DNA sequencing by fredrick sanger’s method
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Fredrick Sanger developed a method for DNA sequencing based on controlled interruption of DNA replication. The Sanger method uses a DNA template, primer, DNA polymerase, normal dNTPs, and dideoxynucleotides (ddNTPs) which terminate DNA strand extension. Electrophoresis is used to separate the DNA fragments of different lengths, which are then read to determine the DNA sequence. The Sanger method was later automated using fluorescent tags and laser detection. While powerful, it is limited to sequencing shorter DNA fragments.
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Sequencing
This document discusses DNA sequencing methods. It provides an overview of DNA and its functions including storing genetic information, self-duplication, and expressing genetic messages. It then describes the Watson and Crick model of DNA structure. The document proceeds to define DNA sequencing as determining the order of nucleotide bases in a DNA section. It outlines early chemical methods like Maxam-Gilbert sequencing and the still common Sanger method using chain termination. Finally, it introduces some modern next-generation sequencing technologies like Illumina and Pyrosequencing.
Lecture 3
This document discusses DNA sequencing techniques. It begins by defining DNA sequencing as determining the order of nucleotides in a DNA molecule. It then summarizes 4 generations of sequencing methods: 1) Sanger sequencing from 1977, the predominant early method; 2) Next generation techniques from 2005-2007 including 454, SOLiD, and Illumina that enabled high-throughput short reads; 3) Third generation methods like PacBio and Oxford Nanopore that enable long reads but with lower accuracy; and 4) Some additional terms used in sequencing like oligonucleotides, plasmids, and cloning vectors.
Replication
1. DNA replication is the process by which a cell makes an identical copy of its DNA during cell division. It is a highly regulated and accurate process that occurs in all living organisms.
2. There are three proposed modes of DNA replication: conservative, semi-conservative, and dispersive. Experiments provided evidence that semi-conservative replication, where each parent strand acts as a template for a new partner strand, is how DNA replication occurs.
3. DNA replication requires several enzymes, including DNA polymerases, to unwind and copy the DNA double helix. In eukaryotes, DNA replication occurs at multiple replication origins along linear chromosomes, while prokaryotes replicate from a single origin on circular chromosomes
Polymerase chain reaction.
The document summarizes polymerase chain reaction (PCR), beginning with its inventor Kary Banks Mullis who was awarded the Nobel Prize in Chemistry for developing the technique. PCR is a laboratory method used to amplify small segments of DNA by using DNA polymerase to copy the target DNA multiple times, resulting in billions of copies. The key steps in PCR involve denaturing the double-stranded DNA, annealing primers to the single strands, and extending the primers to synthesize new strands. PCR has wide applications in research, forensics, genetic testing, and disease diagnosis.
PCR by Dr.Nazir
The document discusses the polymerase chain reaction (PCR) technique developed by Kary Mullis in 1983. PCR allows for rapid copying of specific DNA sequences, producing millions of copies. It works by repeated heating and cooling of the DNA sample in the presence of DNA polymerase, primers that bind to the target sequence, and nucleotides. During each cycle, the DNA strands are separated by heating, then the primers bind and the polymerase enzyme copies the target. This process is repeated many times to exponentially amplify the target DNA sequence. PCR is a sensitive technique that has many applications including disease diagnosis, forensics, and evolutionary studies.
DNA sequencing
DNA sequencing methods have evolved greatly since first being developed in the 1970s. The document discusses the key developments and purposes of DNA sequencing. It describes the original Maxam-Gilbert and Sanger chain termination methods, and how modern sequencing uses improvements to the Sanger technique. DNA sequencing is now used widely in medicine, forensics, agriculture and more to analyze genetic information and detect mutations or genetic disorders.
Illumina (sequencing by synthesis) method
The document outlines the Illumina sequencing by synthesis method in 12 steps: 1) DNA sample preparation and attachment to a flow cell, 2) bridge amplification to clone the DNA fragments, 3) determination of the first base by addition of fluorescently labeled nucleotides and imaging, 4) repeating the process to determine the second and subsequent bases, 5) generating sequenced reads. The sequenced reads are then 6) aligned and analyzed by comparing to a reference sequence to identify differences.
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Sequencing
This document discusses DNA sequencing methods. It provides an overview of DNA and its functions including storing genetic information, self-duplication, and expressing genetic messages. It then describes the Watson and Crick model of DNA structure. The document proceeds to define DNA sequencing as determining the order of nucleotide bases in a DNA section. It outlines early chemical methods like Maxam-Gilbert sequencing and the still common Sanger method using chain termination. Finally, it introduces some modern next-generation sequencing technologies like Illumina and Pyrosequencing.
Lecture 3
This document discusses DNA sequencing techniques. It begins by defining DNA sequencing as determining the order of nucleotides in a DNA molecule. It then summarizes 4 generations of sequencing methods: 1) Sanger sequencing from 1977, the predominant early method; 2) Next generation techniques from 2005-2007 including 454, SOLiD, and Illumina that enabled high-throughput short reads; 3) Third generation methods like PacBio and Oxford Nanopore that enable long reads but with lower accuracy; and 4) Some additional terms used in sequencing like oligonucleotides, plasmids, and cloning vectors.
Replication
1. DNA replication is the process by which a cell makes an identical copy of its DNA during cell division. It is a highly regulated and accurate process that occurs in all living organisms.
2. There are three proposed modes of DNA replication: conservative, semi-conservative, and dispersive. Experiments provided evidence that semi-conservative replication, where each parent strand acts as a template for a new partner strand, is how DNA replication occurs.
3. DNA replication requires several enzymes, including DNA polymerases, to unwind and copy the DNA double helix. In eukaryotes, DNA replication occurs at multiple replication origins along linear chromosomes, while prokaryotes replicate from a single origin on circular chromosomes
Polymerase chain reaction.
The document summarizes polymerase chain reaction (PCR), beginning with its inventor Kary Banks Mullis who was awarded the Nobel Prize in Chemistry for developing the technique. PCR is a laboratory method used to amplify small segments of DNA by using DNA polymerase to copy the target DNA multiple times, resulting in billions of copies. The key steps in PCR involve denaturing the double-stranded DNA, annealing primers to the single strands, and extending the primers to synthesize new strands. PCR has wide applications in research, forensics, genetic testing, and disease diagnosis.
PCR by Dr.Nazir
The document discusses the polymerase chain reaction (PCR) technique developed by Kary Mullis in 1983. PCR allows for rapid copying of specific DNA sequences, producing millions of copies. It works by repeated heating and cooling of the DNA sample in the presence of DNA polymerase, primers that bind to the target sequence, and nucleotides. During each cycle, the DNA strands are separated by heating, then the primers bind and the polymerase enzyme copies the target. This process is repeated many times to exponentially amplify the target DNA sequence. PCR is a sensitive technique that has many applications including disease diagnosis, forensics, and evolutionary studies.
DNA sequencing
DNA sequencing methods have evolved greatly since first being developed in the 1970s. The document discusses the key developments and purposes of DNA sequencing. It describes the original Maxam-Gilbert and Sanger chain termination methods, and how modern sequencing uses improvements to the Sanger technique. DNA sequencing is now used widely in medicine, forensics, agriculture and more to analyze genetic information and detect mutations or genetic disorders.
Illumina (sequencing by synthesis) method
The document outlines the Illumina sequencing by synthesis method in 12 steps: 1) DNA sample preparation and attachment to a flow cell, 2) bridge amplification to clone the DNA fragments, 3) determination of the first base by addition of fluorescently labeled nucleotides and imaging, 4) repeating the process to determine the second and subsequent bases, 5) generating sequenced reads. The sequenced reads are then 6) aligned and analyzed by comparing to a reference sequence to identify differences.
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REPLICATION VS POLYMERASE CHAIN REACTION
PCR allows for targeted amplification of specific DNA fragments. It involves repeated cycles of DNA denaturation by heating and cooling, primer annealing, and extension of the DNA strand by a DNA polymerase. Key steps include separation of double stranded DNA at 95°C, annealing of primers at 40-65°C, and extension of the DNA chain by DNA polymerase at 72°C. PCR amplifies the target DNA sequence up to 100,000-fold, enabling detection of rare DNA sequences. It has largely replaced other methods for DNA analysis and molecular cloning due to its specificity, sensitivity, speed, and ability to quantitatively analyze DNA.
Polymerase chain reaction
Polymerase chain reaction is the process of gene amplification and gene isolation in artificial conditions
Sanger sequencing
Sanger sequencing is a method of DNA sequencing developed by Frederick Sanger in 1977 that was widely used for 25 years. It involves making copies of a DNA region using DNA polymerase and chain-terminating dideoxynucleotides that are labeled with different colored dyes. This produces fragmented DNA of different lengths that can be separated by size to determine the DNA sequence. Sanger sequencing is useful for sequencing single genes or short sequences but is limited to read lengths of 300-1000 base pairs. It has been replaced by next generation sequencing methods for most applications.
Dna sequensing
This document discusses DNA sequencing methods. It describes the Maxam-Gilbert method, which uses chemical degradation of DNA to determine nucleotide sequences. It also describes the Sanger method, the most commonly used modern method, which uses DNA polymerase and dideoxynucleotides to terminate DNA strand elongation at specific positions. The document compares the two main historical sequencing methods and discusses their principles, steps, advantages, and disadvantages.
PCR
PCR exponentially amplifies small fragments of DNA through thermal cycling to generate thousands to millions of copies. Sanger sequencing allows us to read nucleotide sequences and search for mutations, which was crucial for genetic disorder diagnosis. The Cole lab researches genes like ABCA3 that encode proteins involved in surfactant function, and receives patient referrals to sequence genes like ABCA3 and build a database. During the summer, students sequenced 32 ABCA3 exomes from 13 patient referrals.
DNA
This document summarizes DNA replication. It begins by stating that DNA replication is the process by which DNA copies itself. It then describes the three modes of replication: dispersive, conservative, and semiconservative. The document explains that semiconservative replication was proposed by Watson and Crick, and that evidence from Meselson and Stahl supported this model. Finally, it provides an overview of the key proteins involved in DNA replication, including DNA polymerase, primase, ligase, endonucleases, pilot proteins, and helicase.
DNA Sequencing
The document describes the development of DNA sequencing methods. It discusses the Maxam-Gilbert chemical cleavage method from the 1970s that took advantage of chemicals that selectively attack DNA bases. It also discusses Sanger's chain termination method from the 1970s using dideoxynucleotides to terminate DNA synthesis. Finally, it discusses the development of automated fluorescence sequencing in the 1980s using fluorescently labeled dideoxynucleotides, laser detection, and computer base calling.
Watson and crick model of dna
Watson and Crick proposed a model of DNA structure in 1953 as a double helix with two antiparallel strands coiled around the same axis. Each strand consists of alternating deoxyribose and phosphate groups with purine or pyrimidine bases stacked inside. Adenine always pairs with thymine via two hydrogen bonds and guanine pairs with cytosine via three hydrogen bonds. Their model explained experimental X-ray diffraction data and Chargaff's rules of base equivalence. The discovery revolutionized our understanding of genetics and heredity.
Section 1.3 dna technology
The polymerase chain reaction (PCR) is a technique used to quickly make millions of copies of a specific DNA sequence. The process involves:
1. Denaturing the DNA template into single strands by heating.
2. Adding primers that are complementary to the DNA sequences and allowing them to anneal.
3. Using DNA polymerase to synthesize new DNA strands.
4. Repeating the process many times to exponentially amplify the target DNA sequence.
PCR has revolutionized DNA analysis and is used for applications like genetic testing, fingerprinting, cloning, and DNA sequencing. It relies on thermostable DNA polymerases that can function at high temperatures needed for denaturing.
Gene Sequencing
This document discusses several methods for gene sequencing, including Sanger sequencing, 454 technology, and sequence assembly. Sanger sequencing uses DNA polymerase and chain terminating nucleotides to synthesize labeled DNA strands of varying lengths that can then be separated by electrophoresis to determine the sequence. 454 technology attaches DNA fragments to beads and performs emulsion PCR to generate millions of copies for pyrosequencing. Sequence assembly involves combining overlapping sequences from fragmented DNA reads to reconstruct the full genome sequence, as individual reads are usually only 500-1000 base pairs long.
Presentation on dna sequencing
This document provides an overview of DNA sequencing. It begins by defining DNA and its structure. It then explains that DNA sequencing is the process of determining the order of nucleotide bases in a DNA strand. Two common methods of DNA sequencing are described in detail: the Maxam-Gilbert chemical cleavage method and the Sanger dideoxy chain termination method. The document concludes by discussing some applications of DNA sequencing in fields like forensics, medical research, and agriculture.
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Recombinant DNA technology involves isolating DNA from organisms, cutting it with restriction enzymes, ligating the cut DNA fragments into vectors, inserting the recombinant DNA into host cells, culturing the host cells to produce multiple copies, and extracting the desired product. Key steps include isolating pure DNA, cutting source and vector DNA with the same enzyme, ligating the gene of interest into the vector, amplifying the gene using PCR, transforming host cells to incorporate the recombinant DNA, and using selective markers like antibiotic resistance to identify transformed cells.
Chapter 12 notes
This chapter discusses the discovery of DNA and its role as the genetic material. It covers Griffith's experiments showing bacterial transformation, the Hershey-Chase experiment demonstrating that viruses use DNA to infect bacteria, and the role of DNA in storing, copying and transmitting information. The chapter also explains the work of Chargaff, Franklin, Watson and Crick to determine DNA's double-helix structure, including the antiparallel strands, hydrogen bonding and base pairing. Finally, it discusses DNA replication in both prokaryotic and eukaryotic cells.
Chp 12 cornell notes
This chapter discusses the discovery of DNA and its role as the genetic material. It covers Griffith's experiments showing bacterial transformation, the Hershey-Chase experiment demonstrating that DNA carries genetic information in viruses, and Chargaff's rules which helped Watson and Crick determine DNA's double-helix structure. The chapter also addresses DNA replication, including the roles of enzymes and telomeres, and how replication occurs differently in prokaryotes and eukaryotes.
Notes chpt 12
This chapter discusses the discovery of DNA and its role as the genetic material. It covers Griffith's experiments showing bacterial transformation, the Hershey-Chase experiment demonstrating that DNA carries genetic information in viruses, and Chargaff's rules which helped Watson and Crick determine DNA's double-helix structure. The chapter also addresses DNA replication, including the roles of enzymes and telomeres, and how replication occurs differently in prokaryotes and eukaryotes.
Maxam-Gilbert method of DNA sequencing
Maxam-Gilbert sequencing uses chemicals to cut DNA fragments at specific bases, allowing the sequence to be determined. It involves separating DNA strands, radioactively labeling one, then breaking it up in four reactions that specifically cleave at adenine, cytosine, guanine, or thymine. The labeled fragments are run on a gel and their sizes reveal the sequence. Though it directly sequences DNA without cloning, it uses toxic chemicals and radioactivity, has a short read length, and is technically complex.
DND sequencing
This document provides information about DNA sequencing methods. It discusses that DNA sequencing determines the precise order of nucleotides in DNA. The two main conventional methods described are the Maxam-Gilbert chemical degradation method and the Sanger chain termination method. The Maxam-Gilbert method uses chemical modification and cleavage of DNA bases. The Sanger method is a PCR-based method that uses dideoxynucleotides to terminate DNA chain elongation. Both methods produce fragmented DNA of different lengths that can be resolved on a gel for sequence determination.
Dna sequencing
The Sanger method is the standard DNA sequencing technique. It involves using specific enzymes to synthesize DNA fragments of varying lengths that terminate at a particular base, like ddATP, ddTTP, etc. These fragments are then sorted by size using electrophoresis in a gel, where the shorter fragments migrate farther due to their negative charge. Each fragment type is typically tagged with a radioactive probe for identification. Capillary electrophoresis is now commonly used for high-throughput sequencing as it allows for faster separation and analysis of larger DNA samples compared to slab gel electrophoresis.
281 lec30 mol_tech2
This document summarizes techniques for analyzing DNA quality and quantity. It discusses running DNA samples on a gel to qualitatively assess size and quality, and using a spectrophotometer to quantitatively measure DNA amount and purity. Specifically, it covers how DNA moves in a gel based on its charge, how agarose concentration affects separation, staining DNA with ethidium bromide, using fluorescent dyes in capillary electrophoresis, and quantifying DNA with a spectrophotometer or Nanodrop. The goal is to learn fundamental methods for characterizing DNA samples before further analysis.
Polymerase Chain Reaction
Polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA through repeated cycles of heating and cooling. It requires a DNA template, primers, DNA polymerase, nucleotides, and magnesium chloride. The process involves denaturation of the DNA strands, annealing of primers to the complementary sequences, and extension of the primers by DNA polymerase to synthesize new strands. PCR can be used to amplify small amounts of DNA for forensic analysis or isolate a known gene from a genome. However, it is limited to amplifying up to 5kb of known DNA sequences.
Sanger sequencing (DNA sequencing by ENZYMATIC METHOD)
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.
DNA SEQUENCING (1).pptx
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.
Knowledge of DNA sequences has become indispensable for basic biological research, DNA Genographic Projects and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers,characterize antibody repertoire, and can be used to guide patient treatment.[5Having a quick way to sequence DNA allows for faster and more individualized medical care to be administered, and for more organisms to be identified and cataloged.
The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of complete DNA sequences, or genomes, of numerous types and species of life, including the human genome and other complete DNA sequences of many animal, plant, and microbial species.
The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of fluorescence-based sequencing methods with a DNA sequencer, DNA sequencing has become easier and orders of magnitude faster.
DNA sequencing refers to the general laboratory technique for determining the exact sequence of nucleotides, or bases, in a DNA molecule. The sequence of the bases (often referred to by the first letters of their chemical names: A, T, C, and G) encodes the biological information that cells use to develop and operate.Whole Genome Sequencing
•Allows doctors to closely analyze a patient's genes for mutations and health indicators.
•Can detect intellectual disabilities and developmental delays.
•WGS is currently available at Yale for patients in the NICU and PICU.
•Involves Genetics.Sequencing may be utilized to determine the order of nucleotides in small targeted genomic regions or entire genomes. Illumina sequencing enables a wide variety of applications, allowing researchers to ask virtually any question related to the genome, transcriptome, or epigenome of any organism.The spectrum of analysis of NGS can extend from a small number of genes to an entire genome, depending on the goal. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) provide the sequence of DNA bases across the genome and exome, respectively.Capillary electrophoresis (CE) instruments are capable of performing both Sanger sequencing and fragment analysis. Fragment analysis is a method in which DNA fragments are fluorescently labeled, separated by CE, and sized by comparison to an internal standard. sanger and Maxam-Gilbert sequencing technologies were classified
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REPLICATION VS POLYMERASE CHAIN REACTION
PCR allows for targeted amplification of specific DNA fragments. It involves repeated cycles of DNA denaturation by heating and cooling, primer annealing, and extension of the DNA strand by a DNA polymerase. Key steps include separation of double stranded DNA at 95°C, annealing of primers at 40-65°C, and extension of the DNA chain by DNA polymerase at 72°C. PCR amplifies the target DNA sequence up to 100,000-fold, enabling detection of rare DNA sequences. It has largely replaced other methods for DNA analysis and molecular cloning due to its specificity, sensitivity, speed, and ability to quantitatively analyze DNA.
Polymerase chain reaction
Polymerase chain reaction is the process of gene amplification and gene isolation in artificial conditions
Sanger sequencing
Sanger sequencing is a method of DNA sequencing developed by Frederick Sanger in 1977 that was widely used for 25 years. It involves making copies of a DNA region using DNA polymerase and chain-terminating dideoxynucleotides that are labeled with different colored dyes. This produces fragmented DNA of different lengths that can be separated by size to determine the DNA sequence. Sanger sequencing is useful for sequencing single genes or short sequences but is limited to read lengths of 300-1000 base pairs. It has been replaced by next generation sequencing methods for most applications.
Dna sequensing
This document discusses DNA sequencing methods. It describes the Maxam-Gilbert method, which uses chemical degradation of DNA to determine nucleotide sequences. It also describes the Sanger method, the most commonly used modern method, which uses DNA polymerase and dideoxynucleotides to terminate DNA strand elongation at specific positions. The document compares the two main historical sequencing methods and discusses their principles, steps, advantages, and disadvantages.
PCR
PCR exponentially amplifies small fragments of DNA through thermal cycling to generate thousands to millions of copies. Sanger sequencing allows us to read nucleotide sequences and search for mutations, which was crucial for genetic disorder diagnosis. The Cole lab researches genes like ABCA3 that encode proteins involved in surfactant function, and receives patient referrals to sequence genes like ABCA3 and build a database. During the summer, students sequenced 32 ABCA3 exomes from 13 patient referrals.
DNA
This document summarizes DNA replication. It begins by stating that DNA replication is the process by which DNA copies itself. It then describes the three modes of replication: dispersive, conservative, and semiconservative. The document explains that semiconservative replication was proposed by Watson and Crick, and that evidence from Meselson and Stahl supported this model. Finally, it provides an overview of the key proteins involved in DNA replication, including DNA polymerase, primase, ligase, endonucleases, pilot proteins, and helicase.
DNA Sequencing
The document describes the development of DNA sequencing methods. It discusses the Maxam-Gilbert chemical cleavage method from the 1970s that took advantage of chemicals that selectively attack DNA bases. It also discusses Sanger's chain termination method from the 1970s using dideoxynucleotides to terminate DNA synthesis. Finally, it discusses the development of automated fluorescence sequencing in the 1980s using fluorescently labeled dideoxynucleotides, laser detection, and computer base calling.
Watson and crick model of dna
Watson and Crick proposed a model of DNA structure in 1953 as a double helix with two antiparallel strands coiled around the same axis. Each strand consists of alternating deoxyribose and phosphate groups with purine or pyrimidine bases stacked inside. Adenine always pairs with thymine via two hydrogen bonds and guanine pairs with cytosine via three hydrogen bonds. Their model explained experimental X-ray diffraction data and Chargaff's rules of base equivalence. The discovery revolutionized our understanding of genetics and heredity.
Section 1.3 dna technology
The polymerase chain reaction (PCR) is a technique used to quickly make millions of copies of a specific DNA sequence. The process involves:
1. Denaturing the DNA template into single strands by heating.
2. Adding primers that are complementary to the DNA sequences and allowing them to anneal.
3. Using DNA polymerase to synthesize new DNA strands.
4. Repeating the process many times to exponentially amplify the target DNA sequence.
PCR has revolutionized DNA analysis and is used for applications like genetic testing, fingerprinting, cloning, and DNA sequencing. It relies on thermostable DNA polymerases that can function at high temperatures needed for denaturing.
Gene Sequencing
This document discusses several methods for gene sequencing, including Sanger sequencing, 454 technology, and sequence assembly. Sanger sequencing uses DNA polymerase and chain terminating nucleotides to synthesize labeled DNA strands of varying lengths that can then be separated by electrophoresis to determine the sequence. 454 technology attaches DNA fragments to beads and performs emulsion PCR to generate millions of copies for pyrosequencing. Sequence assembly involves combining overlapping sequences from fragmented DNA reads to reconstruct the full genome sequence, as individual reads are usually only 500-1000 base pairs long.
Presentation on dna sequencing
This document provides an overview of DNA sequencing. It begins by defining DNA and its structure. It then explains that DNA sequencing is the process of determining the order of nucleotide bases in a DNA strand. Two common methods of DNA sequencing are described in detail: the Maxam-Gilbert chemical cleavage method and the Sanger dideoxy chain termination method. The document concludes by discussing some applications of DNA sequencing in fields like forensics, medical research, and agriculture.
12th Biology Biotechnology Principles and Processes Part 4
Recombinant DNA technology involves isolating DNA from organisms, cutting it with restriction enzymes, ligating the cut DNA fragments into vectors, inserting the recombinant DNA into host cells, culturing the host cells to produce multiple copies, and extracting the desired product. Key steps include isolating pure DNA, cutting source and vector DNA with the same enzyme, ligating the gene of interest into the vector, amplifying the gene using PCR, transforming host cells to incorporate the recombinant DNA, and using selective markers like antibiotic resistance to identify transformed cells.
Chapter 12 notes
This chapter discusses the discovery of DNA and its role as the genetic material. It covers Griffith's experiments showing bacterial transformation, the Hershey-Chase experiment demonstrating that viruses use DNA to infect bacteria, and the role of DNA in storing, copying and transmitting information. The chapter also explains the work of Chargaff, Franklin, Watson and Crick to determine DNA's double-helix structure, including the antiparallel strands, hydrogen bonding and base pairing. Finally, it discusses DNA replication in both prokaryotic and eukaryotic cells.
Chp 12 cornell notes
This chapter discusses the discovery of DNA and its role as the genetic material. It covers Griffith's experiments showing bacterial transformation, the Hershey-Chase experiment demonstrating that DNA carries genetic information in viruses, and Chargaff's rules which helped Watson and Crick determine DNA's double-helix structure. The chapter also addresses DNA replication, including the roles of enzymes and telomeres, and how replication occurs differently in prokaryotes and eukaryotes.
Notes chpt 12
This chapter discusses the discovery of DNA and its role as the genetic material. It covers Griffith's experiments showing bacterial transformation, the Hershey-Chase experiment demonstrating that DNA carries genetic information in viruses, and Chargaff's rules which helped Watson and Crick determine DNA's double-helix structure. The chapter also addresses DNA replication, including the roles of enzymes and telomeres, and how replication occurs differently in prokaryotes and eukaryotes.
Maxam-Gilbert method of DNA sequencing
Maxam-Gilbert sequencing uses chemicals to cut DNA fragments at specific bases, allowing the sequence to be determined. It involves separating DNA strands, radioactively labeling one, then breaking it up in four reactions that specifically cleave at adenine, cytosine, guanine, or thymine. The labeled fragments are run on a gel and their sizes reveal the sequence. Though it directly sequences DNA without cloning, it uses toxic chemicals and radioactivity, has a short read length, and is technically complex.
DND sequencing
This document provides information about DNA sequencing methods. It discusses that DNA sequencing determines the precise order of nucleotides in DNA. The two main conventional methods described are the Maxam-Gilbert chemical degradation method and the Sanger chain termination method. The Maxam-Gilbert method uses chemical modification and cleavage of DNA bases. The Sanger method is a PCR-based method that uses dideoxynucleotides to terminate DNA chain elongation. Both methods produce fragmented DNA of different lengths that can be resolved on a gel for sequence determination.
Dna sequencing
The Sanger method is the standard DNA sequencing technique. It involves using specific enzymes to synthesize DNA fragments of varying lengths that terminate at a particular base, like ddATP, ddTTP, etc. These fragments are then sorted by size using electrophoresis in a gel, where the shorter fragments migrate farther due to their negative charge. Each fragment type is typically tagged with a radioactive probe for identification. Capillary electrophoresis is now commonly used for high-throughput sequencing as it allows for faster separation and analysis of larger DNA samples compared to slab gel electrophoresis.
281 lec30 mol_tech2
This document summarizes techniques for analyzing DNA quality and quantity. It discusses running DNA samples on a gel to qualitatively assess size and quality, and using a spectrophotometer to quantitatively measure DNA amount and purity. Specifically, it covers how DNA moves in a gel based on its charge, how agarose concentration affects separation, staining DNA with ethidium bromide, using fluorescent dyes in capillary electrophoresis, and quantifying DNA with a spectrophotometer or Nanodrop. The goal is to learn fundamental methods for characterizing DNA samples before further analysis.
Polymerase Chain Reaction
Polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA through repeated cycles of heating and cooling. It requires a DNA template, primers, DNA polymerase, nucleotides, and magnesium chloride. The process involves denaturation of the DNA strands, annealing of primers to the complementary sequences, and extension of the primers by DNA polymerase to synthesize new strands. PCR can be used to amplify small amounts of DNA for forensic analysis or isolate a known gene from a genome. However, it is limited to amplifying up to 5kb of known DNA sequences.
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12th Biology Biotechnology Principles and Processes Part 4
12th Biology Biotechnology Principles and Processes Part 4
Similar to DNA sequencing by fredrick sanger’s method
Sanger sequencing (DNA sequencing by ENZYMATIC METHOD)
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.
DNA SEQUENCING (1).pptx
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.
Knowledge of DNA sequences has become indispensable for basic biological research, DNA Genographic Projects and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers,characterize antibody repertoire, and can be used to guide patient treatment.[5Having a quick way to sequence DNA allows for faster and more individualized medical care to be administered, and for more organisms to be identified and cataloged.
The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of complete DNA sequences, or genomes, of numerous types and species of life, including the human genome and other complete DNA sequences of many animal, plant, and microbial species.
The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of fluorescence-based sequencing methods with a DNA sequencer, DNA sequencing has become easier and orders of magnitude faster.
DNA sequencing refers to the general laboratory technique for determining the exact sequence of nucleotides, or bases, in a DNA molecule. The sequence of the bases (often referred to by the first letters of their chemical names: A, T, C, and G) encodes the biological information that cells use to develop and operate.Whole Genome Sequencing
•Allows doctors to closely analyze a patient's genes for mutations and health indicators.
•Can detect intellectual disabilities and developmental delays.
•WGS is currently available at Yale for patients in the NICU and PICU.
•Involves Genetics.Sequencing may be utilized to determine the order of nucleotides in small targeted genomic regions or entire genomes. Illumina sequencing enables a wide variety of applications, allowing researchers to ask virtually any question related to the genome, transcriptome, or epigenome of any organism.The spectrum of analysis of NGS can extend from a small number of genes to an entire genome, depending on the goal. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) provide the sequence of DNA bases across the genome and exome, respectively.Capillary electrophoresis (CE) instruments are capable of performing both Sanger sequencing and fragment analysis. Fragment analysis is a method in which DNA fragments are fluorescently labeled, separated by CE, and sized by comparison to an internal standard. sanger and Maxam-Gilbert sequencing technologies were classified
Dna sequening
DNA sequencing methods have evolved significantly over time. Early methods like the Maxam-Gilbert chemical method and Sanger chain termination method involved laborious gel electrophoresis. Later developments led to automated Sanger sequencing using fluorescence detection. Next-generation sequencing methods like Illumina sequencing by synthesis and 454 pyrosequencing enabled massively parallel sequencing of many DNA fragments simultaneously. These new methods produce vast amounts of sequence data at lower cost and are used widely in research and applications such as agriculture, medicine, forensics, and more.
Dna Sequencing
The document summarizes DNA sequencing methods. It discusses the DNA double helix structure and how the four nitrogenous bases form complementary pairs between strands. It then describes the two main historical DNA sequencing methods: the Maxam-Gilbert method which uses chemical degradation, and the Sanger method which is based on chain termination using dideoxynucleotides. The Sanger method is now the most common approach and involves sequencing in four separate reactions with one of the four ddNTPs added to each.
DNA Sequencing- Sanger's Method
Fred Sanger developed the chain termination method of DNA sequencing that is still widely used today. It involves making multiple copies of a DNA fragment, conducting separate sequencing reactions with each of the four dideoxynucleotides, and using gel electrophoresis or capillary electrophoresis to determine the sequence based on fragment sizes. While it can sequence fragments up to 900 base pairs, Sanger sequencing is time-consuming and expensive for large genomes. However, it provides high quality sequence data and remains useful for targeted sequencing applications.
Sangers method for DNA Sequencing
In these slides we are describe the method and steps of Sanger's method of DNA sequencing and the various agents which are helpful in this method.
Dna synthesis & sequencing
DNA sequencing involves determining the order of nucleotides (adenine, guanine, cytosine, thymine) in a DNA molecule. There are three main methods: 1) Maxam-Gilbert chemical degradation, 2) Sanger dideoxynucleotide chain termination, and 3) direct sequencing using PCR. Gene synthesis chemically builds DNA base-by-base without a template, and has applications in forensics, agriculture, and solving crimes.
DNA SEQUENCING METHOD
DNA sequencing determines the order of nucleotide bases in a DNA molecule. The first methods were developed in the 1970s by Maxam and Gilbert (chemical method) and Sanger (chain termination/dideoxy method). Sanger's method is now most commonly used and involves DNA synthesis with chain termination by dideoxynucleotides to generate fragments of different lengths that can be separated and read to determine the DNA sequence. DNA sequencing has revolutionized biological sciences by enabling diagnosis of genetic diseases, identification of disease-causing mutations, and mapping of genomes. It provides benefits for medicine, forensics, and agriculture.
Dna sequencing methods
DNA sequencing determines the precise order of nucleotides in a DNA fragment. There are several methods for DNA sequencing, including the chain termination method developed by Sanger, and the Maxam-Gilbert chemical cleavage method. Next generation sequencing is now used, which allows high-throughput sequencing of entire genomes quickly and accurately using automated methods. DNA sequencing has many applications, such as identifying disease-causing genes and mutations.
DND sequencing
This document defines DNA sequencing and describes some common DNA sequencing methods. It explains that DNA sequencing determines the order of the four nucleotide bases that make up DNA. It then describes two basic DNA sequencing methods - Maxam-Gilbert chemical sequencing and Sanger chain termination sequencing. For Sanger sequencing, it provides details on how fluorescent dideoxynucleotides are used to randomly terminate DNA strands during replication, allowing the sequence to be read from the resulting fragments.
DNA sequencing
This document defines DNA sequencing and describes some common DNA sequencing methods. It explains that DNA sequencing determines the order of the four nucleotide bases that make up DNA. It then describes two basic DNA sequencing methods - Maxam-Gilbert chemical sequencing and Sanger chain termination sequencing. For Sanger sequencing, it provides details on how fluorescent dideoxynucleotides are used to randomly terminate DNA strands during replication, allowing the sequence to be read from the resulting fragments.
Dna sequencing methods
DNA sequencing techniques have evolved over time. The dideoxy method developed by Sanger is useful for sequencing short DNA fragments of 500-750bp by terminating the chain with ddNTPs. Whole genome sequencing became possible using shotgun sequencing, which breaks the genome into random fragments that are sequenced and then reassembled. More recently, pyrosequencing was developed for sequencing by synthesis and allows accurate, parallel, and automated sequencing without the need for gel electrophoresis.
DNA sequencing
Its a presentation about DNA sequencing by basic methods and one of the advanced method along with applications of the dna sequencing.
DNA sequencing
This document discusses various methods for DNA sequencing, including both early chemical methods and more recent next-generation sequencing technologies. It describes Frederick Sanger's chain termination method from 1977, which uses DNA polymerase, dNTPs, and ddNTPs. It also describes Maxam-Gilbert sequencing from the same year, which uses chemical cleavage. Next-generation methods discussed include pyrosequencing, sequencing by ligation, sequencing by synthesis, and ion semiconductor sequencing. The document compares the different methods based on read length, accuracy, throughput, run time, and cost.
Dna sequencing techniques
The document discusses techniques for DNA sequencing, including early methods developed in the 1970s by Maxam and Gilbert as well as Sanger. It provides details on how both methods work, such as using specific chemical or enzymatic reactions to generate labeled DNA fragments of different lengths corresponding to nucleotide positions in the sequence. The document also describes how these methods were later automated, using fluorescent tags on dideoxynucleotides and capillary electrophoresis to simultaneously sequence multiple samples in a single gel. This allowed rapid determination of thousands of nucleotides and enabled large genome sequencing projects such as the Human Genome Project.
Sanger’s Method of Sequencing (1).pptx
DNA Sequencing by sanger's method. Uses dideoxy method of sequencing. Human genome project's data is completely basedon this method, look in the ppt for more information of how Frederick Sanger discovered this process of DNA sequencing and which was one of the biggest breakthrough in the field of biology and life sciences. This discovery won nobel prize in the year 1980.
GENE ISOLATION AND SEQUENCING.pdf
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine.
Sanger-Shortgun sequencing.pdf
Sanger Sequencing-chain termination method, “gold standard” for validating DNA sequences, dideoxynucleotide triphosphates , steps of Sanger sequencing.
Shotgun sequencing-Steps, limitation
dna sequencing methods
1. DNA sequencing involves determining the order of nucleotide bases in DNA. The original chain termination or dideoxy method developed by Sanger is still widely used for small DNA segments.
2. Whole genome shotgun sequencing breaks large genomes into fragments that are sequenced and then reassembled, allowing sequencing of entire genomes.
3. Pyrosequencing is a sequencing by synthesis method that uses a bioluminescent reaction to determine nucleotides added, enabling accurate and fast sequencing.
DNA SEQUENCE ANALYSIS.pdf
DNA is the molecule that contains the genetic instructions used in the development and functioning of all known living organisms. A gene is a unit of heredity and consists of a segment of DNA that codes for a protein or RNA molecule. DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule, and includes any method used to determine the order of the four bases - adenine, thymine, guanine, and cytosine. There are two main historical methods for DNA sequencing - the Maxam-Gilbert chemical method and the Sanger dideoxy chain termination method, upon which modern automated sequencing is based using fluorescence detection. DNA sequencing has many applications in fields like medicine, forensics, and agriculture
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Sanger sequencing (DNA sequencing by ENZYMATIC METHOD)
Sanger sequencing (DNA sequencing by ENZYMATIC METHOD)
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Metal ion ion indicators
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With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
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DNA sequencing by fredrick sanger’s method
- 1. DNA Sequencing - Fredrick Sanger’s Method Prepared by- Mr. A.R.Shinde
- 2. • Fredrick Sanger (1977 received the Noble prize) • Based on controlled interruption of DNA replication. • He created a system in which a cloned fragment of DNA is copied, but som of the copies are halted at each base pair along the sequence.
- 3. THE SANGER METHOD Deoxyribo nucleic acid ribose sugar lacks an oxygen atom. “dideoxynucleotide” lacks a 2nd oxygen atom (3’OH) responsible for extension of growing DNA strand.
- 4. REQUIREMENTS i. Template DNA ii. A short primer(about 20 nucleotides) iii. DNA polymerase iv. dNTP nucleotides v. 4 ddNTP nucleotides(i.e. ddG,ddC,ddA,ddT which are radioactively labeled)
- 5. PROCEDURE 4 different reactions.(each containing template DNA,primer,DNApolymerase & dNTPs) Add one type of ddNTP to each. DNA is denatured by heating 2 strands separate. DNA polymerase starts synthesizing a new strand by using one strand as the template. dNTPs & ddNTPs are randomly added to the new chain(but as soon as a ddNTP is added , the chain extension stops) DNA strands of different lengths are obtained.
- 6. Electrophoresis(PAGE). The PAGE is divided into 4 different lanes. Each reaction is then loaded into separate lanes of PAGE containing urea. DNA migrates towards +ve pole of the electric field. Movement(size dependent) fragments separated by length from shortest to longest Blue tracking dye(to track the movement) X-ray film(gives the positions of the DNA bands in the gel). Sequencing gel is read from bottom to top
- 9. Automated DNA sequencing Leroy Hood. Used instead of X-ray film. Each ddNTP was given a colour tag(fluorescently labeled) PAGE was illuminated by laser beam.
- 10. Disadvantages The dideoxy method is good only for500- 750bp reactions. Expensive. Time consuming. The human genome is about 3billion bp. Advantage Use fewer toxic reagents than Maxam-Gilbert method.