Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. Pyrosequencing relies on light detection based on a chain reaction when pyrophosphate is released. Hence, the name pyrosequencing.
Pyrosequencing of the DNA : Genomics and ProteomicsAbhay jha
Pyrosequencing of the DNA is sequencing technique which was one of the suitable method for DNA sequencing. It is very useful for the part of genomics and proteomics which will results into the knowledge of the DNA sequencing.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
Pyrosequencing of the DNA : Genomics and ProteomicsAbhay jha
Pyrosequencing of the DNA is sequencing technique which was one of the suitable method for DNA sequencing. It is very useful for the part of genomics and proteomics which will results into the knowledge of the DNA sequencing.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
The next generation sequencing platform of roche 454creativebiogene1
454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
Origin of Junk DNA Hypothesis
Types of Junk DNA
Mobile DNA Element: Overview
Rate of Transposition, Induction and Defence
Classification of Transposons
Transposable Elements in Bacteria
Mobile Genetic Elements in Eukaryotes
Drosophila Transposons
Human Retrotranspons
Transposons as Mutagens
Genetic Transformation using Transposons
Transposons and Genome Organization
Transposable Elements and Evolution
Transposons and Diseases
The next generation sequencing platform of roche 454creativebiogene1
454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
Origin of Junk DNA Hypothesis
Types of Junk DNA
Mobile DNA Element: Overview
Rate of Transposition, Induction and Defence
Classification of Transposons
Transposable Elements in Bacteria
Mobile Genetic Elements in Eukaryotes
Drosophila Transposons
Human Retrotranspons
Transposons as Mutagens
Genetic Transformation using Transposons
Transposons and Genome Organization
Transposable Elements and Evolution
Transposons and Diseases
Unlock the mysteries of life with our latest episode on DNA sequencing! Join us on a captivating journey into the world of genetics as we delve deep into the fascinating process of decoding the fundamental building blocks of life.
🔍 In this episode, we demystify the complexities of DNA sequencing, exploring the cutting-edge technologies and methodologies that scientists use to unravel the secrets hidden within our genetic code. From the revolutionary Sanger sequencing to the high-throughput wonders of Next-Generation Sequencing (NGS), we break down the techniques that have shaped our understanding of genetics.
🔬 Get ready to witness the incredible precision and innovation behind modern DNA sequencing machines, as we showcase how they read, analyze, and interpret the four-letter alphabet that comprises our genetic information. We'll explore the significance of DNA sequencing in various fields, from medicine and forensics to evolutionary biology and personalized genomics.
🌐 Join us as we interview leading experts in the field, gaining insights into the latest advancements and future possibilities of DNA sequencing technology. Learn about the impact of sequencing on medical diagnostics, disease research, and the development of personalized therapies.
📊 Dive into the world of bioinformatics, where powerful algorithms make sense of the vast amount of data generated by DNA sequencing. Discover how this information is transforming our understanding of human evolution, biodiversity, and the interconnectedness of all living organisms.
👩🔬 Whether you're a science enthusiast, student, or simply curious about the intricacies of life, this episode promises to unravel the wonders of DNA sequencing in an accessible and engaging manner. Don't miss out on this illuminating exploration of the code that defines us all!
In this lecture tried to introduce some basic methods of DNA sequencing like pyrosequencing, sequencing by ligation, sequencing by synthesis and Ion Semiconductor Sequencing
and describe them. Also introduced some new sequencing method (third generation sequencing) like SMRT (Single Molecule Real-Time Sequencing) and GridION.
It contains information about- DNA Sequencing; History and Era sequencing; Next Generation Sequencing- Introduction, Workflow, Illumina/Solexa sequencing, Roche/454 sequencing, Ion Torrent sequencing, ABI-SOLiD sequencing; Comparison between NGS & Sangers and NGS Platforms; Advantages and Applications of NGS; Future Applications of NGS.
The problems attract worldwide attention K/a Global Environmental Problems.
The top three environmental problems are: (1) Greenhouse Effect and Global Warming (2) Depletion of Ozone and (3) Acid Rain.
Aim1: To study the method of genome identification through ENSEMBL browser.
Aim2: To study the method of genome identification through VISTA.
Aim3: To study the method of genome identification through UCSC Genome Browser.
Aim4: To study the method of genome and amino acid sequences through UCSC Genome Browser.
Intracellular Components
We will now begin our discussion of intracellular organelles. As we have mentioned, only eukaryotic cells have intracellular sub-divisions, so our discussion will exclude prokaryotic cells. We will also focus on animal cells, since plant cells have a number of further specialized structures. In this section we will discuss the importance of the cell nucleus, mitochondria, peroxisomes, endoplasmic reticulum, golgi apparatus, and lysosome.
Types of Receptors
Receptors are protein molecules in the target cell or on its surface that bind ligands. There are two types of receptors: internal receptors and cell-surface receptors.
Microbial biomass conversion processes take advantage of the ability of microorganisms to consume and digest biomass and release hydrogen. Depending on the pathway, this research could result in commercial-scale systems in the mid- to long-term timeframe that could be suitable for distributed, semi-central, or central hydrogen production scales, depending on the feedstock used.
The cells derived from root apical and shoot-apical meristems and cambium differentiate and mature to perform specific functions. This act leading to maturation is termed as differentiation. During differentiation, cells undergo few to major structural changes both in their cell walls and protoplasm. The living differentiated cells, that by now have lost the capacity to divide can regain the capacity of division under certain conditions. This phenomenon is termed as dedifferentiation. For example, formation of meristems – interfascicular cambium and cork cambium from fully differentiated parenchyma cells. While doing so, such meristems / tissues are able to divide and produce cells that once again lose the capacity to divide but mature to perform specific functions, i.e., get redifferentiated.
Meat and milk from farmed animals including livestock (cattle, goat and buffalo) and poultry are sources of high quality protein and essential amino acids, minerals, fats and fatty acids, readily available vitamins, small quantities of carbohydrates and other bioactive components.1 The Food and Agriculture Organization (FAO) 2008 estimate shows that meat consumption has grown with increase in population. The average global per capita meat consumption is 42.1 kg/year with 82.9 kg/year in developed and 31.1 kg/year in developing countries in a recommended daily animal-sourced protein per capita of 50 kg per year2. Milk on the other hand is consumed in various forms: liquid, cheese, powder, and cream at a global per capita consumption of 108 kg per person per year which is way below the FAO recommended daily consumption of 200 kg.
Antibodies, also known as immunoglobulins, are secreted by B cells (plasma cells) to neutralize antigens such as bacteria and viruses. The classical representation of an antibody is a Y-shaped molecule composed of four polypeptides-two heavy chains and two light chains. Each tip of the "Y" contains a paratope (a structure analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together with precision. The ability of binding to an antigen has led to their ubiquitous use in a variety of life science and medical science. These antibodies can be classified into two primary types (monoclonal and polyclonal) by the means in which they are created from lymphocytes. Each of them has important role in the immune system, diagnostic exams, and treatments.
There are many characteristics of biological data. All these characteristics make the management of biological information a particularly challenging problem. Here mainly we will focus on characteristics of biological information and multidisciplinary field called bioinformatics. Bioinformatics, now a days has emerged with graduate degree programs in several universities.
Hormones, Proteins, etc. present in blood in minute concentration can be assayed by the recent advanced technique of “Enzyme Immuno Assay” without involving any disadvantage. The basic reaction is the interaction between an antibody and an antigen.
Meat and milk from farmed animals including livestock (cattle, goat and buffalo) and poultry are sources of high quality protein and essential amino acids, minerals, fats and fatty acids, readily available vitamins, small quantities of carbohydrates and other bioactive components.1 The Food and Agriculture Organization (FAO) 2008 estimate shows that meat consumption has grown with increase in population. The average global per capita meat consumption is 42.1 kg/year with 82.9 kg/year in developed and 31.1 kg/year in developing countries in a recommended daily animal-sourced protein per capita of 50 kg per year2. Milk on the other hand is consumed in various forms: liquid, cheese, powder, and cream at a global per capita consumption of 108 kg per person per year which is way below the FAO recommended daily consumption of 200 kg.
In shotgun sequencing the genome is broken randomly into short fragments (1 to 2 kbp long) suitable for sequencing. The fragments are ligated into a suitable vector and then partially sequenced. Around 400–500 bp of sequence can be generated from each fragment in a single sequencing run. In some cases, both ends of a fragment are sequenced. Computerized searching for overlaps between individual sequences then assembles the complete sequence.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Vaccine (L. vacca = cow) is a preparation/suspension or extract of dead/attenuated (weakened) germs of a disease which on inoculation (injection) into a healthy person provides temporary/permanent active/passive immunity by inducing antibodies formation.
Thus antibody provoking agents are called vaccines.
Biological treatment is an important and integral part of any wastewater treatment plant that treats wastewater from either municipality or industry having soluble organic impurities or a mix of the two types of wastewater sources.
The four processes are: (1) Preliminary Treatment (2) Primary Treatment (3) Secondary or Biological Treatment and (4) Tertiary or Advanced Treatment
The genetic variations found in the in vitro cultured cells are collectively referred to as somaclonal variations.
The plants derived from such cells are referred to somaclones. Some authors use the terms calliclones and proto-clones to represent cultures obtained from callus and protoplasts respectively.
The growth of plant cells in vitro is an asexual process involving only mitotic division of cells. Thus, culturing of cells is the method to clone a particular genotype. It is therefore expected that plants arising from a given tissue culture should be the exact copies of the parental plant.
The occurrence of phenotypic variants among the regenerated plants (from tissue cultures) has been known for several years. These variations were earlier dismissed as tissue culture artefacts. The term somaclonal variations was first used by Larkin and Scowcraft (1981) for variations arising due to culture of cells, i.e., variability generated by a tissue culture. This term is now universally accepted.
As described elsewhere the explant used in tissue culture may come from any part of the plant organs or cells. These include leaves, roots, protoplasts, microspores and embryos. Somaclonal variations are reported in all types of plant tissue cultures.
In recent years, the term gametoclonal variations is used for the variations observed in the regenerated plants from gametic cells (e.g., anther cultures). For the plants obtained from protoplast cultures, proto-clonal variations is used.
Solid waste management is a polite term for garbage management. As long as humans have been living in settled communities, solid waste, or garbage, has been an issue, and modern societies generate far more solid waste than early humans ever did.
The chemical compounds produced by plants are collectively referred to as phytochemicals. Biotechnologists have special interest in plant tissue culture for the large scale production of commercially important compounds. These include pharmaceuticals, flavours, fragrances, cosmetics, food additives, feed stocks and antimicrobials.
Most of these products are secondary metabolites— chemical compounds that do not participate in metabolism of plants. Thus, secondary metabolites are not directly needed by plants as they do not perform any physiological function (as is the case with primary metabolites such as amino acids, nucleic acids etc.). Although the native plants are capable of producing the secondary metabolites of commercial interest, tissue culture systems are preferred.
The Roman Empire A Historical Colossus.pdfkaushalkr1407
The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
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Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
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This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
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Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
2. Pyrosequencing
Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in
DNA) based on the "sequencing by synthesis" principle, in which the sequencing is
performed by detecting the nucleotide incorporated by a DNA polymerase. Pyrosequencing
relies on light detection based on a chain reaction when pyrophosphate is released. Hence,
the name pyrosequencing.
The principle of Pyrosequencing was first described in 1993 by Bertil Pettersson, Mathias
Uhlen and Pål Nyren by combining the solid phas sequencing method using streptavidin coated
magnetic beads with recombinant DNA polymerase lacking 3´to 5´exonuclease activity (proof-
reading) and luminescence detection using the firefly luciferase enzyme. A mixture of
three enzymes (DNA polymerase, ATP sulfurylase and firefly luciferase) and a nucleotide (dNTP)
are added to single stranded DNA to be sequenced and the incorporation of nucleotide is
followed by measuring the light emitted. The intensity of the light determines if 0, 1 or more
nucleotides have been incorporated, thus showing how many complementary nucleotides are
present on the template strand. The nucleotide mixture is removed before the next nucleotide
mixture is added. This process is repeated with each of the four nucleotides until the DNA
sequence of the single stranded template is determined.
A second solution-based method for Pyrosequencing was described in 1998 by Mostafa
Ronaghi, Mathias Uhlen and Pål Nyren. In this alternative method, an additional
enzyme apyrase is introduced to remove nucleotides that are not incorporated by the DNA
polymerase. This enabled the enzyme mixture including the DNA polymerase, the luciferase and
the apyrase to be added at the start and kept throughout the procedure, thus providing a simple
set-up suitable for automation. An automated instrument based on this principle was introduced
to the market the following year by the company Pyrosequencing.
3. A third microfluidic variant of the Pyrosequencing method was described in
2005 by Jonathan Rothberg and co-workers at the company 454 Life Sciences. This
alternative approach for Pyrosequencing was based on the original principle of attaching
the DNA to be sequenced to a solid support and they showed that sequencing could be
performed in a highly parallel manner using a microfabricated microarray. This allowed for
high-throughput DNA sequencing and an automated instrument was introduced to the
market. This became the first next generation sequencing instrument starting a new era
in genomics research, with rapidly falling prices for DNA sequencing allowing whole genome
sequencing at affordable prices.
Procedure: "Sequencing by synthesis" involves taking a single strand of the DNA to be
sequenced and then synthesizing its complementary strand enzymatically. The
pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA
synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method
allows sequencing a single strand of DNA by synthesizing the complementary strand along
it, one base pair at a time, and detecting which base was actually added at each step. The
template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially
added and removed from the reaction. Light is produced only when the nucleotide solution
complements the first unpaired base of the template. The sequence of solutions which
produce chemiluminescent signals allows the determination of the sequence of the
template.For the solution-based version of Pyrosequencing, the single-strand DNA (ssDNA)
template is hybridized to a sequencing primer and incubated with the enzymes DNA
polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´
phosphosulfate (APS) and luciferin.
4. The addition of one of the four deoxynucleotide triphosphates (dNTPs) (dATPαS, which is
not a substrate for a luciferase, is added instead of dATP to avoid noise) initiates the
second step. DNA polymerase incorporates the correct, complementary dNTPs onto the
template. This incorporation releases pyrophosphate (PPi).
ATP sulfurylase converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This
ATP acts as a substrate for the luciferase-mediated conversion of luciferin to oxyluciferin
that generates visible light in amounts that are proportional to the amount. The light
produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a
program.
Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can
restart with another nucleotide.
The process can be represented by the following equations:
PPi + APS → ATP + Sulfate (catalyzed by ATP-sulfurylase);
ATP + luciferin + O2 → AMP + PPi + oxyluciferin + CO2 + hv (catalyzed by luciferase);
where:
PPi is pyrophosphate
APS is adenosine 5-phosphosulfate;
ATP is adenosine triphosphate;
O2 is oxygen molecule;
AMP is adenosine monophosphate;
CO2 is carbon dioxide;
hv is light.
5.
6. Limitations
Currently, a limitation of the method is that the lengths of individual reads of DNA
sequence are in the neighborhood of 300-500 nucleotides, shorter than the 800-1000
obtainable with chain termination methods (e.g. Sanger sequencing). This can make the
process of genome assembly more difficult, particularly for sequences containing a large
amount of repetitive DNA. Lack of proof-reading activity limits accuracy of this method.
Commercialization
The company Pyrosequencing AB in Uppsala, Sweden was founded with venture
capital provided by HealthCap in order to commercialize machinery and reagents for
sequencing short stretches of DNA using the pyrosequencing technique. Pyrosequencing
AB was listed on the Stockholm Stock Exchange in 1999. It was renamed to Biotage in
2003. The pyrosequencing business line was acquired by Qiagen in 2008. Pyrosequencing
technology was further licensed to 454 Life Sciences. 454 developed an array-based
pyrosequencing technology which emerged as a platform for large-scale DNA sequencing,
including genome sequencing and metagenomics.
Roche announced the discontinuation of the 454 sequencing platform in 2013 when its
technology became noncompetitive.
Thank you
References: Online notes, notes from research papers and Books by google search Engine