x109 base pairs and roughly 100,000 genes. The structure of the DNA helix is preserved by weak interactions (i.e. hydrogen bonds and hydrophobic interactions established between the stacked base), it is possible to separate the two strands by treatments involving heating, bringing to alkaline pH7.
1.1.2 SOURCES OF DNA
Different sample types used in DNA extractions include8, Whole blood, Buffy coats, Blood clots, Serum, Plasma, and Cell pellets, Mouthwash, Buccal swabs, Cytobrushes, Saliva, Bronchial alveolar lavage, Mouse tails, Plants. Other solid tissues that can be used for DNA extraction include Breast, Prostrate, Kidney, Brain, Placental, Heart, and Muscle. Other sample types include Nails, Paraffin embedded tissue, Polyps, Urine, Feces and sputum. These sample types create great difficulty in isolating DNA37.
1.1.3 DNA EXTRACTION METHODS
2) Column Based
Obtaining high quality genomic DNA is critical for epidemiological studies that aim to evaluate the role of genetic factors in human disease susceptibility. Blood samples are an excellent source of large amounts of genomic DNA. However, epidemiological studies often need alternative sources when study subjects are reluctant to provide a blood sample, when only a self-administered collection protocol is logistically or economically feasible or as backup source of DNA in studies that collect blood samples9.
Whole blood is the common source of human genomic DNA for genetic testing. The disadvantages of using blood include invasive collection, need for a trained phlebotomist, special storage, and time consuming DNA extraction10. Therefore the collection of samples for DNA extraction is a critical procedure as it is time-consuming and may involve ethical aspects. In contrast DNA from buccal cells giving the same result as DNA from blood and may be collected non-invasively from the inside of the cheek by non-technical personnel11.
1.2 BUCCAL CELLS
Buccal cells are the cells from the inner lining of the mouth or cheek. These cells are routinely shed and replaced by new cells. As the old cells die, they accumulate in the saliva in the mouth and can be easily be collected by a simple procedure using mouthwash13. The mean number of epithelial cells per 1 ml of saliva is about 4.3Ã-105, whereas the number of nucleated cells in 1 ml of whole blood is about 4.5-11Ã-105. Moreover the turnover of epithelial cells is quite expensive in mouth; as the surface layer of epithelial cells is replaced on average every 2.7 hr suggesting that there is likely to be intact genomic DNA in saliva samples12.
1.2.1 ADVANTAGES OF BUCCAL CELLS
Extraction of genomic DNA from mouthwashes is extremely useful as a quick, noninvasive technique for collection and isolation of DNA. DNA extracted by this method is used in many applications such as genotyping, detection of disease markers and for comparison to crime scene samples.
Exfoliated buccal mucosa cells are a good source of DNA. Also, sample collection in such cases is non-invasive and can be self-administered. Collecting buccal cells enables researches to better understand the way people process substances that affect cancer and other diseases and to determine why some people who are exposed to certain substances develop to diseases, whereas others exposed to the same substances do not. The material in the buccal cell samples, combined with information on occupational, environmental and dietary factors, allows research to get a more complete assessment of what is affecting the health of human population. The buccal cell sample is being collected to study in differences in genes that may related to how people process disease-causing substances and how the effects of diet, lifestyle, environment, race, and ethnicity age and other factors may be related to these genes. Therefore this study describes a simple and inexpensive protocol to obtain high-quality DNA from buccal cells using mouthwash samples. DNA extracted by this method yields sufficient quantity of DNA for several rounds of PCR amplifications13.
1.2.2 CHARACTERISTICS OF BUCCAL CELLS
Research has shown that sublingual cells correlate well with deep body tissue such as heart tissue taken during bypass surgery and skeletal muscle biopsies. Buccal cells have high correlation between altered mineral levels and path physiological conditions in multiple medical syndromes. Sublingual epithelial cells offer a rapidly renewing, homogenous cell population that reflects current total body intracellular mineral status. Buccal cell has a high cytoplasm to nucleus structure facilitating mineral analysis. Blood and urine levels of mineral and ions do not necessarily reflect what is happening in the working cellular tissues. Cells contain about 99% of the body's magnesium and potassium, while serum contains only 1% of the total. Buccal cells are safe, easy to obtain and used as a smear on specially prepared slides. Fixed specimens have a long life and do not deteriorate in transit14.
Genomic DNA is identical whether it comes from blood cells or cheek cells. Buccal cell is viable alternative to isolation from blood. Buccal cell DNA is used for many diagnostic applications such as epidemiologic studies and paternity testing15. There are several advantages to buccal cell DNA isolation over blood. First no needles are involved, so it is less invasive and painless. It is well studied for young subjects. Buccal cells provide less of a potential hazard to the people who handle samples16.
Repeated sampling is not feasible by blood. With the growing interest in large scale in genomic studies epidemiological studies have become very important in trying to elucidate gene-environment interaction in individuals prone to mutagenic diseases like cancer and cardiovascular diseases. As already known exfoliated buccal cells are good source of DNA and sample collection in such cases are non-invasive and can be self-administered. The average yield is about 30 µg and is sufficient for more than 300 PCR amplifications. It has been observed that good quality high molecular weight genomic DNA can be obtained from exfoliated Buccal cells in the early morning mouthwash samples and that the DNA yield from similar samples decreases during the day, with the very low yields obtained in the late evening. This was due to very few exfoliated cells being present in the oral cavity at the time17. Oral epithelial cells are constantly exfoliated and may be captured through gentle scraping of the oral mucosa or by oral rinsing18.
Small sample size
Ease of collection
Low cost storage
Offers a source for study of exogenous or endogenous compounds other than DNA
Genotyping generally requires 10ng/genotype and with current technology as little as 2.5ng/SNP so that scores to hundreds of genotypes could be obtained from one blood spot
Low DNA yield may not be suitable for whole-genome amplification.
Nonrenewable smaller amplifications
Whole blood anti-coagulated or blood clots
Relatively low-cost storage
Yields large quantities of high quality of genomic DNA
Offers a source for study of exogenous or endogenous compounds other than DNA
Invasive sample collection
106cells = 6µg
Renewable source of DNA
Yields large quantities of high-quality genomic DNA
High cost storage
Does not offer a source for study of exogenous or endogenous compounds other than DNA or RNA.
49.7µg mean;0.2-134 µg range(total mouthwash DNA)
1-2 µg/cytobrush and swab.
32 µg median, 4-196 µg range human DNA in mouthwash.
Noninvasive and easy sample collection. Genotyping generally requires 110ng/genotype and with current technology as little as 2.5ng per SNP for genotyping for getting more genotypes from buccal cell specimen.
Low DNA yield.
Highly variable yield.
Does not offer a source for study of exogenous or compounds other than DNA or RNA.
Bacterial contamination must be addressed.
Mutation refers to a change in DNA structure of a gene. The substances (chemicals) which can induce mutations are collectively known as mutagens. The process of formation of a mutant organism is called mutagenesis1.
1.3.1 Types of mutations
The replacement of one base pair by other results in point mutation. They are of two subtypes.
Transitions: In this case a purine or pyrimidine is replaced by another.
Transversions: These are characterized by replacement of a purine by a pyrimidine or vice versa.
Frame shift Mutations
These occur when or more base pairs are inserted in or deleted from the DNA, respectively, causing insertion or deletion mutations.
Deletion: This occurs when a block of one or more nucleotide pairs is lost from a DNA Molecule.
Insertion: Insertion is addition of one or more nucleotide pairs.
A mutation that changes the wild type allele of a gene to a different allele is called a forward mutation19.
Reverse mutation or reversion:
Mutation can also cause a novel mutant allele to revert back to wild type.
Hydrolysis, Radiation UV and Oxidation can alter the information stored in DNA.
The hydrolysis of a purine base A or G from the deoxyribose phosphate back bone occurs 1000 times an hour in every human cell. Because the resulting apurinic sites cannot specify a complementary base the DNA replication process sometimes introduces a random base opposite the apurinic site causing a mutation in the newly synthesized complementary strand 3 quarter of the time.
The removal of an amino group can change cytosine to uracil, the nitrogenous base found in RNA but not in DNA, and already known U always pairs with A rather than G deamination followed by replication may alter a C-G base to T-A pair in future generation of DNA molecules6. Damaged DNA could mean the failure of important cell processes, or could even lead to cancer and early death20.
1.4 DNA Repair mechanisms
The following structural changes occur in DNA during mutation
Pyrimidine dimers, in which two adjacent pyrimidines on a DNA strand are coupled by additional covalent bonds and thus lose their ability to pair.
Chemical changes of single bases, such as alkylation or deamination, thus causing changes in the pairing properties of the DNA.
Crosslink between the complementary DNA strands, which prevent their separation in replication.
Intercalation of mutagenic agents into the DNA causing frame shift mutations.
Single strand breaks.
Double strand breaks21.
DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. Consequently, the DNA repair process must be constantly active so it can respond rapidly to any damage in the DNA structure. The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states:
An irreversible state of dormancy, known as senescence.
Cell suicide, also known as apoptosis or programmed cell death.
Unregulated cell division, which can lead to the formation of a tumor that is Cancer.
1.5 DNA REPAIR AND CANCER
Inherited mutations that affect DNA repair genes are strongly associated with high cancer risks in humans. Hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. BRCAI and BRCA2, two famous mutations conferring a hugely increased risk of breast cancer on carriers, are both associated with a large number of DNA repair pathways, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming capacity of the cell to repair DNA damage resulting in cell death. Cells that are most rapidly dividing - most typically cancer cells - are preferentially affected. The side effect is that other non-cancerous but rapidly dividing cells such as stem cells in the bone marrow are also affected. Modem cancer treatments attempt to localize the DNA damage to cells and tissues only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body)22.
Cancer is a disease characterized by uncontrolled multiplication and spread of abnormal growth of the cells. It is one of the major causes of death in the developed nations. One in the three people will be diagnosed with cancer during their life time, with lung and bowel cancer comprising the largest category, closely followed by breast and prostate cancer. At first glance incidence figures for the past 100 years or so give the impression that the disease is increasing in developed countries, but cancer is largely a disease of later life, and with advances in public health and medical science many more people now live to an age where they are more liable to contract to cancer.
Cancer refers to the hyper proliferation of cells that have lost the ability to be controlled by normal cell signals. Cancer cells have the ability to proliferate independent of their environment and are capable of metastasizing, or colonizing other tissues in the body.
1.6.1 The Special Characteristics of Cancer Cells
Cancer cells manifest, to varying degrees. There are four characteristics that distinguish them from normal cells. These are
Differentiation and loss of function
1.6.2 Causes of Cancer
The incidence, geographic distribution, and behavior of specific types of cancer are related to multiple factors, including sex, age, race, genetic predisposition and exposure to environmental carcinogens. Out of these factors, environmental exposure is probably most important. Exposure to ionizing radiation has been well established to be a significant risk factor for a number of cancers, including acute leukemia, thyroid cancer, breast cancer, lung cancer, soft tissue sarcoma, and basal skin cancers. Chemical carcinogens (particularly those in tobacco smoke) as well as azo dyes, aflatoxins, asbestos, benzene and radon have been clearly implicated in cancer induction in humans and animals. Viruses have been implicated as the etiologic agents of several human cancers. Expression of viruses-induced neoplasia probably also depends on additional host and environmental factors that modulate the transformation process.
1.6.3 Types of Cancer
Carcinoma : Malignant tumors that arises from epithelial cells
Melanoma : Cancerous growth of melanocytes, skin epithelial cell that produce the pigment melanin
Sarcoma : Cancer arising from muscle cell or connective tissue
Osteogenic Sarcoma : The most frequent type of childhood cancer
that destroys normal bone tissue.
Leukemia : It is a cancer of blood forming organs characterized by rapid growth of abnormal leucocytes
1.7 ORAL CANCER51
Oral cancer is the 11th most common cancer in the world, accounting for 267,000 new cases and 128,000 deaths annually around the year 2000, of which two-thirds are observed in developing countries. A high incidence of oral cancer is observed in the Indian subcontinent, which accounts for a third of the world burden. A high prevalence of betel quid (with or without tobacco) chewing, smoking and alcohol drinking is responsible for the high risk of oral cancer in India. Primary prevention, by avoidance of tobacco and alcohol, early detection and appropriate treatment are important control measures24. The etiology of oral cancer, specifically cancer of the tongue and mouth is well established and predominantly involves the use of tobacco and alcohol. the prevalence of the disease depends upon the exposure to the etiological agents such as: Cigarette, cigar and pipe smoking are the main forms of tobacco use and the effect of tobacco is known, not only to be dose and time dependent but, also, to act synergistically with the intake of alcohol to multiply disease risk. In addition, factors such as dietary deficiencies, in particular vitamins A and C, iron and certain trace elements, are thought to be associated with oral cancer25.
1.7.1 TOBACCO CHEWING AND SMOKING
Smoking and tobacco chewing is probably the most obvious factor for adverse mortality and is perhaps less obvious is that smoking inflicts extensive DNA damage. Tobacco smoke contains over two hundred chemicals known to cause cancer, known as carcinogens. The precise mechanism whereby cancer starts is not fully understood. These DNA mutations are permanent and forever increase the likelihood for developing lung cancer. Tobacco smoke also contains chemicals in a group called the polycyclic aromatic hydrocarbons which can lead to specific genetic mutations in a gene known as 'p53'. 'p53' plays an important role in suppressing tumors and significantly, mutations in this gene are present in around half of all major human tumors26. Tobacco smoking is the most important and well documented cause of cancer currently known. Epidemiological associations have been found for lung, mouth, pharynx, esophagus, kidney, bladder, and pancreas and cervix cancer. The relationship with cancer of the mouth, pharynx, esophagus and lung is easily explained by their direct contact with smoke. In the light of the fact that over 90% cancers involve epithelial cells and that DNA damage is considered a crucial mechanism in cancer development, the evaluation of DNA damage in buccal epithelial cells may thus provide a good biomarker of early damage in target tissues20.
Worldwide, tobacco kills one human being every six seconds.
That works out to 560 people every hour, 13,440people per day and 49 lakh People per annum.
Tobacco kills 15 times as many people as suicides, murder or manslaughter.
1.7.2 p53 gene28
p53 protein was first identified in 1979 as a transformation-related protein and a cellular protein which accumulates in the nuclei of cancer cells and binds tightly to the simian virus 40 (SV402) large T antigen. The gene encoding p53 was initially found to have weak oncogenic activity as the p53 protein was observed to be overexpressed in mouse and human tumor cells. In subsequent studies, p53 became widely recognized as a tumor suppressor, and the p53 gene became probably the most common site for genetic alterations in human cancers. Subsequent research with wt p53 clearly demonstrated that p53 was a major "guardian of the genome". The biological consequences of p53 activity include cell-cycle regulation, induction of apoptosis, development, differentiation, gene amplification, DNA recombination, chromosomal segregation, and cellular senescence. Presently, p53 is known to play a key role in practically all types of human cancers, and the mutation or loss of the p53 gene can be identified in more than 50% of all human cancer cases worldwide. This significant involvement in oncogenesis extends far beyond the simple role in viral transformation p53 was suspected of playing in earlier investigation.
The structure of p53
Human p53 is a nuclear phosphor-protein of MW 53 k Da, encoded by a 20-Kb gene containing 11 exons and 10 introns, which is located on the small arm of chromosome 17. This gene belongs to a highly conserved gene family containing at least two other members, p63 and p73.
The physiological functions of p53
As a tumor suppressor, p53 is essential for preventing inappropriate cell proliferation and maintaining genome integrity following genotoxic stress. Following various intracellular and extracellular stimuli, such as DNA damage, heat shock, hypoxia, and oncogene overexpression, wt p53 is activated and emerges as a pivotal regulatory protein which triggers diverse biological responses, both at the level of a single cell as well as in the whole organism. p53 activation involves an increase in overall p53 protein level as well as qualitative changes in the protein through extensive posttranslational modification, thus resulting in activation of p53-targeted genes.
Mutation of p53
The p53 gene is often found to be genetically altered in tumors, and is one of the most frequently inactivated genes in human cancers. Aberrant stimulation of cell proliferation leads to DNA replication stress, DNA DSBs, genomic instability, activation of the DNA damage checkpoint, and ultimately p53-dependent apoptosis. p53 dependent apoptosis suppresses expansion of pre-cancerous lesions (p53 tumor suppressor function) and provides selective pressure for p53 inactivation. The function of p53 tumor suppressor in cancers can be lost by various mechanisms, including lesions that prevent activation of p53, mutations within the p53 gene itself or mutations of downstream mediators of p53's function. Acquired mutations (more than 18,000 mutations have been identified) in the p53 gene are found in all major types of human cancers. Approximately half of all human tumors have a mutation or loss in the p53 gene leading to inactivation of its function.
1.8 AGAROSE GEL ELECTROPHORESIS
Agarose is a linear polymer composed of alternating residues of D- and L - galactose joined by alpha- (1-3) and beta-(1-4) glycosidic linkages. The L-galactose residue has an anhydrous bridge between the three and six positions.
Figure 1(3): Structure of Agarose
Chains of agarose form helical fibers that aggregate into super coiled structures with a radius of 20-3Onm. Gelation of agarose results in a three-dimensional mesh of channels whose diameters range from 50nm to 200nm.
1.8.1 THE RATE OF MIGRATION OF DNA THROUGH AGAROSE GELS
Figure 1(4): Agarose gel formation.
The following factors determine the rate of migration of DNA through agarose gels
(i) The molecular size of the DNA:
Molecules of double-stranded DNA migrate through gel matrices at rates that are inversely proportional to the log10 of the number of base pairs. Larger molecules migrate more slowly because of greater frictional drag and because they wore their way through the pores of the gel less efficiently than smaller molecules.
(ii) The concentration of agarose:
A linear DNA fragment of a given size migrates at different rates through gels containing different concentrations of agarose. There is a linear relationship between the logarithm of the electrophoretic mobility of the DNA and the gel concentration.
(iii) The conformation of the DNA:
Super helical circular (form I), nicked circular (form II), and linear (form III)
DNAs migrate through agarose gels at different rates. The relative mobilities of the three forms depend primarily on the concentration and type of agarose used to make the gel, but they are also influenced by the strength of the applied current, the ionic strength of the buffer, and the density of super helical twists in the form I DNA. Under some conditions, form I DNA migrates faster than form III DNA; under other conditions, the order is reversed. In most cases, the best way to distinguish between the different conformational forms of DNA is simply to include in the gel a sample of untreated circular DNA and a sample of the same DNA that has been linearised by digestion with a restriction enzyme that cleaves the DNA in only one place.
(iv) The presence of ethidium bromide in gel and electrophoresis buffer:
Intercalation of ethidium bromide causes a decrease in the negative charge of the double stranded DNA and an increase in both its stiffness and length. The rate of migration of the linear DNA dye complex through gels is consequently retarded by a factor approximately 15%. The most convenient and commonly used method to visualize DNA in agarose gels is staining with the fluorescent dye ethidium bromide which contains a tricyclic planar group that intercalates between the stacked bases of DNA. Ethidium bromide binds to DNA with little or no sequence preference. At saturation in solutions of high ionic strength, approximately one ethidium molecule is intercalated per 2.5 bp. After insertion into the helix, the dye lies perpendicular to the helical axis and makes Vander Waals contacts with the base pairs above and below. The fixed position of the planar group and its close proximity to the bases causes dye bound to DNA to display an increased fluorescent yield compared to that of dye in free solution. UV radiation at 254nm is absorbed by the DNA and transmitted to the dye radiation at 302nm and 366nm is absorbed by the bound dye itself. Ethidium bromide can be used to detect both single and double stranded nucleic acids. However, the affinity of the dye for single stranded nucleic acid is relatively low and the fluorescent yield is comparatively poor.
Ethidium bromide is prepared as a stock solution of 10mg/ml in water, which is stored at room temperature in dark bottles or bottles wrapped in aluminum foil. The dye is usually incorporated into agarose gels and electrophoresis buffers at a concentration of 0.5µg/ml. Although the electrophoretic mobility of linear double stranded DNA is reduced by 15% in the presence of the dye, the ability to examine the agarose gels directly under UV illumination during or at the end of the run is a great advantage. However, sharper DNA bands are obtained when electrophoresis is carried out in the absence of ethidium bromide. During restriction digestion the agarose gel should be run in the absence of ethidium bromide and stained after electrophoresis is complete. Staining is accomplished by immersing the gel in electrophoresis buffer or water containing ethidium bromide for 30-45 minutes at room temperature. De-staining is not usually required. However, detection of very small amounts (< l0ng) of DNA is made easier if the background fluorescence caused by unbound ethidium bromide is reduced by soaking the stained gel in water or l mm MgS04 for 20 minutes at room temperature.
(v) The applied voltage:
At low voltages, the rate of migration of linear DNA fragments is proportional to the voltage applied. However, as the strength of the electric field is raised, the mobility of high-molecular weight fragments increases differentially. Thus, the effective range of separation in agarose gels decreases as the voltage is increased. To obtain maximum resolution of DNA fragments >2kb in size, agarose gels should be run at no more than 5-8V/cm.
(vi) Types of agaroses
The two major classes of agarose are standard agaroses and low-melting temperature agaroses. A third and growing class consists of intermediate Melting/gelling temperature agaroses, exhibiting properties of each of the two major classes.
(vii) The electrophoresis buffer
The electrophoretic mobility of DNA is affected by the composition and ionic strength of the electrophoresis buffer. In the absence of ions electrical conductivity is minimal and DNA migrates slowly, if at all, in buffer of high ionic strength electrical conductance is very efficient and significant amounts of heat are generated, even when moderate voltages are applied. In the worst case, the DNA denatures.
1.8.1 TYPES OF ELECTROPHORESIS BUFFER
Several different buffers are available for electrophoresis of native, double stranded DNA. These contain Tris-acetate and EDTA (pH 8.0; TAE) (also called TE buffer), Tris borate (TBE) or Tris-phosphate (TPE) at a concentration of 50mM (pH 7.5-7.8). Electrophoresis buffers are usually made up as concentrated solutions and stored at room temperature. All these buffers work well, and the choice among them is largely a matter of personal preference. TAE has the lowest buffering capacity of the three and will become exhausted if electrophoresis is carried out for prolonged periods of time. When this happens, the anodic portion of the gel becomes acidic and bromophenol blue migrating through the gel toward the anode changes in color from bluish-purple to yellow. This change begins at pH 4.6 and is complete at pH 3.0. Exhaustion of TAE can be avoided by periodic replacement of the buffer during electrophoresis or by recirculation of the buffer between the two reservoirs. Both TBE and TPE are slightly more expensive than TAE, but they have significantly higher buffering capacity.
Gel loading buffers are mixed with the samples before loading in to the slots of the gel. These buffers serve three purposes. They increase the density of the sample thereby simplifying the loading process and they contain dyes that in an electric field, move toward the anode at predictable rates, bromophenol blue migrates through agarose gels approximately 2.2 fold faster than xylene cyanol FF, independent of agarose concentration. Bromophenol blue migrates through agarose gels run in 0.5X TBE approximately the same rate as the linear double stranded DNA 300bp in length, whereas Xylene cyanol FF migrates at approximately the same rate as linear double-stranded DNA 4kb in length. These relationships are not significantly affected by the concentration of agarose in the gel over the range of 0.5 - 1.4%.
The DNA fragments that results from restriction enzyme cutting are easily separated and displayed by electrophoresis through agarose gels29.
1.9 POLYMERASE CHAIN REACTION (PCR)
Polymerase chain reaction is an in-vitro technique for generating large quantities of a specified DNA. Perceptibly, PCR is a cell-free amplification technique for synthesizing multiple identical copies (billions) of any DNA of interest. PCR is now considered as a basic tool for the molecular biologist19. PCR was first proposed by H. Ghobind Khorana in 1970's and developed in 1984 by Karry Mullis and co-workers at Cetus to the amplification of human Î²-globin DNA and to the parental diagnosis of sickle cell anemia29.
1.9.1 VARIENTS OF PCR
Semi nested PCR
Nested PCR Multiplex-PCR
Quantitative PCR (Q-PCR)
Reverse Transcription PCR (RT-PCR)
Thermal asymmetric interlaced PCR (TAIL-PCR) 22, 38.
1.9.2 PRINCIPLES OF SIMPLE PCR
PCR is carried out in a single tube kept in a thermal cylinder which is programmable to set alternate heating and cooling. The DNA to be amplified, reagents, two types of oligonucleotide primers, deoxyribonucleotides and Taq polymerase are added into the tube. Over this mixture a thin film of mineral oil is poured to prevent evaporation of the reaction mixture during thermal cycles. The tube is then kept inside the thermal cylinder. This is the experimental set up for PCR32.
An important property of PCR particularly in diagnostic application is the capacity to amplify a target sequence from crude DNA preparations as well as from degraded DNA templates.
PCR in short is denaturation of the template by heat, annealing of the oligonucleotide primers to the single stranded target sequence and extension of the annealed primers by a thermo stable DNA polymerase33.
The essential components of PCR are listed below:
A DNA template for the polymerase to copy: In case of mammalian g DNA up to 1.0 µg of DNA is utilized/reaction an amount that contains approximately 3x105copies of a single copy autosomal gene.
A thermostable DNA polymerase to catalyze template dependent synthesis of DNA. Taq Polymerase (0.5-2.5U).
A pair of synthetic oligonucleotide to prime DNA synthesis. Primers should be selected with a random base distribution, and with CG content similar to that of fragment being amplified. Primers with stretches of polypurines polypyrimidines or other unusual sequence should be avoided. In particularly avoiding primers with 3'end overlaps will reduce the incidence of primer dimer.
dNTP 200-250 µM of each dNTPs are recommended for Taq polymerase in reactions containing 1.5mM MgCl2.
Buffer to maintain pH: Tris-HCI (pH 8.3-8.8). Monovalent cations: Standard PCR buffer contains 50mM KCI and works well or amplification of segments of DNA >500 bp in length. 1.5 mM MgCl2 is optimal (200 µM each dNTP). Generally excess Mg2+ may results in the accumulation of non-specific amplification products and insufficient Mg2+will reduce the yield.
Thermo stable DNA polymerases: This is isolated from two classes of organisms, the thermophilic and hyperthermophilic eubacteria Archaebacteria. Sometimes cocktails are preferred.
In PCR each cycle contains following stages:
ds DNA template denature at a temperature i.e., determined in part by their G+C content. The higher the proportion of G+C, the higher the temperature required to separate the strands of template DNA. The longer DNA molecules the longer time required to separate. If temperature is short or time is short, only AT rich regions of the template DNA will be denature. When the temperature is reduced later in the PCR cycle the template DNA will re anneal into a fully native condition. This is carried out at 94-95 °c which is the highest temperature the enzyme can endure for 30 or more cycles. Higher temperature may be required to denature template that are rich in G+C content. DNA polymerases isolated from Archae are more heat tolerant than Taq.
If Annealing temperature is too high the oligonucleotide primers anneal poorly, yield is also low. If temperature is low nonspecific annealing of primers may occur, resulting in unwanted amplification.
Extension of oligonucleotide primers:
72-78 °c is the optimum temperature for extension. The polymerization rate of Taq polymerase in approximately 2000 nucleotides/min and carried for 1 min for every 1000 bp of product. Result of PCR is not altered by using 3 times longer extension time.
The standard PCR is typically done in a 50 or I00 µl volume an in addition to the sample DNA contains 50 mM KCI, 10 mM Tris HCI (pH 8.4), 1.5mM MgCl2,100 µg/ml gelatin, 0.25 µM of each primer, 200 µM of each deoxy nucleotide triphosphate (dATP, dCTP, dGTP, dTTP) and 2.5 U of Taq polymerase.
Variation whatever may be its cause and however it may be limited, is the essential phenomenon of evolution. The readiest way, then of solving the problem of evolution is to study the facts of variation William Bateson (1894). The term polymorphism has been defined as a 'Mendelian trait' that exists in the population in at least 2 phenotypes, neither of which occurs at a frequency of less than 1%. Some DNA polymorphisms are neutral single base pair changes detected by virtue of the consequent introduction or removal of restriction enzyme recognition.
These are variations in DNA sequence between individuals. There are about 60,000 polymorphisms in human genome34. RFLPs are not rare being distributed throughout the genome at a frequency of between 1/200 and 1/1000 bp. Not unexpectedly, the vast majority of polymorphisms occurs in introns or intergenic regions rather than within coding sequences and may thus be expected to be neutral with respect to fitness. Those polymorphisms that occur either in coding regions or in the promoter region may however affect whether the structure or function of the gene product or the expression of the genes and may have the potential to be of phenotypic or even pathological significance. Restriction enzymes are named based on the bacteria in which they are isolated in the following manner:
E Escherichia (genus)
Co coli (species)
R RY13 (strain)
I First identified Order ID'd in bacterium
Average fragment size
Estimated number of
5' --G ATTC---3'
3'--CTT AA G--5'
5' ---G GATCC---3
Table 1(2): Restriction enzymes and their property
1.11 RESTRICTION FRAGMENT LENGTH POLYMORPHISM19, 32
RFLP is a laboratory technique used to amplify unknown (random) DNA segments. A DNA molecule can be cut into different fragments by a group of enzymes called Restriction Endonucleases. These fragments are called polymorphisms (literally means many forms).
RFLP was the very first technology employed for the detection of polymorphism, based on the DNA different sequences. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and recombinations of genomic DNA.
A RFLP represents a stretch of DNA that serves as a marker for mapping a specified gene. RFLPs are located randomly throughout a person's chromosomes and have no apparent function.
DNA polymorphisms offer a number of advantages for mapping genomes firstly number of DNA markers already exceeds that of suitable protein markers, secondly a DNA sequences does not necessarily have to express a protein in order to be identified by polymorphism cleavage sites. DNA polymorphisms can of course occur in any DNA sequence particularly in introns.
1.11.1 RFLPs in the diagnosis of diseases
RFLPs are especially useful for identifying genetic defects in humans and can be exploited for diagnostic purposes as long as the DNA alterations involved do not occur several times, and are associated with single genes. Most of the RFLPs known today appear to have occurred randomly and bear no relation to neighboring gene35.
If the RFLP lies within or even close to the locus of a gene that causes a particular disease, it is possible to trace the defective gene by the analysis of RFLP in DNA. The person's cellular DNA is isolated and treated with restriction enzymes. The DNA fragments so obtained are separated by electrophoresis. The RFLP patterns of the disease suspected individuals can be human g compared with that of normal people. By this approach it is possible to determine whether the individual has the marker RFLP and the disease gene. With 95% certainty, RFLPs can detect single gene based diseases.
1.11.2 Mutation specific RFLPs36
In some single gene disorder the mutation responsible eliminates a restriction enzyme recognition site. This direct approach has been used in sickle cell disease using the Restriction Mst II.
Variation in the nucleotide sequence of the human genome is common, occurred approximately once every 200 bp. These single base pair differences in DNA nucleotide sequences are inherited in a Mendelian co dominant manner and have no phenotypic effects as they usually occur in intergenic non-coding DNA. If a difference in DNA sequence occurs within the nucleotide recognition sequence of a restriction enzyme the DNA fragments produced by that RE will be of different lengths in different people. This can be recognized by the altered mobility of the restriction fragments on gel electrophoresis.