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How Would I Isolate and Amplify a Viral Enzyme?

How Would I Isolate and Amplify a Viral Enzyme?


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what procedures would I use to isolate and amplify integrase? If I am trying to study the integrase enzyme which is found in HIV how would I 1) destroy the viral capsule to release its contents. 2) separate Integrase from other enzymes and the viral DNA. 3) make copies of the enzyme for testing. Any help would be greatly appreciated!!


Each virus would contain extremely low amounts of enzyme (probably a few individual molecules, at best); collecting and concentrating enough viruses to extract a significant amount of enzyme would be long and difficult, if at all possible.

The usual strategy in research laboratories is to clone the nucleic acid sequence coding for this protein (either by direct amplification from an HIV-infected cell, or by synthesizing a piece of DNA in vitro) and integrate it in an expression vector, that is, a larger piece of DNA, usually circular, suitable for integration in a host cell (human, or even bacterial) and carrying the sequences necessary for persistence and gene expression. This way you can produce thousands of copies of the enzyme you are interested in in each cell, and you can then purify a significant amount of integrase to study it.

To make purification easier, it is common practice to add to the gene a sequence coding for a "purification tag", that is, a few extra amino acids that have special properties that make the final protein easier to purify. Stretches of 6-8 histidine residues are very commonly used. The assumption is that the protein carrying this tag will have an activity similar to the natural enzyme, after purification.


A RADICA Approach to Viral Diagnostics

The COVID-19 pandemic has spurred a wave of new technologies for rapid viral diagnostics, given just how critical such innovations are in managing infectious disease outbreaks. Now, researchers have developed the RApid DIgital Crispr Approach, or RADICA, a molecular testing platform four times faster than the conventional polymerase chain reaction (PCR) method. The study was published in the journal Biomaterials.

The PCR test is among the most accurate and reliable means of detecting genetic material from a pathogenic organism to diagnose infections. This process takes around four hours and requires the use of specialized reagents and a thermal cycler in a diagnostic laboratory.

In contrast, RADICA can quantify viral nucleic acids in under an hour, using a simple water bath&mdashan inexpensive, standard piece of lab equipment. The team from the Singapore-MIT Alliance for Research and Technology (SMART) validated RADICA using two viral models: SARS-CoV-2 and the Epstein-Barr virus. According to the researchers, the technique is flexible and can be applied to identifying the presence of other viruses in biological samples and cell culture supernatants.

In the RADICA protocol, DNA or RNA from the sample is divided into thousands of individual 15 microlitre reactions. The Cas12a enzyme is then used to identify and amplify the presence of any viral nucleic acid, subsequently emitting a fluorescent signal. The number of reactions that glow positive reveals the copy number of viruses in the sample.

"This is the first reported method of detecting nucleic acids to utilize the sensitivity of isothermal amplification and specificity of CRISPR based detection in a digital format - allowing rapid and specific amplification of DNA without the time consuming and costly need for thermal cycling," said Xiaolin Wu, one of the inventors of RADICA. "RADICA offers four times faster absolute quantification compared to conventional digital PCR methods."


The PCR Technique

The polymerase chain reaction (PCR) was made possible by the discovery of thermophiles and thermophilic polymerase enzymes (enzymes that maintain structural integrity and functionality after heating at high temperatures). The steps involved in the PCR technique are as follows:

  • A mixture is created, with optimized concentrations of the DNA template, polymerase enzyme, primers, and dNTPs. The ability to heat the mixture without denaturing the enzyme allows for denaturing of the double helix of DNA sample at temperatures in the range of 94 degrees Celsius.
  • Following denaturation, the sample is cooled to a more moderate range, around 54 degrees, which facilitates the annealing (binding) of the primers to the single-stranded DNA templates.
  • In the third step of the cycle, the sample is reheated to 72 degrees, the ideal temperature for Taq DNA Polymerase, for elongation. During elongation, DNA polymerase uses the original single strand of DNA as a template to add complementary dNTPs to the 3’ ends of each primer and generate a section of double-stranded DNA in the region of the gene of interest.
  • Primers that have annealed to DNA sequences that are not an exact match do not remain annealed at 72 degrees, thus limiting elongation to the gene of interest.

This process of denaturing, annealing and elongation are repeated multiple (30-40) times, thereby increasing exponentially the number of copies of the desired gene in the mixture. Although this process would be quite tedious if performed manually, samples can be prepared and incubated in a programmable Thermocycler, now commonplace in most molecular laboratories, and a complete PCR reaction can be performed in 3-4 hours.

Each denaturing step stops the elongation process of the previous cycle, thus truncating the new strand of DNA and keeping it to approximately the size of the desired gene. The duration of the elongation cycle can be made longer or shorter depending on the size of the gene of interest, but eventually, through repeated cycles of PCR, the majority of templates will be restricted to the size of the gene of interest alone, as they will have been generated from products of both of the primers.

There are several different factors for successful PCR that can be manipulated to enhance the results. The most widely used method to test for the presence of PCR product is agarose gel electrophoresis. Which is used to separate DNA fragments based on size and charge. The fragments are then visualized using dyes or radioisotopes.


Abstract

As the most abundant biological entities with incredible diversity, bacteriophages (also known as phages) have been recognized as an important source of molecular machines for the development of genetic-engineering tools. At the same time, phages are crucial for establishing and improving basic theories of molecular biology. Studies on phages provide rich sources of essential elements for synthetic circuit design as well as powerful support for the improvement of directed evolution platforms. Therefore, phages play a vital role in the development of new technologies and central scientific concepts. After the RNA world hypothesis was proposed and developed, novel biological functions of RNA continue to be discovered. RNA and its related elements are widely used in many fields such as metabolic engineering and medical diagnosis, and their versatility led to a major role of RNA in synthetic biology. Further development of RNA-based technologies will advance synthetic biological tools as well as provide verification of the RNA world hypothesis. Most synthetic biology efforts are based on reconstructing existing biological systems, understanding fundamental biological processes, and developing new technologies. RNA-based technologies derived from phages will offer abundant sources for synthetic biological components. Moreover, phages as well as RNA have high impact on biological evolution, which is pivotal for understanding the origin of life, building artificial life-forms, and precisely reprogramming biological systems. This review discusses phage-derived RNA-based technologies terms of phage components, the phage lifecycle, and interactions between phages and bacteria. The significance of RNA-based technology derived from phages for synthetic biology and for understanding the earliest stages of biological evolution will be highlighted.


Data supporting improved characteristics of EquiPhi29 DNA Polymerase

In studies comparing other commercially available versions of Phi29 DNA polymerases, EquiPhi29 DNA polymerase demonstrated the lowest bias when amplifying targets with GC-rich content (Figure 1) and delivered the highest yield of a target sequence whether from DNA plasmid (Figure 2) or whole genomic DNA (Figure 3) within 2 hours.

Figure 1. EquiPhi29 DNA Polymerase demonstrated low GC bias when amplifying 3 bacterial genomes. A mixture of bacterial genomes with low-GC (S. aureus, 33% GC), moderate-GC (E. coli, 51% GC), and high-GC (P. aeruginosa, 68% GC) content was amplified using EquiPhi29 and Phi29 DNA polymerases as well as a DNA polymerase from another supplier. For each genome, the GC content of the reference genome, in 100 bp windows indicated in gray, was plotted versus the coverage normalized to the unamplified genome mix, indicated in green. In the absence of sequencing bias, all windows should be equally distributed close to the normalized coverage of 1, indicated in light blue. The normalized coverage obtained after amplification using different polymerases is shown. EquiPhi29 DNA Polymerase amplifies DNA with the lowest GC bias across all GC contents when compared to other DNA polymerases (EquiPhi29 DNA Polymerase is indicated in yellow).

Figure 2. EquiPhi29 DNA Polymerase delivered high plasmid DNA yields with faster reaction times than other suppliers’ products. Amplification of 0.5 ng of pUC19 plasmid DNA was carried out using EquiPhi29 DNA Polymerase, Phi29 DNA Polymerase, and DNA polymerases from other suppliers. The DNA products were purified using magnetic beads and quantified using the Qubit dsDNA BR Assay Kit. The recommended reaction temperature for EquiPhi29 DNA Polymerase is 42°C, delivering the highest yield after 2 hr of incubation.

Figure 3. EquiPhi29 DNA Polymerase delivered high genomic DNA yields with faster reaction times than other suppliers’ products. Amplification of 0.5 ng of human genomic DNA was carried out using EquiPhi29 and Phi29 DNA polymerases as well as DNA polymerases from other suppliers. The DNA products were purified using magnetic beads and quantified using the Qubit dsDNA BR Assay Kit. The recommended reaction temperature for EquiPhi29 DNA Polymerase is 42°C however, higher yields can be obtained after a 4 hr incubation at 30°C.

Phi29-type polymerases produce the best template for downstream assays from long intact DNA stretches. Therefore, it is important to denature DNA carefully but completely. The two most common methods include heat denaturation at 95°C and alkaline DNA denaturation. Heat denaturation carries the risk of DNA breakage, whereas alkaline denaturation may be incomplete and inconvenient. Intact but well-separated stretches of starting DNA ensure lower bias and higher yields. The more double-stranded DNA in a sample, the lower the performance of the WGA reaction.

Using the Phi29-type polymerases, the genome is amplified during multiple displacement amplification (MDA) reaction, which starts by binding of random primers to multiple sites of denatured DNA. The polymerase amplification involves strand displacement, therefore additional priming events occur on each displaced strand yielding a branched DNA product of up to 70 kb. The reaction takes place at a constant temperature.

Phi29-type polymerases are capable of replicating DNA from minute starting amounts without dissociating from the genomic DNA template (the average product length is greater than 10 kb). This feature makes it a great candidate for whole genome amplification from single cells. The larger the amount of DNA, and therefore the copy number of the genome, the more likely a specific locus will be detected after whole genome amplification.


A Level Biology - Gene Technologies:

Check Where this Lesson fits into your Exam Specification!

A Level Biology the polymerase chain reaction (PCR)

00:00 Introduction | Learning Outcomes

00:56 The Polymerase Chain Reaction

01:46 Why is PCR called PCR? / 1. "Polymerase"

02:48 What are Thermostable DNA Polymerase Enzymes?

04:05 Primers and annealing

04:38 What you need to know for your A level exams.

05:00 PCR - Step 1. Denaturation

05:30 PCR - Step 2. Annealing

05:50 PCR - Step 3. Polymerisation

06:23 PCR Amplifies DNA Exponentially with each cycle.

07:02 Applications of PCR (environmental, medical and forensic).


Introducing recombinant DNA into cells other than bacteria

Agrobacterium tumefaciens and the Ti plasmid

Agrobacterium tumefaciens is a plant pathogen that causes tumor formation called crown gall disease. The bacterium contains a plasmid known as the Ti (tumor inducing) plasmid, which inserts bacterial DNA into the host plant genome. Scientists utilize this natural process to do genetic engineering of plants by inserting foreign DNA into the Ti plasmid and removing the genes necessary for disease, allowing for the production of transgenic plants.

Gene gun

A gene gun uses very small metal particles (microprojectiles) coated with the recombinant DNA, which are blasted at plant or animal tissue at a high velocity. If the DNA is transformed or taken up by the cell&rsquos DNA, the genes are expressed.

Viral vectors

For a viral vector, virulence genes from a virus can be removed and foreign DNA inserted, allowing the virus capsid to be used as a mechanism for shuttling genetic material into a plant or animal cell. Marker genes are typically added that allow for identification of the cells that took up the genes.


Respiratory viral pathogens, quickly caught on-site!

Researchers in South Korea developed a plasmonic isothermal recombinase polymerase amplification (RPA) array chip, the world's first plasmoinc isothermal PCR technology which can detect 8 types of pathogens (4 bacteria and 4 viruses) that cause acute respiratory infectious diseases in 30 minutes, led by Dr. Sung-Gyu Park and Dr. Ho Sang Jung of the Korea Institute of Materials Science (KIMS, President Jung-Hwan Lee) and by Dr. Min-Young Lee and Dr. Ayoung Woo of Samsung Medical Center. KIMS is a government-funded research institute under the Ministry of Science and ICT.

* PCR(Polymerase Chain Reaction): A test method to amplify and detect nucleic acids target

The current detection technology for COVID-19 is impossible to analyze on-site as it takes about 4 hours or more to be confirmed after specimen collection, making it difficult to isolate the infectee as soon as possible.

To solve this problem, the researchers combined isothermal PCR technology with 3D Au nanostructured substrate which can amplify the fluorescence signal of RPA products with DNA amplicons and sucessfully detected bacterial DNA and viral RNA within 30 minutes.

In addition, the research team also developed a 3D plasmonic array chip for multiplex molecular detections: a chip that can simultaneously analyze 8 pathogens(4 bacteria and 4 viruses).

* 4 bacteria: Streptococcus pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae, Mycoplasma pneumoniae
* 4 viruses: Coronavirus 229E, OC42, NL63(Coronavirus 229E, OC43, NL63), Human metapneumovirus

The "multiplex diagnosis technology for acute respiratory infections" was also confirmed to be valid for clinical specimens collected by nasopharyngeal swabs. The team is planning to perform the reliability test of medical devices through large-scale clinical trials on COVID-19 infectees and applying for approval from the Ministry of Food and Drug Safety.

The "3D plasmonic nanomaterials technology for enhancing optical signal" of KIMS has already been patented in Korea, the US, and China, and the "on-site rapid pathogen detection technology" has been applied for a domestic patent jointly with Samsung Medical Center.

"We developed a medical device that can detect pathogens in half an hour on-site, by developing core plasmonic nanomaterials which enable ultra-sensitive pathogene diagnosis of more than 10 types of respiratory viral pathogens. The on-site molecular diagnostic devices can be prevalent rapidly as we actively research with Samsung Medical Center and domestic diagnostic device companies." said Dr. Sung-Gyu Park, a principal research scientist of KIMS.

Jung-hwan Lee, the president of KIMS said, "KIMS consistently supports to commercialize the on-site molecular diagnosis technology for respiratory infectious disease and ultrasensitive drug detection sensor technology which are based on the 3D highly sensitive plasmonic nanomaterials. We will do our utmost so that our research outcomes contribute to the quality of life and safe society."

This research was supported by the Fundamental Research Program of the Korea Institute of Materials Science (KIMS) and funded by Nano Plasmonic In Vitro Diagnostic Research Center of the Ministry of Science and ICT, and the alchemist project of the Ministry of Trade, Industry and Energy.

Also, the technology was published in Biosensors and Bioelectronics (IF:10.257), the principal international journal in the field of analytical chemistry.

* Research paper title: Rapid and sensitive multiplex molecular diagnosis of respiratory pathogens using plasmonic isothermal RPA array chip

The research team was selected as a national R&D excellence in 2020 by developing an ultra-sensitive detection sensor for sepsis through a 3D nano-biosensor chip.

KIMS is a non-profit government-funded research institute under the Ministry of Science and ICT (MSIT) of the Republic of Korea. As the only institute specializing in comprehensive materials technologies in Korea, KIMS has contributed to Korean industry by carrying out a wide range of activities related to materials science including R&D, inspection, testing&evaluation, and technology support.

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.


Talk Overview

Jennifer Doudna: Genome Engineering with CRISPR-Cas9: Birth of a Breakthrough Technology

Jennifer Doudna tells the story of how studying the way bacteria fight viral infection turned into a genomic engineering technology that has transformed molecular biology research. In 2013, Doudna and her colleagues developed the CRISPR-Cas9 gene expression system that, when introduced into animal cells, makes site-specific changes to intact genomes. CRISPR-Cas9 is more precise, more efficient, and less expensive than other genome editing tools and, as a result, has facilitated a wide range of studies that were previously unachievable.


Large-Scale Fermentations

Large-scale fermentations are key to the production of numerous products ranging from food to pharmaceutical items.

Learning Objectives

Describe fermentation and its applications to produce food, alcoholic beverages, fuel and recombinant products such as insulin

Key Takeaways

Key Points

  • Large-scale fermentations are utilized to create massive quantities of ethanol which are used for food production, alcohol production, and even gasoline production.
  • Fermentation is characterized by the metabolic processes that are used to transfer electrons released from nutrients to molecules obtained from the breakdown of those same nutrients.
  • Fermentation utilizes numerous organic compounds, such as sugars, as endogenous electron acceptors to promote the electron transfer that occurs.

Key Terms

  • oxidation: A reaction in which the atoms of an element lose electrons and the valence of the element increases.
  • amylase: A type of digestive enzyme capable of breaking down complex carbohydrates into simple sugars.

Fermentation includes the processes by which energy is extracted from the oxidation of organic compounds. The oxidation of organic compounds occurs by utilizing an endogenous electron acceptor to transfer electrons released from nutrients to molecules obtained from the breakdown of these same nutrients.

Common types of fermentation: These are common types of fermentation utilized in eukaryotic cells.

There are various types of fermentation which occur at the industrial level such as ethanol fermentation and fermentation processes used to produce food and wine. The ability to utilize the fermentation process in anaerobic conditions is critical to organisms which demand ATP production by glycolysis. Fermentation can be carried out in aerobic conditions as well, as in the case of yeast cells which prefer fermentation to oxidative phosphorylation. The following is a brief overview of a few types of the large-scale fermentations utilized by industries in production creation.

Ethanol Fermentation

Ethanol fermentation is used to produce ethanol for use in food, alcoholic beverages, and both fuel and industry. The process of ethanol fermentation occurs when sugars are converted into cellular energy. The sugars which are most often used include glucose, fructose, and sucrose. These sugars are converted into cellular energy and produce both ethanol and carbon dioxide as waste products. Yeast is the most commonly used organism to produce ethanol via the fermentation process for beer, wine, and alcoholic drink production. As stated previously, despite abundant amounts of oxygen which may be present, yeast prefer to utilize fermentation. Hence, the use of yeast on a large-scale to produce ethanol and carbon dioxide occurs in an anaerobic environment.

The ethanol which is produced can then be used in bread production. Yeast will convert the sugars present in the dough to cellular energy and produce both ethanol and carbon dioxide in the process. The ethanol will evaporate and the carbon dioxide will expand the dough. In regards to alcohol production, yeast will induce fermentation and produce ethanol. Specifically, in wine-making, the yeast will convert the sugars present in the grapes. In beer and additional alcohol such as vodka or whiskey, the yeast will convert the sugars produced as a result of the conversion of grain starches to sugar by amylase. Additionally, yeast fermentation is utilized to mass produce ethanol which is added to gasoline. The major source of sugar utilized for ethanol production in the US is currently corn however, crops such as sugarcane or sugar beets can be used as well.

Fermentation in grapes: This is a photograph of grapes undergoing fermentation during the wine-making process.

Recombinant Products

Fermentation is also utilized in the mass production of various recombinant products. These recombinant products include numerous pharmaceuticals such as insulin and hepatitis B vaccine. Insulin, produced by the pancreas, serves as a central regulator of carbohydrate and fat metabolism and is responsible for the regulation of glucose levels in the blood. Insulin is used medically to treat individuals diagnosed with diabetes mellitus. Specifically, individuals with type 1 diabetes are unable to produce insulin and those with type 2 diabetes often develop insulin resistance where the hormone is no longer effective.

The increase in individuals diagnosed with diabetes mellitus has resulted in an increase in demand for external insulin. The mass production of insulin is performed by utilizing both recombinant DNA technology and fermentation processes. E. coli, which has been genetically altered to produce proinsulin, is grown to a large amount to produce sufficient amounts in a fermentation broth. The proinsulin is then isolated via disruption of the cell and purified. There is further enzymatic reactions that occur to then convert the proinsulin to crude insulin which can be further altered for use as a medicinal compound.

An additional recombinant product that utilizes the fermentation process to be produced is the hepatitis B vaccine. The hepatitis B vaccine is developed to specifically target the hepatitis B virus infection. The creation of this vaccine utilizes both recombinant DNA technology and fermentation. A gene, HBV, which is specific for hepatitis B virus, is inserted into the genome of the organism yeast. The yeast is used to grow the HBV gene in large amounts and then harvested and purified. The process of fermentation is utilized to grow the yeast, thus promoting the production of large amounts of the HBV protein which was genetically added to the genome.



Comments:

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    Nada Syo take note !!!!

  2. Ball

    Bravo, your idea it is brilliant

  3. JoJomuro

    all this is dynamic and very positive

  4. Norberto

    Sorry, but this is not exactly what I need.



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