...Polymerase Chain reaction (PCR) Name of student Name of Institution Abstract Polymerase chain reaction (PCR) is greatly used in molecular genetics. It entails amplification of a single DNA strand into millions of similar DNA fragments. It involves three stages in each cycle. It is repeated to about 30 cycles. This method is vital as it is used in various processes such molecular identification, genetic engineering, and sequencing. The three stages in each cycle have varying duration and temperature. A thermal cycler is involved in the regulation of temperature in various stages. Over time, various modifications have been done to PCR technique so that it can be applied in specific roles. The PCR has been of aid in the diagnosis of diseases and other numerous applications. In the near future, PCR will be advanced and perhaps replaced by better techniques. Nevertheless, PCR will remain critical for future advancements in molecular genetics. Introduction Polymerase chain reaction (PCR) is broadly employed by scientists in biochemistry and molecular biology. Thus, its essence cannot be underestimated in the development of genetic analysis and gene manipulation. The technique was established by Karry Mullis in the early 1980s. It entails amplification of a single or several DNA fragments into millions of identical copies of DNA. The process is done by repeated number...
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...The polymerase chain reaction is a laboratory process in which a specific sequence of deoxyribonucleic acid (DNA) is amplified producing many copies of the specific DNA sequence. However, their must be components such as (DNA template, primers, DNA polymerase, deoxyribonucleotide triphosphates (dNTP’s), buffer solution, and magnesium chloride salt solution) are required to carry out the process which undergoes through three major stages to make the copies of DNA segment. First stage is denaturation, after that annealing, then extension. However, this can be done if and only if the 3’ and 5’ ends are known, this helps in initiating DNA synthesis in which it is ensured that two short oligonucleotides acts as primer will anneal onto DNA strands. Polymerase chain reaction process is used as a diagnostic and research tool due to the fact that it can be done within a few hours which makes it a rapid assay....
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...2014-2018 Global Polymerase Chain Reaction (PCR) Market EMISPDF us-thermo-url from 208.89.140.11 on 2014-11-24 17:09:41 GMT. DownloadPDF. technavio insights Downloaded by us-thermo-url from 208.89.140.11 at 2014-11-24 17:09:41 GMT. EMIS. Unauthorized Distribution Prohibited. 2014-2018 Global Polymerase Chain Reaction (PCR) Market Table of Contents 01. Executive Summary.......................................... 1 02. List of Abbreviations ......................................... 2 03. Scope of the Report.......................................... 3 03.1 Market Overview .......................................................... 3 03.2 Product Offerings .......................................................... 3 04. Market Research Methodology ..................... 6 04.1 Market Research Process .......................................... 6 04.2 Research Methodology .............................................. 6 05. Introduction ....................................................... 8 06. Market Landscape ........................................... 9 06.1 Market Overview .......................................................... 9 06.2 Market Size and Forecast........................................... 9 06.3 PCR Market in US ........................................................ 10 06.3.1 Mark et Size and Forecast .................................................. 10 06.4 PCR Market in Europe......................................
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...CURRICULUM VITAE NAME: BERNARD NDINI MWENDWA PHONE NUMBER: 0789921182/ 0707343489/0727609248 E-MAIL: benardmwendwa.bm@gmail.com/benardndini@yahoo.com ADDRESS: 16-90214 DOB: 11/7/1992 GENDER: MALE NATIONALITY: KENYAN ID NUMBER: 29808279 RELIGION: CHRISTIAN MARITAL STATUS: SINGLE LANGUAGES: ENGLISH, KISWAHILI (both spoken and written) SUMMARY A hard-working and motivated BSC Biochemistry and Molecular Biology graduate with proven communication, organization and numeracy skills seeking to gain relevant experience to diversify and excel in varying fields. Looking to apply solid knowledge of biochemistry and molecular biology practices to setting and building on skills developed during course work studies. Eager to share the knowledge I have gained. Pro-active and keen to learn, ready to back up the knowledge I have gained with relevant experience .Wishing to make a positive contribution to production and research institutions. EDUCATION BACKGROUND 2012-2015: BSC BIOCHEMISTRY (MOLECULAR BIOLOGY) JOMO KENYATTA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY, SECOND CLASS HONOURS (UPPER DIVISION) Jan 2007 –Nov 2010: KENYA CERTIFICATE OF SECONDARY EDUCATION St JOSEPH’S MUTITO BOYS SECONDARY SCHOOL GRADE ATTAINED:...
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...Eyeless mutation gene located within the second intron of Drosophila melanogaster Justin Lazarus Genetic 300 Abstract The following experiment was conduct over a several week time span to determine and identify the mutation that is causing the eyeless mutation within the Drosophila melanogaster fruit flies. The experiment included genome sequencing and comparison between the Drosophila melanogaster wild type and the Drosophila melanogaster eyeless type. After combining the two different phenotypes. We determined that we were unable to visualize the mutation at a chromosomal level, as both wild-type and eyeless flies looked similar. The experiment involved electrophoresis and Polymerase Chain Reaction (PCR) through which we were able to isolate and amplify the needed DNA eyeless DNA. The difference between the wild-type Drosophila melanogaster and the eyeless Drosophila melanogaster is approximately only 500-nucleotide base pairs. As we see the eyeless phenotype is approximately 3000 base pairs in length while the wild-type phenotype is approximately 2500 nucleotides base pairs in length, a difference of about 500 base pairs. After completing nucleotide sequencing and comparing our data on the blast website, we determined that the eyeless mutation has being interest exons two and three, but more specifically the mutation itself was located within the second intron at base pairs 8264 to 9212. Introduction In the early 20th century scientists had already been acquainted with...
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...POLYMERASE CHAIN REACTION (PCR) PCR stands for the Polymerase Chain Reaction and was developed in 1987 by Kary Mullis (which won him a Nobel Prize) and associates. With this technique it is possible to make virtually unlimited copies of a single DNA molecule even though it is initially present in a mixture containing many different DNA molecules. It is used to amplify a specific DNA (target) sequence lying between known positions (flanks) on a double-stranded (ds) DNA molecule. The polymerase chain reaction can be used to amplify both double and single stranded DNA. In order to perform PCR, one must know at least a portion of the sequence of the target DNA molecule that has to be copied. Generally, PCR amplifies small DNA targets 100-1000 base pairs (bp) long. It is technically difficult to amplify targets >5000 bp long. A pair of single stranded oligonucleotide primers, which have DNA sequences complementary to the flanking regions of the target sequence, must be synthesized. The primers are complementary to either end of the target sequence but lie on opposite strands. The primers are usually 20-30 nucleotides long and bind to complementary flanking region at 3' end. Requirements: Thermal cycler (thermocycler) PCR amplification mix typically containing: Sample dsDNA with a target sequence Thermostable DNA polymerase Two oligonucleotide primers Deoxynucleotide triphosphates (dNTPs) Reaction buffer containing magnesium ions and other components Procedure: 1. The DNA molecule...
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...copies of the gene responsible for the fluorescence are needed. Perform the Polymerase Chain Reaction (PCR) and analyze the PCR reactions via agarose gel electrophoresis. NOTE: Read and all the instructions carefully before starting your experiment. Facilitators will guide you on the use of PCR machine and agarose gel electrophoresis. Be cautious when there is a need to handle items/equipments and the hazardous reagent, Ethidium Bromide (EB) in the EB room. Materials: Agarose gel electrophoresis set DNA ladder (marker) DNA loading dye DNA samples (labelled as DNA 1 and DNA 2; DNA 2 is obtained from P3) Deionised water Ice in tub Biohazard bin Disposable pipette tips Gloves Microcentrifuge (table top) Micropipettes (P1000, P100, P20) Microcentrifuge tubes (1.5ml) Paper Towels PCR machine PCR master mix (Taq Polymerase, MgCl2, dNTPs, buffer) Primers (labelled as Primer 1 and Primer 2) Methods: LAB 1: AM session (9.15am to 12noon) Part A: The Polymerase Chain Reaction (PCR) As a team, you will be using TWO DNA samples to perform TWO PCR reactions; DNA 1 will be given and DNA 2 which is obtained from your P3. NOTE: If you have TWO DNA samples from P3, choose the ONE with better purity or quantity. 1. Refer to Table 1 and complete the components and volumes needed to make PCR master mix. 2. For TWO PCR reactions, you need to prepare PCR master mix for 2.5 reactions (on ice). Pipette the solutions 1, 2, 3, 4 into a sterile microcentrifuge...
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...Maheetha Bharadwaj Retrieved from the Progeria Research Foundation Website: progeriaresearchfoundation.org Table of Contents Introduction and History Research of Erikkson et al. (2003) Causes: Mechanisms of mRNA Splicing Truncated Lamina A: Progerin Reverse Transcriptase Polymerase Chain Reaction Effects Treatment: Macro and Molecular Conclusion Bibiography Introduction and History The Hutchinson-Gilford Progeria Syndrome (Progeria): fatal disease that causes rapid aging Only 1 in 8 million have this disease First appearance: Hutchinson(1886) Second appearance: Gilford (1904) Only roughly 60 cases have been reported since: (Debrusk 1972, Brown et al. 1985 & 1986) Erikkson et al. 2003—f rst research done to show actual i causes and effects Research of Erikkson et. al (2003) 1. Out of 23 progeria, he found that 20 had a de novo mutation in LMNA gene(codes for nuclear Lamina A) 2. 18 out of 20: GGCGGT 1 out of 20: GGCAGC 1 out of 20: GAGAAG The causes of Progeria in the other three cases are unknown 3. Creation of splice site Mechanisms of mRNA Splicing Introns vs. Exons Donor acceptor pairs: GT-AG, GC-AG, AT-AC, and GT-GG. (Fong et al. 2006). The base pair progressions, GT, GC, and AT have the potential to set of a splice site. The splice site signals the excision of genetic information, leading to a deletion of 150 bps, and 50 amino acids. Retrieved from: http://progeria2010researchproject...
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...Part 1 - Sample Preparation The key to a successful identification is to start with a "good" sample. Many pathogenic bacteria do not grow well on solid culture medium, making identification by traditional means difficult. (Why?) Even poorly growing bacterial cultures, however, can be identified with the methods used in this lab. In this lab, you will act as a pathologist or perhaps a pathology lab technician at a well-equipped research hospital. Your task is to identify a bacterial sample received from a clinician. Assuming that you have managed to grow bacterial colonies on a solid medium culture dish, the first step is to pick up a single colony and drop it into a microcentrifuge tube. The process of extracting bacterial DNA consists of dissolving the cell wall with a digestive buffer (in the white-capped bottle) available as a commercial kit. The buffer contains proteolytic enzymes that "eat" the cell wall. This step may take several hours. Since we will be using other enzymes in the next step, we need to get rid of the proteolytic enzymes before we can proceed. The enzymes are denatured by heating the sample in a water bath at 100°C. Next, the cellular debris is spun down in the centrifuge and appears as a solid deposit (pellet) at the bottom of the tube. The DNA is contained in the supernatant (the liquid), which is then transferred to the PCR tube. Why / Back to Part 1Note: Limitations of the traditional methods of identification Over the years...
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...EXAMINATION OF THE TAS2R38 GENE AND ITS SPECIFIC NUCLEOTIDE DIFFERENTIATIONS TO DETERMINE ABILITY TO TASTE PHENYLTHIOCARBAMIDE INTRODUCTION Although humans are essentially genetically identical as a whole, there are some minute variances in our gene coding that allow for differences in our interactions with the world. These genetic modifications may have extensive detrimental effects, small effects, or no apparent effect at all. A few of these alterations can even affect our senses. In this lab, we examine how a discovery by a scientist gives us insight into how a relative dissimilarity between humans can affect the ability or inability to taste certain chemicals. Scientist Arthur Fox learned that the chemical phenylthiocarbamide, or PTC, could be tasted by certain people while others could not (Dolan DNA Learning Center 2006). When this was revealed, it was inferred that the ability or inability to taste this substance may be genetically related. It was also possible that there was a specific gene that coded for this capability. The gene that was found to encode for the capacity to taste PTC is named the TASR38 gene (Dolan DNA Learning Center 2006). However, it is not just the gene itself that causes differences in the ability to taste this substance, but the differences of coding within certain locations of this gene. These distinctions in gene coding across human populations at nucleotide positions 145, 785, and 886 are called single nucleotide polymorphisms (SNPs)...
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...ABSTRACT I TABLE OF CONTENTS II ACKNOWLEDGEMENTS V LIST OF TABLES VI LIST OF FIGURES VII LIST OF ABBREVIATIONS VIII 1 INTRODUCTION 1 1.1 HEPATITIS C VIRUS 1 1.1.1 DISCOVERY 1 1.1.2 EPIDEMIOLOGY 2 1.1.3 PATHOGENESIS 2 1.1.4 TREATMENT 3 1.2 MOLECULAR BIOLOGY 3 1.2.1 STRUCTURE OF GENOME 3 1.2.2 GENETIC VARIATION 6 1.2.3 GENOTYPIC DIFFERENCES 8 1.3 RNA DEPENDENT RNA POLYMERASE ACTIVITY 9 1.3.1 POLYMERASE FUNCTION 9 1.3.2 MODEL SYSTEMS OF HCV REPLICATION 11 1.3.3 GENOTYPE SPECIFIC STUDIES 11 1.3.4 BIOCHEMICAL PROPERTIES 12 1.4 KUNJIN VIRUS RNA DEPENDENT RNA POLYMERASE 13 1.5 CONCLUSION 15 1.6 AIMS AND HYPOTHESIS 16 2 MATERIALS AND METHODS 17 2.1 HCV POSITIVE SERA SAMPLES 17 2.2 RNA EXTRACTION 17 2.3 CDNA SYNTHESIS 17 2.4 HCV PRIMER DESIGN AND USAGE 18 2.5 NESTED POLYMERASE CHAIN REACTION (NPCR) 21 2.5.1 REACTION AND CYCLING CONDITIONS 21 2.5.2 PCR PRODUCT PURIFICATION 22 2.6 AGAROSE GEL VISUALISATION 22 2.7 DNA SEQUENCING 22 2.8 DNA SEQUENCE AND PHYLOGENETIC ANALYSIS 23 2.9 KUNJIN VIRUS PLASMID 23 2.10 KUN PRIMER DESIGN AND USAGE 23 2.11 CLONING PCR PRODUCTS 24 2.11.1 RESTRICTION DIGEST 24 2.11.2 LIGATION 24 2.11.3 TRANSFORMATION 24 2.11.4 COLONY PCR 24 2.12...
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...PCR also known as polymerase chain reaction, it was developed by Kary Mullis in 1983 in order to magnify small segments of DNA. PCR is a technique used to produce millions of copies of a particular DNA sequence in less amount of time than previous methods. It became important in the identification of bacteria and virus, diagnosis of disease, genetic manipulation, forensic science, cloning, and in other fields. PCR takes advantage of different temperatures while it mimics the natural process of DNA replication, its main components are two primers, segments of single DNA built in laboratories that are complementary to the DNA that is going to be copied; DNA polymerase, which synthesize the DNA segment most used is Taq polymerase; and nucleotides the foundation of the DNA molecule. The method of PCR has three important steps denaturing, annealing, and elongation. The first step is the denaturing of the DNA, denature consist of the disruption of the hydrogen bonds between nitrogenous base pairs, which provide the DNA double strand structure. This denaturing targets the DNA by heating the sample to 94 °- 96 ° C during several minutes, the result is two single DNA strands. The seconds step is the annealing step, the temperature is lowered to 50°- 65° C for several minutes, this allows left and right primers that were added to the mix to base pair to their complementary sequence. The main function of the primers is to enclose the region of DNA that would be amplified; this is...
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...ABSTRACT I TABLE OF CONTENTS II ACKNOWLEDGEMENTS V LIST OF TABLES VI LIST OF FIGURES VII LIST OF ABBREVIATIONS VIII 1 INTRODUCTION 1 1.1 HEPATITIS C VIRUS 1 1.1.1 DISCOVERY 1 1.1.2 EPIDEMIOLOGY 2 1.1.3 PATHOGENESIS 2 1.1.4 TREATMENT 3 1.2 MOLECULAR BIOLOGY 3 1.2.1 STRUCTURE OF GENOME 3 1.2.2 GENETIC VARIATION 6 1.2.3 GENOTYPIC DIFFERENCES 8 1.3 RNA DEPENDENT RNA POLYMERASE ACTIVITY 9 1.3.1 POLYMERASE FUNCTION 9 1.3.2 MODEL SYSTEMS OF HCV REPLICATION 11 1.3.3 GENOTYPE SPECIFIC STUDIES 11 1.3.4 BIOCHEMICAL PROPERTIES 12 1.4 KUNJIN VIRUS RNA DEPENDENT RNA POLYMERASE 13 1.5 CONCLUSION 15 1.6 AIMS AND HYPOTHESIS 16 2 MATERIALS AND METHODS 17 2.1 HCV POSITIVE SERA SAMPLES 17 2.2 RNA EXTRACTION 17 2.3 CDNA SYNTHESIS 17 2.4 HCV PRIMER DESIGN AND USAGE 18 2.5 NESTED POLYMERASE CHAIN REACTION (NPCR) 21 2.5.1 REACTION AND CYCLING CONDITIONS 21 2.5.2 PCR PRODUCT PURIFICATION 22 2.6 AGAROSE GEL VISUALISATION 22 2.7 DNA SEQUENCING 22 2.8 DNA SEQUENCE AND PHYLOGENETIC ANALYSIS 23 2.9 KUNJIN VIRUS PLASMID 23 2.10 KUN PRIMER DESIGN AND USAGE 23 2.11 CLONING PCR PRODUCTS 24 2.11.1 RESTRICTION DIGEST 24 2.11.2 LIGATION 24 2.11.3 TRANSFORMATION 24 2.11.4 COLONY PCR 24 2.12...
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...this experiment was to determine 1. Taster Phenotype, 2. Isolate DNA from each individual 3. Determine Taster Genotype Hypothesis: Ho: If I am a taster, then my genotype for PTC taster must be either TT (homozygous dominant) or Tt (heterozygous) The traits of being able to taste versus the inability to taste follows the mendelian inheritance. In mendelian inheritance when two heterozygous dominant parents were mated, genotypic ratios were….. Methods: This experiment aimed to investigate the allele frequency of the PTC taster gene (TAS2R38) in a small population, represented by the students in class. To determine the genotypic profile of the students PTC gene,a sample of DNA was extracted using Chelex resin. Polymerase chain reaction was then used to amplify then digest the gene of TAS2R38 gene with a restriction enzyme. Digested DNA will be cut into 2 fragments 239 bp and 64 bp in length. (Figure 2) This discrepancy in length allowed us to profile the alleles using gel electrophoresis: non-tasters have only the uncut fragment, taster homozygotes the...The enzyme could either cut the enzyme or leave it whole producing a restriction fragment polymorphism that can be separated by agarose gel...
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...BIOTERRORISM Name: 1. Introduction Historically, infectious disease outbreaks brought about by microbial species against human beings have caused far more mortality rates than war itself. Numerous cases of disease outbreaks in the past have been a major concern to health authorities, although such concerns have been partially addressed in the recent past. Examples of disease outbreaks in the past include: the infamous Bubonic Plague of the 14th century in Europe that led to the death of approximately a quarter of the continent’s population (approximately 25 million people); the influenza pandemic of 1918 to 1919 that led to the death of 21 million people; and the death of 95% of Pre-Columbian Native American people by measles, plague, small pox, influenza, and typhoid (Magner, 2009). Although there has been some response to such epidemics in the recent past, naturally occurring infections still remain the Achilles’ heel of today’s health systems. 2. Terrorism Versus Bioterrorism Despite the dark past in healthcare systems, current issues of the use of biological agents as means of mass destruction is alarming. Most of the countries across the globe are now faced with the daunting task of terrorism control, since this is one area where biological agents find a lot of use. According to Forst, terrorism may be defined as a “premeditated and unlawful use of violence [on] non-combatant populations having symbolic significance….” (Forst, 2009). Forst mentions that...
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