INTRODUCTION

Hepatitis B virus (HBV) is one of the major global public health problems. HBV infection is the 10th leading cause of death and HBV related hepatocellular carcinoma (HCC) is the 5th most frequent cancer worldwide. In India, HBsAg prevalence among general population ranges from 2% to 8%, placing India in intermediate HBV endemicity zone and the number of HBV carriers is estimated to be 50 million, forming the second largest global pool of chronic HBV infections (Datta S. 2008). The extensive use of HBV vaccine and availability of therapeutic drugs has reduced the incident of HBV infection and its chronicity. HBV is diagnosed using commercially available serological test which distinguish acute, self-limited infections from chronic infection. Despite of extensive use, these serologic test shows false-negative test like in chronic HBV carriers, the HBsAg level may be below the detecting limit (Jilg W. et al,1995); infected individuals with HBV mutants, circulating low levels of viral proteins may escape detection by screening assay. Nucleic acid testing for HBV-DNA is increasingly being used to quantify HBV viral load and measure the effectiveness of therapeutic agents. Current HBV DNA assays like signal amplification assays and detection based on nested PCR can detect 105-106 genome copies and as few as 102-103genomes copies respectively (Sablon E., Shapiro F., 2005). New developments in HBV DNA testing, the Roche COBAS(R) TaqMan (R), approved by the U.S. Food & Drug Administration, that has decrease the number of handling steps, reduced chances of contamination, increase throughput and the accuracy of quantification can detect the WHO HBV International Standard in plasma and serum as low as 3.5 IU/ml and 3.4 IU/ml respectively (FDA approved First Hepatitis B Viral Load Test, 2009). The former serological test due to its false negative results and latter PCR test which are expensive necessitate an emergency for development of accurate and cost effective assay. The aim of present work is to construct a set of standards using conventional PCR which is sensitive, reliable, versatile and cost effective method for accurate quantitation of HBV DNA in patients sera and attempts had been taken to calculate viral load for better management of the disease. This new assay could be an important step for clinical application of PCR while designing a molecular based kit to detect HBV DNA.

REVIEW OF LITERATURE
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HEPATITIS Hepatitis implies inflammation of the liver characterized by the presence of inflammatory cells in the tissue of the organ. Hepatitis B is an infectious illness caused by Hepatitis B Virus (HBV) which infects the liver of hominoidae, including humans, and causes an inflammation called hepatitis.

HBV Hepatitis B Virus (HBV) is the prototype member of a steadily growing family of viruses called hepadnaviruses. Hepadnaviruses are found in both mammals (orthohepadnaviruses) and birds (avihepadnaviruses). The HBV genome after cloning and sequencing showed similarities with the viruses discovered in woodchucks (Marmota monax), ground squirrels (Spermophilus beecheyi) and pekin duck (Anas domesticus). Also numerous new viruses that are similar to HBV were found in mammals and birds and have been cloned. Table 1 and 2 gives list of hepadnaviruses in mammals and birds along with their host (Schaefer S., 2007).

Orthohepadnaviruses and their host

Host Hepatitis B Virus Man Homo sapiens sapiens Chimpanzee Hepatitis B Virus Chimpanzee Pan troglodytes Gibbon Hepatitis B Virus White handed gibbon Hylobates lar Orangutan Hepatitis B Virus Orangutan Pongo pygmacus pygmacus Gorillla Hepatitis B Virus Gorilla Gorilla gorilla Woolly Monkey Hepatitis B Virus Woolly Monkey Lagothrix lagotricha Woodchuck Hepatitis Virus Woodchuck Marmota monax Ground Squirrel Hepatitis Virus Ground Squirrel Spermophilus beecheyi Arctic Squirrel Hepatitis Virus Arctic Squirrel Spermophylus parryi kennicotti TABLE 1: Orthohepadnaviruses and their host 2

Avihepadnaviruses and their host

Host Duck Hepatitis B Virus Pekin duck DHBV Anas domesticus Grey Teal Hepatitis B Virus Grey Teal GTHBV Anas gibberifrons gracilis Heron Hepatitis B Virus Heron HHBV Adrea cinerea Maned Duck Hepatitis B Virus Maned Duck MDHBV Chenonetta jubata Ross Goose Hepatitis Virus Ross Goose RGHV Anser rossi Snow Goose Hepatitis B Virus Snow Goose SGHBV Anser caerulescens Stork Hepatitis B Virus White Stork STHBV Ciconia ciconia Crane Hepatitis B Virus Demoisella cranes CHBV Anthropoides virgo Grey crowned cranes Balearica regulorum TABLE 2: Avihepadnaviruses and their host HBV Genotypes and Subgenotypes Human HBV can be grouped into eight genotypes (based on more than 8% difference). There are several attempts have been made to reconstruct the evolution of hepadnaviruses where the rate of synonymous substitution for HBV was found to be 4.5710 -5 per site per year. Duck Hepatitis B Virus (DHBV) has been proposed to have diverged about 30,000 years ago from a common ancestor while Ground Squirrel Hepatitis Virus (GSHV) and Woodchuck Hepatitis Virus (WHV) should have diverged about 10,000 years ago from HBV and the HBV serotypes would have been separated about 3000 years ago. Still as it is not cleared about estimating the mutation rate of HBV calculating the time point for separation of HBV genotypes or hepadnaviral species becomes difficult. A prototypic HBV genome may have length of 3215bp, as found in HBV genotypes B, C, F and H. Table 3 gives the fundamental properties of genomes and differences between HBV genotypes where HBV genotype G is 66bp longer than genotype D. Also, with the help of extensive phylogenetic analysis have shown that HBV genotypes are further subdivided into subgenotypes as shown in Table 4 they differ by at least 4% (Schaefer S., 2007).

Fundamental properties of genomes and differences between HBV genotypes

Genotypes Genome length in bp A 3221 B 3215 C 3215 D 3182 E 3212 F 3215 G 3248 ORF-diffrences Insertion of aa 153 and 154 in HBc Deletion of aa 1-11 in preS1 Deletion of aa 11 in preS1 Insertion of 12 aa in HBc Deletion of aa 11 in preS1

H 3215 TABLE 3: Fundamental properties of genomes and differences between HBV genotypes

HBV subgenotypes and geograhic prevalence

A Subgenotype A1 A2 A3 (A4) (A5) B1 B2 B3 B4 B5 C1 Geographic origin Africa, (Asia, South America) Europe Gabon, Cameroon Mali Nigeria Bj Japan Ba Asia without Japan Indonesia, Philippines Vietnam Philippines Cs South East Asia (Vietnam, Myanmar, Thailand, Southern China) C2 Ce Far East (Korea, Japan, Northern China) C3 Micronesia C4 Australia C5 Philippines, Vietnam D1 Mongolia, Belarus, Europe D2 India D3 South Africa, East India, Serbia D4 Australia D5 East India F1 South and Central America F2 South America F3 Bolivia F4 Argentina TABLE 4: HBV subgenotypes and geographic prevalence Synonyms Aa, A Ae, A-A Ac

Having evolved distinctly in specific geo-ethnic populations, HBV genotypes/subgenotypes have a distinct geographical distribution pattern (Schaefer S., 2007).

FIGURE 1: Geographic distribution of HBV genotypes and subgenotypes HBV Genome HBV belongs to Hepadnaviridae family, having double-stranded enveloped DNA. The size is 42nm which replicates in the liver and causes hepatic dysfunction. HBsAg is found on surface of the virus and is also produced in excess amounts. It circulates in the blood in two forms 22nm spherical and tubular particles. The inner core of the virus contains HBcAg, HBeAg, a partially double-stranded DNA and DNA-dependent DNA polymerase (Figure 2 of HBV particle and surface antigen). HBsAg persist for variable periods and can be identified in serum 30-60 days after exposure to HBV.

FIGURE 2: HBV particle and surface antigen HBV is partially double-stranded circular DNA with 3200bp in its genome. It contains overlapping genes on minus strand that code for structural proteins (pre-S, surface and core) and replication related proteins (polymerase and X protein). The plus strand is shorter and variable in length. Figure 3 shows the Structure and Organization of the HBV genome.

FIGURE 3: Structure and organization of the HBV genome.

HBcAg is essential for viral packaging and is an integral part of the nucleocapsid. It can be detected in liver tissue in patients with acute or chronic HBV infection. HBeAg is a soluble protein not essential for viral replication and can be detected in the serum of patients with high virus titers. The surface/pre-S gene encodes for the virus envelope. The major 6

protein that forms the HBsAg particles is the smallest gene product (SHBs). The middle protein (MHBs), which contains the pre-S2 component, and the large surface protein (LHBs), which contains pre-S1, are also incorporated into HBsAg particles but are found in larger proportions in the intact virus particles. The specialized functions of these proteins have been the subject of intense study. The pre-S proteins play an important role in the attachment of HBV to hepatocytes. HBV replication begins with binding of the virus to the cell surface and penetration. The virus is transported without processing to the nucleus, where the relaxed circular DNA is converted to a covalently closed circular DNA (cccDNA), which in turn acts as the template for viral RNA synthesis. HBV DNA does not integrate into the host genome during the normal course of replication. Transcription results in RNA of various sizes. The 3.5-kb genomic RNA serves as a template for reverse transcription and DNA synthesis, which produces an open circular DNA molecule. The mature core particles are packed into HBsAg and pre-S proteins in the endoplasmic reticulum and exported from the cell. A pool of cccDNA is maintained in the nuclei by the transport of newly synthesized HBV DNA back to the nucleus. Because HBsAg can inhibit the formation of cccDNA, this may represent negative feedback to HBV replication (Mahoney F., 1999). Classical Serotypes Classically, HBV strains were distinguished by the presence of two pairs of mutually exclusive serotype determinants 'd'/'y' and 'w'/'r', in the HBsAg along with the main antigenic determinant 'a', which led to the description of 4 serotypes, namely adw, adr, ayw or ayr. Additional serotypes were subsequently characterized leading to the description of nine serotypes namely ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq+ and adrq- and a distinct geographical pattern for the distribution of serotypes was also documented. However, with the advent of molecular biological techniques and advanced computational methods for the phylogenetic analysis of complete viral genome sequences, HBV genotypes and subgenotypes have been described, that have largely replaced the classical serotype based classification of HBV strains (Datta S., 2008). PATHOLOGY

HBV causes both acute and chronic liver disease. The pattern of liver injury is characterized by hepatocellular destruction, regeneration, and inflammatory infiltrates. Although these pathologic changes typically resolve completely after viral clearance, chronic infection is often accompanied by progressive fibrosis. Because chronic hepatitis B takes decades to progress to end-stage liver disease, it is important to assess not only the pattern of injury but also the histologic severity and stage of hepatitis B (Fields B., 2001). Acute Hepatitis The average incubation period for newly infected persons who develop acute hepatitis is 90 days (range: 60-150 days). The development of symptoms is age dependent of a new HBV infection. Over 90% of perinatal HBV infections are asymptomatic, while typical manifestation are noted in 5-15% of newly infected young children (1-5 years of age) and 3350 % of older children, adolescents and adults. Persons with acute hepatitis B can show signs and symptoms that include nausea, abdominal pain, vomiting, fever, jaundice, dark urine, change in stool color and hepatomegaly or splenomegaly. The first serologic markers to become detectable in persons with acute HBV infection are HBsAg and antibodies to hepatitis B core antigen. In the 612 months after infection, immunoglobulin M antibodies to hepatitis B core antigen become undetectable. Total antibodies to hepatitis B core antigen persist for life and are found in persons with chronic infection as well as those who recover from infection. In persons who recover from HBV infection, HBsAg is eliminated from the blood, and antibody to hepatitis B surface antigen (anti-HBs) develops during convalescence. The presence of anti-HBs indicates immunity to HBV infection. Most persons who recover from natural infection (resolved infection) will be positive for both anti-HBs and antibodies to hepatitis B core antigen, but anti-HBs becomes undetectable in some over time. Immunosuppressed persons can develop reactivation of previously resolved HBV infection. Resolved acute infection is not a risk factor for subsequent cirrhosis or hepatocellular carcinoma (Shepard C., et al 2006). Chronic HBV Infection Chronic HBV infection is defined as either the presence of HBsAg in the serum for at least 6 months or the presence of HBsAg in a person who tests negative for immunoglobulin M antibodies to hepatitis B core antigen. Unlike persons who recover from acute HBV infection, persons with chronic HBV infection do not develop anti-HBs, and HBsAg typically 8

persists for decades. HBeAg, a marker of high viral replication activity which correlates with greater infectivity, is also usually present in the early phases of illness. For many persons with chronic infection, HBeAg becomes undetectable at some point (usually a decade or more) after the acute infection; this change usually indicates a decrease in viral replication. Approximately 1525 percent of persons infected with HBV as infants or young children, develop chronic infection die prematurely from cirrhosis or hepatocellular carcinoma. Extrahepatic complications can also occur, including polyarteritis nodosa, membranous glomerulonephritis, and membranoproliferative glomerulonephritis. Persons with chronic HBV infection should receive periodic medical evaluation, and some authorities recommend regular screening for hepatocellular carcinoma using a-fetoprotein or ultrasonography. Recently approved therapeutic agents for treatment of chronic hepatitis B are now being used to achieve sustained suppression of HBV replication and remission of liver disease for some patients. However, adverse events associated with treatment, expense, development of antiviral resistance, and low rates of HBsAg clearance remain barriers to treatment for many patients with chronic infection (Shepard C., et al 2006). MODES OF TRANSMISSION Transmission of hepatitis B virus results from exposure to infectious blood or body fluids containing blood. Possible forms of transmission include unprotected sexual contact, blood transfusions, re-use of contaminated needles and syringes, and vertical transmission from mother to child during childbirth. A mother who is positive for HBsAg confers a 20% risk of passing the infection to her offsprings at the time of birth. This risk is as high as 90% if the mother is also positive for HBeAg. HBV can be transmitted between family members within household, possibly by contact of nonintact skin or mucous membrane with secretions or saliva containing HBV (Petersen N. et al 1976) (Hepatitis B - the facts: IDEAS Victorian Government Health Information, Australia).

EPIDEMIOLOGY OF HBV INFECTIONS IN INDIA According to the WHO report on prevention of HBV in India (World Health Organization 2002), HBsAg prevalence among general population ranges from 0.1% to 11.7%, being between 2% to 8% in most studies. HBsAg prevalence rate among blood donors ranged from 1% to 4.7%. With the exception of higher HBsAg positivity in some 9

North Eastern states (~7%), no substantial geographical variation was apparent in other parts of India. Considering, on an average, HBsAg carrier rate of 5%, the total number of HBV carriers in the country was estimated to be about 50 million that forms nearly 15% of the entire pool of HBV carriers in the world and is the second largest pool of chronic HBV infections in the world (World Health Organization 2002). Using conservative prevalence estimates of different HBV seromarkers for estimating the number of HBV infections and serious disease outcomes in population, it was predicted that over 9 million are estimated to acquire HBV infection during their lifetime, an estimated 1,507,000 will develop chronic HBV infection, and nearly 200,000 will die of acute or chronic consequences of HBV infection (World Health Organization 2002), which clearly indicates an impending danger (Datta S. 2008).

REASERCH NEWS One in 12 people worldwide are grappling chronic Hepatitis B or Hepatitis C. It is sad that, even though prevalence of Hepatitis beats even that of HIV or cancer, the awareness of this condition is not up to the mark. The theme for World Hepatitis Day 2010, This is Hepatitis is an extension of the 2009 theme - Am I Number 12? (Savitha www.medindia.net/news). Hepatitis B virus (HBV) is one of the major global public health problems. HBV infection is the 10th leading cause of death and HBV related hepatocellular carcinoma (HCC) is the 5th most frequent cancer worldwide. About 30 percent of the world's population has serological evidence of current or past infection with HBV. Of these, an estimated 350 million are chronically infected with HBV and approximately 1 million persons die annually from HBV-related chronic liver diseases, including severe complications such as liver cirrhosis (LC) and HCC (Hepatitis B. Fact sheets WHO). HBV is distributed worldwide, but its prevalence varies significantly between different populations of the world. Based on the prevalence of HBV surface antigen (HBsAg) carrier rate in the general population, sub-Saharan African, East Asian and Alaskan populations are classified as having high HBV endemicity (HBsAg carriage > 8%), while the populations of southern parts of Eastern and Central Europe, the Amazon basin, the Middle East, and the Indian subcontinent are classified as intermediate HBV endemicity (HBsAg carriage 27%), 10

and the populations in western and northern Europe, North America, and Australia are classified as low HBV endemic (HBsAg carriage < 2%) regions (Lavanchy D., 2004).

NEED FOR DIAGNOSIS HBV infection may result in subclinical or asymptomatic infection, acute self-limited hepatitis, or fulminant hepatitis requiring liver transplantation. Persons infected with HBV may also develop chronic HBV infection, which can lead to cirrhosis or hepatocellular carcinoma. The likelihood that newly infected persons will develop chronic HBV infection is dependent on their age at the time of infection. More than 90 percent of infected infants, 25 50 percent of children infected between 1 and 5 years of age, and 610 percent of acutely infected older children and adults develop chronic infection. Immunosuppressed persons (e.g., hemodialysis patients and persons with human immunodeficiency virus infection) are also at higher risk of developing chronic infection. Because of the inverse association between age and risk of chronic infection, persons infected as children assume a disproportionately large burden of morbidity and mortality attributable to HBV. Up to 25 percent of infants and older children who acquire HBV eventually develop HBV related hepatocellular carcinoma or cirrhosis. Adults who have had chronic HBV infection since childhood develop primary hepatocellular carcinoma at a rate of 5 percent per decade, which is 100300 times the rate among uninfected persons (Shepard C. et al 2006). DIAGNOSTIC METHOD A number of assays are available to differentiate acute from chronic HBV infection, to evaluate relative infectivity and prognosis, and to assess the immune status of the patient. Among the many commercially licensed assays offered, the radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are the most sensitive and specific. These assays can detect HBsAg in the 0.2 to 0.7 ng/ml range, and anti-HBs at a level of 1 mIU/ml. Within limits, the level of reactivity represents an approximation of the concentration of antigen or antibody present in a specimen. However, comparisons of results from one sample to another are usually meaningless unless paired samples are tested concurrently. Dilutions are often required because the capacity of commercial solid-phase systems is easily exceeded at relatively low concentrations. For example, most patients with acute HBV have HBsAg concentrations of 200g/mL or more, but the HBsAg assays reach saturation above 1 g/mL. Similarly, anti-HBs assays reach a plateau at concentrations that approach values of 300 to 11

500 mIU/mL. Reactivities that are close to the cutoff values of the assay may signify a falsepositive result (Fields B., 2001). Molecular Tests for HBV Quantification: Available Assays and Performance Characteristics First-generation assays for HBV DNA quantification in peripheral blood (usually serum or plasma) were based on solution hybridization technology and measured HBV DNA in picograms per milliliter. Solution hybridization was successful due to high viral loads in many patients with chronic hepatitis B. This assay was relatively insensitive (approximately 5.0 log10 copies/ml), and its linearity ranged from 5.0 to 10.0 log 10 copies/ml. Adaptation of advanced molecular technologies, such as signal and target amplification, led to the development of second-generation assays with enhanced sensitivity (as low as 200 copies/ml).The latest generation HBV quantification assays utilize real-time PCR and have improved analytical performance characteristics, including low limits of detection, broad linear ranges, and excellent precision (Figure 4). These advances have been demonstrated to translate into better clinical performance as manifested by better detection of low virus concentrations and elimination of specimen dilution for a large proportion of specimens with high viral loads. Real-time PCR assays have utilized a number of different methods for extraction of HBV DNA. Automated extraction methods with COBAS AmpliPrep (Roche Diagnostics) and MagNAPure (Roche Diagnostics) have been reported for RealArt HBV LC PCR reagents (Artus HBV PCR; QIAGEN Diagnostics) and COBAS TaqMan reagents. Manual column methods have also been reported for use with COBAS TaqMan reagents (Valsamakis A., 2007).

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FIGURE 4: Ranges of HBV DNA assays

ADVANTAGES AND DISADVANTAGES OF HBV DNA TESTING (Sablon E., Shapiro R., 2005) Advantages Earliest indicator of infectivity. Can help monitor effectiveness of antiviral therapy. Can help assess ongoing disease activity in chronic infections. Can indicate and confirm emergence of antiviral resistance. 13

Direct marker and gold standard for HBV viral replication. Useful marker of infectivity in presence of precore/core promoter mutants. Confirmation of spontaneous remission or co-infection.

Disadvantages Definitive role in patient management still to be clarified. Not yet recommended for routine evaluation. Not well standardized. Wide variation in test sensitivities. No gold standard among different methodologies. Relatively slower to detect drug resistance. Detection of low viral levels of uncertain clinical significance. Cut-off levels for inactive disease unclear. Threshold levels for progressive liver disease unknown.

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The aim of present work was to construct a set of standards to quantitate HBV DNA in patients sera using conventional PCR. DNA PURIFICATION FROM HBsAg POSITIVE BLOOD SAMPLES USING 1-2-3 DNA PURIFICATION PROTOCOL Principle: Blood DNA Isolation is based on silica-gel-membrane technology yielding high-purity nucleic acids most suitable for molecular biology like restriction digestion, ligation, labeling, amplification, radioactive and fluorescent sequencing.

FIGURE 5: Structure of Silica-Gel Materials In this silica based DNA purification systems, DNA is first released by chaotropic lysis buffer distruption followed by binding to the silica membrane in the presence of high concentration of chaotropic salts. Washes with ethanol will remove salts and other contaminants from the silica membrane binding DNA. The purified DNA is then eluted by low ionic solution such as water and TE buffer. Proteinase K, Reagent 1, and 100% Ethanol used in first step helps in lysis of protein, cell wall components with release of nucleic acids in the solution. Nucleic acids get attached on the silica-gel-membrane in the presence of chaotropic salts, while other components are drained off after centrifugation. Colunmwash step is being carried out using Reagent 2 for removal of proteins and divalent cations. After a wsah step, pure nucleic acids are eluted with Reagent 3 under low or no salt conditions I small volume. Advantages: Purification of nucleic acids with silica-gel membrane products is fast, convenient and economical. No time-consuming steps involved as in phenol-chloroform extractions, or alcohol or PEG precipitation.

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PCR AMPLIFICATION The polymerase chain reaction (PCR) is a technique widely used in molecular biology, microbiology, genetics, diagnostics, clinical laboratories, forensic science, environmental science, hereditary studies, paternity testing, and many other applications. The name, polymerase chain reaction, comes from the DNA polymerase used to amplify (replicate many times) a piece of DNA by in vitro enzymatic replication. The original molecule or molecules of DNA are replicated by the DNA polymerase enzyme, thus doubling the number of DNA molecules. Then each of these molecules is replicated in a second "cycle" of replication, resulting in four times the number of the original molecules. Again, each of these molecules is replicated in a third cycle of replication. This process is known as a "chain reaction" in which the original DNA template is exponentially amplified. With PCR it is possible to amplify a single piece of DNA, or a very small number of pieces of DNA, over many cycles, generating millions of copies of the original DNA molecule. PCR has been extensively modified to perform a wide array of genetic manipulations, diagnostic tests, and for many other uses. The method was first proposed in the early 1970s by H.Ghobind Khorana and his colleagues as a strategy to lessen the labor involved in chemical synthesis of genes. The technique was independently conceived 15 years later, given its present name, and put into practice by Kary Mullis and coworkers at Cetus Corporation, who described invitro amplification of single-copy mammalian genes using the Klenow fragment of Escherichia coli DNA Polymerase I. The use of a thermostable polymerase from Thermus aquaticus greatly increased the efficiency of PCR and opened the door to automation of the method. In addition to its simplicity, PCR is robust, speedy, and most of all, flexible. Essential Components of Polymerase Chain Reactions PCRs contain seven essential components:
A thermostable DNA polymerase to catalyze template-dependent synthesis of

DNA. A wide choice of enzymes is now available that vary in their fidelity, efficiency and ability to synthesize large DNA products. For routine PCRs, Taq polymerase (0.5-2.5 units per standard 25-50l reaction) remains the enzyme of choice. Taq (T.aquaticus) DNA

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polymerase, the first isolated and best understood of the thermostable DNA polymerase remains the workhorse of PCR in most laboratories.
A pair of synthetic oligonucleotides to prime DNA synthesis. Of the many factors

that influence the efficiency and specificity of the amplification reactions, none are more crucial than the design of oligonucleotide primers. Careful design of primers is required to obtain the desired products in high yield, to suppress amplification of unwanted sequences, and to facilitate subsequent manipulation of the amplified product. PCR primers should be 10-24 nucleotides in length. The GC content should be 40%-60%. The primer should not be self-complementary or complementary to any other primer in the reaction mixture, to prevent primer-dimer and hairpin formation. Melting temperatures of primer pairs should not differ by more than 5C, so that the GC content and length must be chosen accordingly. The melting and annealing temperatures of a primer are estimated as follows: if the primer is shorter than 25 nucleotides, the approximate melting temperature is calculated with the formula: Tm = 4 (G + C) + 2 (A + T). The annealing temperature should be about 5C lower than the melting temperature.
Deoxynucleoside triphosphates (dNTPs). Standard PCRs contain equimolar

amounts of dATP, dTTP, dCTP, dGTP. Concentrations of 200-250M of each dNTP are recommended for Taq polymerase in reactions containing 1.5mM MgCl2. In a 50l reaction, these amounts should allow synthesis of ~6-6.5g of DNA, which should be sufficient even for multiplex reactions in which eight or more primer pairs are used at the same time.
Divalents cations. All thermostable DNA polymerases require free divalent cations-

usually Mg2+, for activity. Some polymerases will also work, albeit less efficiently with buffers containing Mn2+. Calcium ions are quite ineffective. Because dNTPs and oligonucleotides bind Mg2+ the molar concentration of the cations must exceed the molar concentration of phosphate groups contributed by dNTPs plus primers. Although a concentration of 1.5 mM Mg2+ is routinely used, increasing the concentration of Mg2+ to 4.5 mM or 6 mM has been reported to decrease nonspecific priming in some cases and to increase it in others. Optimization can be achieved by comparing the yield obtained from a series of ten PCRs containing concentrations of Mg2+ ranging from 0.5 mM to 5.0 mM, in 0.5 mM increments. Preparations of template should not contain significant amounts of chelating agents such as EDTA or negatively charged ions, such as PO43-, which sequester Mg2+.

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 Buffer to maintain pH. Tris-Cl, adjusted to a pH between 8.3 and 8.8 at a room

temperature, is included in standard PCRs at a concentration of 10 mM. When incubated at 72C, the pH of the reaction mixture drops by more than a half unit, producing a buffer whose pH is ~7.2.
Monovalent cations. Standard PCR buffer contains 50 mM KCl and works well for

amplification of segments of DNA > 500 bp in length. Raising the KCl concentration to ~70100 mM often improves the yield of shorter DNA segments.
Template DNA. Template DNA containing target sequence can be added to PCR in

single-or double-stranded form. Closed circular DNA templates are amplified slightly less efficiently than linear DNAs. The typical amounts of yeast, bacterial, and plasmid DNAs used per reaction are 10ng. 1ng, and 1pg, respectively. Programming Polymerase Chain Reactions PCR is an iterative process, consisting of three elements: denaturation of the template by heat, annealing of the oligonucleotide primers to single-stranded target sequence(s), and extension of the annealed primers by a thermostable DNA polymerase.
Denaturation. Double-stranded DNA templates denature at a temperature that is

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 the DNA molecules, the greater the time required at the chosen denaturation temperature to separate the two strands completely. If the temperature of denaturation is too low or if the time is too short, only AT-rich regions of the template DNA will be denatured. When the temperature is reduce later in the PCR cycle, the template DNA will reanneal into a fully native condition. In PCR catalyzed by Taq DNA polymerase, denaturation is carried out at 94-95C, which is the highest temperature that the enzyme can endure for 30 or more cycles without sustaining excessive damage. Recommended denaturation for 45 seconds at 94-95C for routine amplification of linear DNA templates whose contents of G+C are 55% or less. Higher temperatures may be required to denature template and/ or target DNAs that are rich in G+C (>55%).
Annealing of primers to template DNA. The temperature used for annealing step

(Ta) is critical. If the annealing temperature is too high, the oligonucleotide primers anneal poorly, if at all, to the template and the yield of amplified DNA is very low. If the annealing 18

temperature is too low, nonspecific annealing of primers may occur, resulting in the amplification of unwanted segments of DNA. Annealing is usually carried out 3-5C lower than the calculated melting temperature at which the oligonucleotide primers dissociate from the templates.
Extension of oligonucleotide primers. It is carried out at or near the optimal

temperature for DNA synthesis catalyzed by the thermostable polymerase, which in the case of Taq DNA polymerase is 72-78C. In the first two cycles, extension from one primer proceeds beyond the sequence complementary to the binding site of the other primer. In the next cycle, the first molecules are produced whose length is equal to the segment of DNA delimited by by the binding sites of primers. From the third cycles onwards, this segment of DNA is amplified geometrically, whereas longer amplification products accumulate arithmetically. The polymerization rate of Taq polymerase is ~2000 nucleotides/minutes at the optimal temperature (72-78C) and as a thumb rule extension is carried out for 1 minute for every 1000 bp of product. For the last cycle of PCR, an extension time that is three times longer in the previous cycles, allows completion of all amplified products.
Number of cycles. The number of cycles required for amplification depends on the

number of copies of template DNA present at the beginning of the reaction and the efficiency of primer extension and amplification. Once established in geometric phase, the reaction proceeds until one of the components becomes limiting. At this point the yield of specific amplification products should be maximal, whereas nonspecific amplification products should be barely detectable, if at all. This is generally the case after ~30 cycles in PCRs containing ~105 copies of the target sequence and Taq DNA polymerase (efficiency ~0.7). At least 25 cycles are required to achieve acceptable levels of amplification of single-copy target sequences in mammalian DNA templates (Sambrook J., Russell D., 2001).

DETECTION IS CARRIED OUT USING AGAROSE GEL ELECTROPHORESIS The main uses of agarose gels are analysis of the size and conformation of nucleic acids in a sample, quantification of DNA and Separation and Extraction of DNA fragments. Here Agarose Gel Electrophoresis is used to analyze the nucleic acid from Isolated HBV samples by 1-2-3 DNA Purification Protocol after PCR Amplification. A molecular marker is

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run and compared with it. Also, it is used to separate and extract the analyzed desired DNA amplicons for futher purification and cloning into pGEM-T Easy Vector. PURIFICATION OF HBV DNA AMPLICONS FROM AGAROSE GEL The HBV PCR product to be ligated was purified using QIAquick gel extraction kit protocol using a microcentrifuge. For gel extraction or cleanup of DNA (70 bp to 10 kb) from enzymatic reactions are used. Advantages: Up to 95% recovery of ready-to-use DNA Fast procedure Cleanup of DNA up to 10 kb in three easy steps Gel loading dye for convenient sample analysis Principle: QIAquick Kits contain a silica membrane assembly for binding of DNA in high-salt buffer and elution with low-salt buffer or water. The purification procedure removes primers, nucleotides, enzymes, mineral oil, salts, agarose, ethidium bromide, and other impurities from DNA samples. Silica-membrane technology eliminates the problems and inconvenience associated with loose resins and slurries. Specialized binding buffers are optimized for specific applications and promote selective adsorption of DNA molecules within particular size ranges. Procedure: The QIAquick system uses a simple bind-wash-elute procedure. Gel slices are dissolved in a buffer containing a pH indicator, allowing easy determination of the optimal pH for DNA binding, and the mixture is applied to the QIAquick spin column. Nucleic acids adsorb to the silica membrane in the high-salt conditions provided by the buffer. Impurities are washed away and pure DNA is eluted with a small volume of low-salt buffer provided or water, ready to use in all subsequent applications. Applications: DNA fragments purified with the QIAquick System are ready for direct use in all applications, including: Radioactive and fluorescent sequencing Microarray analysis 20

Ligation and transformation Restriction digestion Labeling Microinjection PCR In vitro transcription

(QIAquick Spin Handbook)

LIGATION OF THE HBV PCR PRODUCT Cloning PCR products into T vectors The single 3 Adenosyl extension generated by Taq DNA polymerase provides a highly efficient method to clone HBV PCR products into a vector (T vector) containing a complementary unpaired 3 thymidyl residue. T vectors may be created by the following three methods:
Digest a vector with restriction enzymes such as XcmI, HphI, and MboII that

generates 3-terminal unpaired deoxythymidine residues. Use terminal transferase and dideoxyTTP to add a single protruding T residue to the 3-termini of a linearized vestor.
Use the template-independent terminal transferase activity of Taq DNA polymerase to

catalyze the addition of a T residue to the terminal 3- hydroxyl groups of a linearized vector. Alternatively, T vectors can be purchased ready-made from many commercial suppliers as components of cloning kits (e.g., pCR-Script [SK+] from Strategene; pCRII in the TA Cloning kit from Invitrogen; pGEM_T from Promega) (Sambrook J., Russell D., 2001) Vector Features: (From Technical Manual of Promega)
T-Overhangs for Easy PCR Cloning: The pGEM-T Easy Vector is linearized

vector with a single 3-terminal thymidine at both ends. The T-overhangs at the insertion site 21

greatly improve the efficiency of ligation of PCR products by preventing recircularization of the vector and providing a compatible overhang for PCR products generated by certain thermostable polymerases
Blue/White Selection of Recombinants: The PGEM-T Easy Vector are high-copy-

number vectors containing T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the -peptide coding region of the enzyme -galactosidase. Insertional inactivation of the -peptide allows identification of recombinants by blue/white screening on indicator plates.
Choice of Restriction Sites for Release of Insert: The PGEM-T Easy Vectors

contain numerous restriction sites within the multiple cloning regions. The PGEM-T Easy Vector multiple cloning region is flanked by recognition sites for the restriction enzymes EcoRI, BstZI and NotI, providing three single-enzyme digestions for release of the insert. A double-digestion may be used to release the insert from a vector.
Rapid Ligation: The PGEM-T Easy Vector Systems are supplied with 2X Rapid

Ligation Buffer. Ligation reactions using this buffer may be incubated for 1 hour at room temperature. The incubation period may be extended to increase the number of colonies after transformation. Generally, an overnight incubation at 4C produces the maximum number of transformants.

FIGURE 6: pGEM-T Easy Vector 22

TRANSFORMATIONS REACTIONS

USING

pGEM-T

EASY

VECTOR

LIGATION

Bacterial transformation is a process by which a recipient cell acquires genes from a free DNA molecule in surrounding medium. Transformation begins with uptake of DNA fragment from surrounding medium by a recipient cell and terminates with recombinant exchange of part of DNA from donor with homologous segment of recipient chromosomes. Since ability of cells to uptake DNA is limited, certain conditions are created such that uptake is greatly enhanced. A population of such cells is called competent cells. Competency is a definite physiological state and hence can be induced by certain growth factors. The usual technique involves a shift down in which cells are transferred from a relatively rich medium to a nutritionally poor medium resulting in appearance of variable number of competent cell in culture. Treating bacteria with ice-cold conditions and briefly heating it also induces cells into a transient competent state during which recipient bacteria can take up DNAs derived from a variety of sources. Competency is seen to arrive at a specific stage of growth of culture i.e. late log phase and depends on culture conditions. Initially a small fraction of cell becomes competent and excretes one or more protein(s) called competence factor, which converts remainder of cells into a competent one. The development of competence may require only a few minutes and can be maintained for some time. The appearance of competence can be enhanced or antagonized by presence or absence of certain amino acids such as arginine, glutamate depending upon growth conditions. Competent cells undergo a lot of changes in both cell wall and cell membrane. The cell wall becomes more porous resulting in leakage of enzymes into the medium. There is an increase in autolytic enzyme activity within competent cells and increase in DNA degrading activity at cell surface. A change in surface charge of cell has been suggested due to binding of competence factor which is positively charged. The positive charged should make it easier for the negatively charged DNA molecule to bind to the cell surface. Ultra structural changes such as increase in the mesosome number occur in competent cells. The structures have an effect of bringing cell surface closer to centre of cells, the location of nucleoid. It has been suggested that mesosomes represent the transport mechanisms of transforming DNA fragment. Thus in an intact cell DNA does not bind preferentially to middle or tip of cell rather than disturbing itself randomly over the cell surface. 23

DNA uptake is not species specific i.e. foreign DNA from any source can be taken up by recipient cell. E.g. here pGEM-T Easy Vector with insert of pGEM-T Easy Vector Ligation Reactions in it is used in transformation. In 1973, Cohen, Chan, Hsus method was shown to work for plasmid DNA. Once inside the bacterial cell, (the phage or plasmid) DNA replicates and expresses markers such as antibiotic resistance that allow selection of transformed cells, conferring their survival in presence of the antibiotic. The ability of bacteria to take up DNA is short lived. After exposure to the agents that enhance uptake, most strains of bacteria remain in a competent state for only 1-2days.

ISOLATION OF PLASMID FROM TRANSFORMANTS Alkaline lysis was first described by Birnboim and Doly in 1979 and has, with a few modifications, been the preferred method for plasmid DNA extraction from bacteria ever since.
Cell Growth and Harvesting: The procedure starts with the growth of the bacterial

cell culture harboring plasmid. When sufficient growth has been achieved, the cells are pelleted by centrifugation to remove them from the growth medium.
Re-suspension: The pellet is then re-suspended in a Solution 1 containing Tris,

EDTA, glucose and RNase A. Divalent cations (Mg2+, Ca2+) are essential for DNase activity and the integrity of the bacterial cell wall. EDTA chelates divalent cations in the solution preventing DNases from damaging the plasmid and also helps by destabilizing the cell wall. Glucose maintains the osmotic pressure so the cells dont burst and RNase A is included to degrade cellular RNA when the cells are lysed.
Lysis: The lysis buffer (Solution 2) contains sodium hydroxide (NaOH) and the

detergent Sodium Dodecyl (lauryl) Sulfate (SDS). SDS is there to solubilize the cell membrane. NaOH helps to break down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and your plasmid, to single stranded DNA (ssDNA). This process is called denaturation and is central part of the procedure, which is

24

why its called alkaline lysis. SDS also denatures most of the proteins in the cells, which helps with the separation of the proteins from the plasmid later in the process. It is important during this step to make sure that the re-suspension and lysis buffers are well mixed, although not too vigorously.
Neutralization: Addition of potassium acetate (Solution 3) returns decreases the

alkalinity of the mixture. Under these conditions the hydrogen bonding between the bases of the single stranded DNA can be re-established, so the ssDNA can re-nature to dsDNA. This is the selective part. While it is easy for the the small circular plasmid DNA to re-nature it is impossible to properly anneal those huge gDNA stretches. This is why its important to be gentle during the lysis step because vigorous mixing or vortexing will shear the gDNA producing shorter stretches that can re-anneal and contaminate your plasmid prep. While the double-stranded plasmid can dissolve easily in solution, the single stranded genomic DNA, the SDS and the denatured cellular proteins stick together through hydrophobic interactions to form a white precipitate. The precipitate can easily be separated from the plasmid DNA solution by centrifugation.
Cleaning and concentration: Now plasmid DNA has been separated from the

majority of the cell debris but is in a solution containing lots of salt, EDTA, RNase and residual cellular proteins and debris, so its not much use for downstream applications. The next step is to clean up the solution and concentrate the plasmid DNA. There are several ways to do this including phenol/chloroform extraction followed by ethanol precipitation and affinity chromotography-based methods using a support that preferentially binds to the plasmid DNA under certain conditions of salt or pH, but releases it under other conditions. Phenol/Chloroform extraction: Phenol chloroform extraction, normally followed by ethanol precipitation, is the traditional method to remove protein from a DNA sample. In this procedure, the DNA solution is mixed with phenol and chloroform. The water-soluble DNA partitions into the aqueous phase, while the proteins are denatured by the organic solvents and stay in the organic phase. The aqueous phase containing the protein-free DNA can then be collected. Advantages: A cheap and effective way to remove proteins from DNA solutions. 25

Disadvantages: Slow compared to most modern methods, there is a risk of phenol/chloroform carry-over into the final sample (which could inhibit downstream enzymatic reactions), chloroform and phenol are both hazardous chemicals.

SCREENING IS CARRIED OUT BY PERFORMING PCR (1-2-3HepB amplification kit) DETECTION USING AGAROSE GEL ELECTROPHORESIS The HBV Cloned Plasmid DNA Isolated is screened by running Agarose Gel Electrophoresis after PCR amplification. A molecular marker is run to screen it.

CALCULATING CONCENTRATION OF UNDILUTED HBV CLONED PLASMID DNA BY NANODROP 1000 Instrument Description: The Thermo Scientific NanoDrop 1000 Spectrophotometer measures 1 ul samples with high accuracy and reproducibility. The full spectrum (220nm750nm) spectrophotometer utilizes a patented sample retention technology that employs surface tension alone to hold the sample in place. This eliminates the need for cumbersome cuvettes and other sample containment devices and allows for clean up in seconds. In addition, the NanoDrop 1000 Spectrophotometer has the capability to measure highly concentrated samples without dilution (50X higher concentration than the samples measured by a standard cuvette spectrophotometer). Operation: A 1 ul sample is pipetted onto the end of a fiber optic cable (the receiving fiber). A second fiber optic cable (the source fiber) is then brought into contact with the liquid sample causing the liquid to bridge the gap between the fiber optic ends. The gap is controlled to both 1mm and 0.2 mm paths. A pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array is used to analyze the light after passing through the sample. The instrument is controlled by PC based software, and the data is logged in an archive file on the PC. Applications: UV/VIS spectrophotometry is simple for samples as small as 1 ul using the NanoDrop 1000 Spectrophotometer. The small sample requirement and ease of use make the NanoDrop 1000 Spectrophotometer ideally suited for measuring: 26

Nucleic acid concentration and purity of nucleic acid samples up to 3700 ng/ul (dsDNA) without dilution Fluorescent dye labeling density of nucleic acid microarray samples Purified protein analysis (A280) up to 100 mg/ml (BSA) Expanded spectrum measurement and quantitation of fluorescent dye labeled proteins, conjugates, and metalloproteins Bradford Assay analysis of protein BCA Assay analysis of protein Lowry Assay analysis of protein Pierce Protein 660 nm Protein Assay Cell density measurements General UV-Vis spectrophotometry (NanoDrop 1000 Spectrophotometer V3.7 Users Manual)

HBV POSITIVE CONTROL DETECTION LIMIT EXPERIMENT. The detection limit experiment is intended to estimate the lowest concentration of HBV DNA from Isolated Plasmid that can be measured. This low concentration limit is obviously of interest in designing a molecular based kit to quantitate HBV DNA where the presence of the HBV DNA is already confirmed. So, here 10 log dilutions are carried out of HBV Cloned Plasmid DNA and also 10 log dilution of HBV Blood DNA Isolated samples. VIRAL LOAD CALCULATION Viral load Calculation is done to calculate the number of HBV DNA copies/ml, from the HBV Cloned DNA plasmid and this with the help of detection limit experiment would help to estimate the viral load present in HBV Blood DNA Isolated samples while designing a molecular based assay. This calculation is based on the assumption that the average weight of a base pair (bp) is 650 Daltons. This means that one mole of a bp weighs 650 g and that the molecular weigth of any double stranded DNA template can be estimate by taking the product of its length (in bp) and 650. The inverse of the molecular weight is the number of moles of template present in one gram of material. Using Avogradros number, 6.0221023 molecules/mole, the number of molecules of the template per gram can be calculated: mol/g molecules/mol = molecules/g 27

Finally, the number of molecules or number of copies of template in the sample can be estimated by multiplying by 1109 to convert to ng and then multiplying by the amount of template (in ng). This calculator requires the user to input the amount of a template present (in ngs) and the length of the template (in bp) and with this information the number of copies of the template is calculated. The formula used is: Number of copies = (amount 6.0221023) / (length 1109 650) Number = (ng number/mole) / (bp ng/g g/mole of bp) (Staroscik A.)

MATERIALS AND METHODS

DNA PURIFICATION FROM HBsAg POSITIVE BLOOD SAMPLES Serologically HBsAg positive samples were obtained from K J Somaiya Medical Colleges serology laboratory. DNA from blood was isolated using 1-2-3 DNA Purification Protocol. Materials: Samples: Serologically HBsAg positive blood samples Chemicals and Reagents: Absolute alcohol, Reagent 1, Reagent 2, Reagent 3 Miscellaneous: Blood lysis tube, Column containing Collection tube, Centrifuge, 1ml droppers, Microdroppers, Gloves etc. Method:

The Laminar Hood was cleaned and UV irradiated before carrying out Blood DNA Isolation. (The tubes were numbered whenever needed to avoid confusion).

Gloves are worn before proceeding. 0.2ml of blood was taken from HBsAg positive sample to Blood lysis tube containing proteinase K using the 1ml dropper. 28

0.2ml Reagent 1 was added to the Blood lysis tube. (Care had been taken that the tip of the dropper was not touched to the mouth of Blood lysis tube).

The Blood lysis tubes were vortex for 15sec and then incubated at 55C for 10mins. After incubation, 0.2ml of absolute alcohol was added and allowed to vortex again for 15sec.

Lysed blood was transferred to the column with collection tube using 1ml dropper. Further centrifugation was carried out at 6500 g for 1min. The collection tube was discarded and column was placed in fresh wash tube to which 0.5ml of Reagent 2 was added. (Absolute alcohol should be added to Reagent 2 before use.).

The tubes were centrifuged at 6500 g for 1min. The above washing step was performed again with Reagent 2 and centrifugation was carried out at highest speed.

The collection tube from previous washing step was discarded and a new elution tube was placed to which 0.2ml of Reagent 3 was added.

The tubes were kept at room temperature for 5mins and further centrifuged at 6500 g for 1min. Elution tube contains eluted DNA, as eluted from column.

The isolated DNA from HBsAg positive Blood samples were either used directly or

stored at -20C.

29

FIGURE 7: DNA Purification Protocol

PCR AMPLIFICATION PCR Amplification setup was carried out for the HBV Blood DNA Isolated samples. Materials: Samples: HBV Blood DNA Isolated samples Chemicals and Reagents: Reagent A, Reagent B 30

Miscellaneous: HBV amplification tubes containing Reaction Mixture, Microdroppers, Chiller racks Gloves, etc. Amplification Setup

The Laminar Hood was cleaned and UV irradiated before carrying out PCR amplification. (The tubes were numbered to avoid confusion). Gloves are worn before proceeding. The HBV amplification tubes containing Reaction Mixture were briefly spin and placed on chiller rack.

The tubes were labeled as one negative control and sample no. Reagent A and B tubes were removed, thawed and briefly spinned. All HBV amplification tubes were opened to which 1 drop of Reagent A was added using the microdropper.

Then all tubes were closed except the tube which is labeled as negative control. 1 drop of reagent B is added to the negative control amplification tube using microdropper and closed tightly.

Everything was removed from the laminar hood except the amplification tubes. The tubes containing HBV DNA sample were then brought and placed in separate rack. Individual tube containing HBV DNA sample and HBV amplification tube was opened a 1 drop of DNA is added to the HBV amplification tube using microdropper.

Both the tubes were closed and same procedure was repeated for next samples. Tubes containing HBV DNA samples were removed from the laminar hood. The pair of gloves are removed and new pair is wore. The HBV amplification tubes were taken to the thermocycler machine and PCR cycle was run.

31

HBV PCR Program STEPS TEMPERATURE TIME CYCLES DENATURATION 94C 40sec 36 ANNEALING 58C 40sec POLYMERIZATION 72C 40sec Final extension for 10mins at 72C and then finalhold at 4C TABLE 5: HBV PCR Program

DETECTION IS CARRIED OUT USING AGAROSE GEL ELECTROPHORESIS An aliquot of the above HBV amplification reaction (from 3.2) was analyzed on an agarose gel before using in the ligation reaction to verify that the reaction produced the desired product. Materials: Samples: HBV Amplification tubes (from 3.2) Chemicals and Reagents: 1% Agarose, 50X TAE buffer, 1X TAE buffer, Gel loading buffer, 10mg/ml ethidium bromide, 100bp DNA ladder from Invitrogen Miscellaneous: Agarose gel electrophoresis unit, Micropipettes with tips, Gloves, UV Transilluminator, etc. Method: Gloves are worn before proceeding. 1% Agarose was prepared with 1X TAE by boiling. The molten agar was allowed to cool to 45C and then 10mg/ml Ethidium bromide was added. Then poured into gel casting plate, having a gel comb. The gel was allowed to set and after solidification comb was gently removed. Gel was placed in electrophoresis unit and submerged in 1X TAE buffer. 10l gel loading dye was used for each PCR detection tubes. The samples were loaded along with 100bp DNA ladder from Invitrogen. 32

The electrophoresis unit was run at 150V and after the run, gel was observed using UV transilluminator.

PURIFICATION OF HBV DNA AMPLICONS FROM AGAROSE GEL The HBV PCR product to be ligated was purified using QIAquick gel extraction kit protocol using a microcentrifuge. Materials: Samples: Agarose gel (from 3.3) Chemicals and Reagents: Buffer QG, Buffer PE, Buffer EB, 3M Sodium Acetate, Isopropanol, QIAquick spin column with collection tube, Miscellaneous: 1.5ml sterile eppendrof tubes, Scalpel, Micropipettes with tips etc. Method: QIAquick Gel Extraction Kit Protocol Using a Microcentrifuge

The DNA fragment was excised from the Agarose gel (from 3.3) with a clean, sharp scalpel and the size of the gel slice was minimized by removing extra agarose. The gel slice was weighed in 1.5ml vial and 3 volumes of Buffer QG were added to 1 volume of gel (100 mg ~ 100 l). Incubation was carried out at 50C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mixing by vortexing the tube every 23 min was done during the incubation. (Solubilize agarose completely).

After the gel slice has dissolved completely, the color of the mixture was checked i.e. yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture was orange or violet, 10 l of 3M sodium acetate, pH 5.0 was added and mix. The color of the mixture will turn to yellow. (The adsorption of DNA to the QIAquick membrane is efficient only at pH 7.5. Buffer QG contains a pH indicator which is yellow at pH 7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.)

Then, 1 gel volume of isopropanol was added to the sample and mixed. QIAquick spin column was placed in a provided 2 ml collection tube.

33

The sample was then added to the QIAquick column so that DNA can bind to the column, and centrifuged for 1 min. (The maximum volume of the column reservoir is 800 l. For sample volumes of more than 800 l, simply load and spin again).

The flow-through was discarded and QIAquick column was placed back in the same collection tube. 0.75ml of Buffer PE was added to wash QIAquick column and centrifuged for 1 min. The flow-through was discarded and the QIAquick column was centrifuged additionally for 1 min at 10,000 x g (~13,000 rpm). The QIAquick column was then placed into a clean 1.5 ml microcentrifuge tube. To elute DNA, 50 l of Buffer EB (10 mM TrisCl, pH 8.5) or H2O was added to the center of the QIAquick membrane and centrifugation was carried out for 1 min at maximum speed. Alternatively, for increased DNA concentration, add 30 l elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.

34

FIGURE 8: QIAquick Gel Extraction Kit Protocol Using a Microcentrifuge

LIGATION OF THE HBV PCR PRODUCT The purified DNA of HBV from step 3.4 was further used for ligation. Materials: Sample: Purified HBV DNA (from 3.4) Chemicals and Reagents: pGEM-T Easy Vector, 10X Ligation Buffer, Ligase enzyme, 10mM dATP Miscellaneous: Micropipettes with tips and Sterile eppendrof tubes Method: 35

Briefly centrifugation of the pGEM-T Easy Vector and Purified DNA of HBV (from 3.4) was carried out to collect the contents at the bottom of the tubes.

Ligation reaction was set up as described below.

SR No. 1 2 3 4 5

REACTION COMPONENTS 10X Ligation Buffer pGEM-T Easy Vector Purified DNA of HBV from step 3.4 Ligase enzyme 10mM dATP TABLE 6: Ligation reaction 1l 1l 6l 1l 1l

Vortex the 10X Ligation Buffer vigorously before each use. The reactions was mixed by pipetting and incubated overnight at 16C.

TRANSFORMATIONS REACTIONS

USING

pGEM-T

EASY

VECTOR

LIGATION

The pGEM-T Easy Vector ligation reactions (from 3.5) were used for carrying out transformation. Materials: Sample: The pGEM-T Easy Vector ligation reactions (from 3.5.) Strain: E.coli DH5 Chemicals and Reagents: Luria Bertani (LB) broth, LB agar plates, Ampicillin stock, 50mM CaCl2 Miscellaneous: Sterile eppendrof tubes, Ice bath, Water bath, Shaker, Incubator, Micropipettes, Pipettes, Spreader, etc. Method: Preparation of Competent Cells (Day 1)

36

Inoculation of one colony of E.coli DH5 strain from prestreaked plate (Streak E.coli DH5 culture on plain LB agar plate to isolate single colony) was carried out to 10ml LB broth.

Overnight incubation at 37C, on a shaker (150 rpm) was done. 200l of above culture was taken and inoculated in 20ml of plain LB. Incubation of above culture was done till O.D. of 0.3(600nm) was obtained. After getting O.D. above culture was centrifuged at 4C at 3000 rpm for 10min. Above cells were resuspended in 10ml ice cold 50mM CaCl2 solution and kept in ice bath for 1 hr.

This above culture was then centrifuge at 4C at 3000 rpm for 10min. The cells were then again resuspended in 2-3ml of ice cold 50mM CaCl2 solution. The competent cells were used directly for transformation or incubated on ice bath for overnight. This incubation increases transformation efficiency by 5 times.

Transformation (Day 2) The competent cells were thawed on ice for 5mins. Ligation product from 3.5 was added to 100l of competent cells and kept on ice for 30min with regular tapping after 5mins interval. After incubation heat shock at 42C for 90sec was given. Snap chill on ice immediately for 1-2mins. 1ml plain LB medium was then added and kept in an incubator shaker for 1hr at 37C. After 1hr, spread plate was done on LB agar plate with Ampicillin (50g/ml) as a selection marker. From above culture 100l-200l was used and spreaded aseptically on LB Ampicillin (50g/ml) agar plates. 37

The vial was centrifuged and the supernatant was discarded, leaving about 200l in the vial.

The cells are resuspended and the whole 200l of the culture was used for spreading on LB Ampicillin (50g/ml) agar plates.

The LB Ampicillin (50g/ml) agar plates were incubated overnight at 37C and then the plates were observed.

ISOLATION OF PLASMID FROM TRANSFORMANTS (from 3.6) Materials: Sample: Transformants (from 3.6) Chemicals and Reagents: LB broth, Ampicillin stock, RNase, Solution 1, Solution 2, Solution 3, Phenol:Chloroform:Isoamyl Alcohol(25:24:1), Isopropanol, 70% Ethanol, 1X Tris-EDTA Miscellaneous: Sterile eppendrof tubes, Micropipettes with tips Method:

1 colony of transformant from 3.6.2.2 was inoculated in 2ml Luria Bertani broth with Ampicillin (50g/ml) and incubated at 37C for overnight on shaker. On next day, 1.5ml overnight culture was transfered into eppendrof tube and centrifuged at 12000g for 30sec at 4C. The supernatant was decanted and resuspension of the pellet in 200l of ice cold Solution 1 and 5l of RNase (25mg/ml) was done and vortexed vigorously. 200l of Solution 2 was added and mixed by inverting and then incubated at room temperature for 3-5mins. Further 200l of Solution 3 was added and mixed by inverting and keeping on ice for 35mins. The above vial was centrifuged at 12000g for 5mins at 4C. Supernatant was then transferred to fresh eppendrof and equal volume of phenol:chloroform:isoamyl alcohol(25:24:1) was added. Centrifugation at 12000g for 5mins at room temperature was done. Supernatant was then transferred in fresh eppendrof and 0.8 volume of isopropanol added. 38

Again centrifuged at 12000g for 30mins at 4C. The supernatant was decanted and 200l of 70% ethanol was added. Centrifuged at 12000g for 20mins at 4C. The supernatant was decanted and the pellet was dried. The pellet was lastly resuspended in 10l of TE, incubated at 37C to homogenize for 10mins.

SCREENING IS CARRIED OUT BY PERFORMING PCR (1-2-3HepB amplification kit) DETECTION USING AGAROSE GEL ELECTROPHORESIS (as described in 3.3)

CALCULATING CONCENTRATION OF UNDILUTED HBV CLONED PLASMID DNA BY NANODROP 1000 Materials: Sample: Isolated Plasmid (from 3.7) Chemicals and Reagents: Sterile Distilled water Miscellaneous: Nanodrop1000, Micropipette with sterile tips Method:

Firstly, the computer and Nanodrop1000 was switched on. The operating software ND-1000 is selected and opened, with the sampling arm in the The sampling arm is opened, the upper and lower pedestal is cleaned with deionised water and wiped with tissue paper (this is done to avoid samples carryover). With the help of 0-2l pipette, firstly 2l deionised water was loaded onto the lower measurement pedestal and lowering the sampling arm into the down position; to ensure measurement pedestal surfaces are cleaned and then OK was clicked.

down position. Nucleic acid application module is selected.

The instrument is ready to use only when the message Initializing Spectrometer-please wait appears. Before taking sample measurement, blank measurement was taken by loading the blank buffer (TE) onto the lower measurement pedestal and lowering the sampling arm into the down position, clicked on Blank button which gives a straight line on the screen.

39

When the measurement was complete the blanking buffer was wiped from both the pedestals using the tissue paper. Then, measured button was clicked to initiate sample measurement. On the screen Sample type DNA-50 is selected. This was carried out in the same manner as Blank buffer being used. On the screen we get a graph with 260/280, 260/230, concentration of nucleic acid readings.

When the measurement was complete the sample buffer was wiped from both the pedestals using the tissue paper. Also was cleaned with deionised water and wiped.

DILUTION OF HBV CLONED PLASMID DNA FOR POSITIVE CONTROL DETECTION LIMIT EXPERIMENT. Materials: Sample: Isolated HBV Cloned Plasmid DNA (from 3.7) Chemicals and Reagents: 1X Tris-EDTA Miscellaneous: Sterile eppendrof tubes, Micropipettes with tips Method: The isolated HBV cloned plasmid DNA is further made to a stock of 1:1000 dilution which was used for Positive Control Detection Limit Experiment.

HBV POSITIVE CONTROL DETECTION LIMIT EXPERIMENT. Materials: Stock: 1:1000 Diluted HBV Cloned Plasmid DNA (from 3.10) Chemicals and Reagents: 1X Tris-EDTA, Reagent A, Reagent B Miscellaneous: HBV amplification tubes containing Reaction Mixture, Sterile eppendrof tubes, Micropipettes with tips, Microdroppers, chiller racks, gloves, etc. Method:

10 log dilution from 1:1000 stock HBV Cloned Plasmid DNA and PCR was performed by 1-2-3HepB amplification kit (from 3.2) & detection using AGE (as described in 3.3).

40

PCR set up for 36 cycles SR No. 1 2 3 4 5 6 WELL No. 1 2 3 4 5 6 REACTION REAGENT MIXTURE A (15L) (15L) CONDITIONS Negative control Positive control(1:104) 1:105 1:106 1:107 1:108 Tap the tubes, mix it and place them in thermo cycler. Set the PCR with appropriate condition. For detection AGE is performed

TABLE 7: 10 log dilution of HBV Cloned Plasmid DNA Also, 10 log dilution was carried out of HBV Blood DNA Isolated sample(serologically positive sample) PCR set up for 36 cycles SR No. 1 2 3 4 5 6 WELL No. 1 2 3 4 5 6 REACTION REAGENT MIXTURE A (15L) (15L) Negative control Tap the tubes, Positive 4 mix it and place control(1:10 ) them in thermo HBV cycler. sample(undiluted) Set the PCR HBV with sample(1:10) appropriate HBV condition. sample(1:102) For detection HBV 3 AGE is sample(1:10 ) 41 CONDITIONS

HBV sample(1:104)

performed

TABLE 8: 10 log dilution of HBV Blood DNA Isolated Sample DETECTION IS CARRIED OUT USING AGAROSE GEL ELECTROPHORESIS (as described in 3.3)
FOR MATETIALS USED IN ABOVE METHODS REFER APPENDIX

RESULTS
Construction of Standards for Quantitation of HBV DNA in patients sera was carried out using Conventional PCR with specific primers included in 1-2-3HepB amplification kit. In brief, the PCR was carried out in a final volume of 40 l containing 15 l HBV Blood DNA Isolated samples. The HBV amplification was carried out for 36 cycles (40 seconds at 94C, 40 seconds at 58C, 40 seconds at 72C). A final extension step was performed for 5 min at 72C. The PCR products were electrophoresed on 1% agarose gel and stained with ethidium bromide. A molecular standard in the form of 100bp ladder is run along with samples. The desired 513bp HBV amplicons are aligned with 500bp molecular marker as the primers specifically amplified HBV DNA present in HBV Blood DNA Isolated samples. In the figure 9 lane 1 is 100bp molecular marker, lane 2 is negative control (does not contain template DNA) with no band and lane 3,4 with HBV Blood DNA Isolated samples respectively showing amplified product in the range of 500bp. The desired 513bp amplicons is used for construction of HBV positive control standards for desiging a molecular based kit to detect and quantitate HBV DNA. Before ligation, the desired 513bp HBV amplicons containing band is excised from the Agarose gel for purification using QIAquick gel extraction kit protocol using a microcentrifuge. The purified HBV DNA (6 l) was added to the ligation reaction components to make a final volume of 10 l and incubated overnight at 16C. The overnight incubated pGEM-T Easy Vector ligation reactions were used for carrying out transformation.

42

FIGURE 9: Detection of HBV Blood DNA Isolated samples by AGE after PCR Amplification Lane 1: 100bp Molecular marker Lane 2: Negative control Lane 3: Sample 1 Lane 4: Sample 2

43

A mini prep isolation of HBV cloned plasmid DNA was carried out as mentioned in step 3.7of Materials and methods. The plasmid DNA was checked for the presence of the cloned HBV amplicon by performing PCR using 1-2-3HepB amplification kit and analysed using Agarose Gel Electrophoresis (AGE). A molecular marker of 100bp is run along with HBV cloned plasmid DNA. The desired 513bp HBV cloned plasmid DNA amplicon is aligned with 500bp molecular marker as the primers specifically amplified HBV Cloned Plamid DNA present in Isolated Plasmid obtained from transformants. In the figure10 lane 1 is HBV Cloned Plasmid DNA aligning in the range of 500bp band of 100bp molecular marker, lane 2 is negative control (does not contain template DNA) with no band and lane 3 is 100bp molecular marker.

FIGURE 10: Screening of HBV cloned plasmid by AGE after PCR Amplification Lane 1: HBV cloned plasmid Lane 2: Negative control Lane 3: 100bp Molecular marker

44

The concentration of undiluted HBV Cloned Plasmid DNA was calculated using NanoDrop 1000 which was found to be 0.49g/l. Further, dilution of HBV Cloned Plasmid DNA was done to prepare 1:1000 stock, this was used to carry out Detection limit experiment. The detection limit experiment is intended to estimate the lowest concentration of HBV DNA from Isolated Plasmid that can be measured. This low concentration limit is obviously of interest in designing a molecular based kit to quantitate HBV DNA where the presence of the HBV DNA is already confirmed. PCR Amplification of 10 log dilution from 1:1000 stock HBV Cloned Plasmid DNA for HBV positive control detection limit experiment and 10 log dilution of HBV Blood DNA Isolated samples was carried out using 1-2-3HepB amplification kit and detected using Agarose Gel Electrophoresis (AGE). In the figure 11 lane 1 is negative control (does not contain template DNA) with no band, lane 2 is positive control having HBV cloned plasmid DNA 1:104 diluted, lane 3-6 contains 10 log dilution of HBV cloned plasmid DNA from 1:105 to 1:108. The band intensity decreases as dilution of HBV cloned plasmid DNA increases. No band is seen in lane 6 having HBV cloned plasmid DNA (1:108 diluted).

FIGURE 11: HBV Positive Control Detection Limit Experiment (10 log Dilution of HBV cloned plasmid) 45

Lane 1: Negative control Lane 2: Positive Control as HBV cloned plasmid DNA(1:104 diluted) Lane 3: HBV cloned plasmid DNA (1:105 diluted) Lane 4: HBV cloned plasmid DNA (1:106 diluted) Lane 5: HBV cloned plasmid DNA (1:107 diluted) Lane 6: HBV cloned plasmid DNA (1:108 diluted)

LANE No. 1 2 3 4 5 6

BAND Negative control Positive control(1:104) 1:105 1:106 1:107 1:108 +++ +++ ++ + -

TABLE 9: Results of HBV Positive Control Detection Limit Experiment (10 log Dilution of HBV cloned plasmid) KEY: + : Negative : Less intense

++ : Moderate intense +++ : Highly intense

10 log dilution was also carried for HBV Blood DNA Isolated samples to detect the lower concentration of HBV DNA present in the sera of a patients. In the figure 12 lane 1 is negative control (does not contain template DNA) with no band, lane 2 is positive control having HBV cloned plasmid DNA 1:104 diluted, lane 3-6 contains 10 log dilution of HBV Blood DNA Isolated Sample from undiluted to 1:104. The band intensity decreases as dilution 46

of HBV cloned plasmid DNA increases. No band is seen in lane 5-7 having HBV Blood DNA Isolated Sample from 1:102 to 1:104.

FIGURE 12: 10 log Dilution of HBV Blood DNA Isolated Sample Lane 1: Negative control Lane 2: Positive Control as HBV cloned plasmid DNA (1:104 diluted) Lane 3: HBV Blood DNA Isolated Sample (undiluted) Lane 4: HBV Blood DNA Isolated Sample (1:10 diluted) Lane 5: HBV Blood DNA Isolated Sample (1:102 diluted) Lane 6: HBV Blood DNA Isolated Sample (1:103 diluted) Lane 7: HBV Blood DNA Isolated Sample (1:104 diluted) 47

LANE No. 1 2 3 4 5 6 7

BAND Negative control Positive control(1:104) HBV sample(undiluted) HBV sample(1:10) HBV sample(1:102) HBV sample(1:103) HBV sample(1:104) +++ ++ + -

TABLE 10: 10 log Dilution of HBV Blood DNA Isolated Sample

KEY: + ++ : Negative : Less intense : Moderate intense

+++ : Highly intense

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CALCULATIONS: Concentration of undiluted plasmid: 0.49g/l by NanoDrop 1000. In HBV Positive Control Detection Limit Experiment 15l of 1:104 dilution is used in 40 l Reaction Mixture, so the template concentration would be 0.000018375ug or 0.018375 ng. To determine the number of copies of a HBV DNA the formula (URI Genomics & Sequencing Center) used is: Number of copies = (amount in ng * 6.022x1023) / (length * 1x109 * 650) = ( 0.018375 * 6.022 1023 ) / ( 513 * 1 109 * 650 ) = 0.11065425 1023 / 333450 109 = 3.32 107 Therefore, 3.321011 no. of HBV viral DNA copies is present in undiluted HBV Cloned Plasmid DNA. So, the low concentration of HBV DNA is detected in 1:107 dilution ie 3.32104 HBV DNA copies can be detected. Also, the HBV Blood DNA Isolated Sample (1:10 diluted) showed a low intense band, which is equivalent to the intensity of the 1:107 dilution of the control DNA shown in figure 11. So, 1:10 dilution of the HBV Blood DNA Isolated Sample contains 3.32104 HBV DNA copies, i.e. HBV Blood DNA Isolated Sample has 3.32105 HBV DNA copies in 15ul, or 2.213107 HBV DNA copies per ml of blood.

49

DISCUSSION
HBV DNA was cloned and further detection limit experiment is carried out to construct a set of standards to quantitate HBV DNA using conventional PCR. The number of viral copies when calculated was found to give sensitivity as low as 2.212107 HBV DNA copies per ml of blood by using a conventional PCR. The sensitivity by using conventional PCR was found to be similar with Hybrid Capture II (Digene) and also it is 10 4 to 105 times sensitive than Solution hybridization (Alexandra Valsamakis). Although conventional PCR is used in present study, the primers are specifically design that after 36 cycles of PCR, it gives enough amplicons of 513bp desired product which can be analysed by electrophoretic DNA separation and staining with ethidium bromide. Conventional PCR can be accepted as the most specific and sensitive technique over commercially available serological assays as

The HBV cloned Plasmid DNA which is 10 log diluted till 1:10 8, can be detected upto 1:107 dilution.

Also, the HBV Blood DNA Isolated Sample which is 10 log diluted till 1:10 4, can be detected upto 1:10 dilution.

PCR is a method known to be used since 10 years for detection of HBV DNA. Besides its accuracy and sensitivity, standardization is still a need of day. The main problem in the detection of HBV DNA is false positivity which is overcome by avoiding contamination and implementing stringent precautions like DNA Purification of HBsAg positive blood sample is carried out in different working place using 1-2-3 DNA Purification protocol which is easy to use; reaction mixtures for PCR are made in DNA and RNA free working space and also setting up a PCR.

50

CONCLUSION
Quantification of HBV DNA has become the most direct and reliable method for the accurate management of hepatitis B as antiviral therapy has dramatically improved the prognosis of HBV disease in active carriers. Moreover, HBV DNA level is an important prognostic factor for HBV-related hepatocellular carcinoma. Highly sensitive and reproducible molecular tests like conventional PCR to monitor viral load in sera of patients and assess the efficacy of the treatment. The present study shows that by using conventional PCR which is a sensitive, reliable, versatile, and cost effective method for HBV DNA in sera, allowing for a rapid and accurate quantification of HBV DNA levels. The system may further improve the management of acute and chronic HBV infection, the assessment of antiviral therapy, the emergence of drug resistance. Therefore a new assay using HBV positive control standard can be developed by using conventional PCR that can be considered as an important step for the clinical application of PCR while designing a molecular based kit to detect HBV DNA.

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APPENDIX (RECIPE FOR MATERIALS AND METHOD)

TABLE 11: DNA Purification Protocol CHEMICAL ENZYME REAGENT Alcohol Absolute Blood lysis tube Reagent 1 Reagent 2 Reagent 3

TABLE 12: Amplification Setup REAGENT HBV amplification Reaction Mixture Reagents A Reagents B tubes containing

TABLE 13: Detection is carried out Using Agarose Gel Electrophoresis CHEMICAL 1% Agarose 50X TAE Tris base 0.5M EDTA pH 8.0 Glacial acetic acid Distilled water to nearly Adjust pH to 8.5 Bring final volume to 1 L. Do no autoclave. For 1000ml 242.0 gm 100.0 ml 57.1 ml 1000.0 ml

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1X TAE For 50 ml 50X TAE 1 ml Distilled water 49 ml Gel loading Dye Ethidium Bromide For 100ml gel 10mg/ml, aqueous 10l 100bp DNA ladder from Invitrogen

TABLE 14: Transformations using pGEM-T Easy Vector REAGENT MEDIA 50mM CaCl2 LB Broth For 100ml Yeast Extract 0.5 gm Tryptone 1.0 gm NaCl 1.0 gm Distilled H2O 80.0 ml Adjust pH to 7.5 &bring volume to 100 ml. Autoclave. LB Ampicillin Agar Plate For 100ml Yeast Extract 0.5 gm Tryptone 1.0 gm NaCl 1.0 gm Distilled H2O 80.0 ml Adjust pH to 7.5 & bring volume to 100 ml. Agar 2% 2.0 gm Mix. Autoclave. After sufficient cooling add 100l of 100mg/ml Ampicillin stock (resulting concentration 50ug/ml) and pour the plates. LB Agar Plate For 100ml

Yeast Extract 0.5 gm Tryptone 1.0 gm NaCl 1.0 gm Distilled H2O 80.0 ml Adjust pH to 7.5 & bring volume to 100 ml. Agar 2% 2.0 gm Mix. Autoclave. After sufficient cooling pour the plates. 55

TABLE 15: Plasmid Isolation REAGENT Solution 1 50mM Glycine 25mM Tris-Cl (pH 8) 10mM EDTA (pH 8) Solution 2 (prepare fresh) 0.2N NaOH 1% SDS Solution 3 (pH 5.5) 5M Potassium acetate-50ml Glacial acetic acid-11.5ml Water-38.5ml Ampicillin Stock (50mg/ml) Working (50g/ml)

ANTIBIOTIC

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