This document discusses the methodology for DNA sequencing using the chain termination method. It explains that chain termination sequencing involves using DNA polymerase to synthesize DNA strands until a dideoxynucleotide is incorporated, which terminates strand elongation. The resulting fragments of different lengths are then separated by gel electrophoresis to determine the DNA sequence. It also discusses some considerations for the method, such as the need for a single-stranded DNA template and the role of the primer in determining the region of the template that will be sequenced.
The document discusses techniques for characterizing and using cloned DNA fragments, including gel electrophoresis to separate vector and cloned DNA, sequencing cloned DNA using the dideoxy chain termination method, and subcloning DNA fragments into vectors. It focuses on the basic Sanger dideoxy chain termination method for sequencing cloned DNA fragments, which involves synthesizing truncated daughter strands using dideoxyribonucleotides to terminate DNA synthesis and reading the sequence based on fragment size separation.
1. Pyrosequencing is a DNA sequencing technique based on detecting pyrophosphate release upon nucleotide incorporation, unlike Sanger sequencing which uses chain termination.
2. In pyrosequencing, only one of the four possible nucleotides is added at a time so light emission determines which nucleotide is incorporated on the template.
3. Next generation sequencing includes 454 pyrosequencing which was the first commercially successful technique and works by parallelizing pyrosequencing on many DNA fragments attached to beads in a picotiter plate.
This document discusses several types of PCR techniques and their applications. It begins by explaining standard PCR and its development. It then describes several specialized PCR techniques including allele-specific PCR, asymmetric PCR, assembly PCR, hot-start PCR, helicase-dependent amplification, in situ PCR, inverse PCR, ligation-mediated PCR, and multiplex ligation-dependent probe amplification. Each technique is explained and examples of its uses and applications are provided.
DNA is made up of a sugar-phosphate backbone and four nucleobases - adenine, guanine, cytosine, and thymine. The bases pair up (A with T, C with G) and the paired bases stack to form the double helix structure of DNA. Early DNA sequencing methods, like Sanger sequencing, determined sequences by chain termination with dideoxynucleotides. Next generation sequencing uses reversible terminators and sequencing by synthesis to sequence millions of DNA fragments in parallel. Third generation sequencers can now sequence single DNA molecules in real time.
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.
This presentation summarizes three topics: polymerase chain reaction (PCR), antisense therapy, and the complement system. PCR is an in vitro technique used to amplify a specific DNA sequence and was invented by Kary Mullis in 1983. It involves DNA denaturation, annealing of primers, and extension of the DNA sequence. Applications include genome mapping, disease diagnosis, and forensics. Antisense therapy inhibits gene expression by using single-stranded oligonucleotides to block transcription. It is used to treat various cancers and infections. The complement system consists of serum proteins that help activate the innate immune response. It can be activated via the classical or alternative pathway and acts through mechanisms like opsonization, inflammation, and
The chain-termination method developed by Frederick Sanger and coworkers in 1977. This method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers.
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.
The document discusses techniques for characterizing and using cloned DNA fragments, including gel electrophoresis to separate vector and cloned DNA, sequencing cloned DNA using the dideoxy chain termination method, and subcloning DNA fragments into vectors. It focuses on the basic Sanger dideoxy chain termination method for sequencing cloned DNA fragments, which involves synthesizing truncated daughter strands using dideoxyribonucleotides to terminate DNA synthesis and reading the sequence based on fragment size separation.
1. Pyrosequencing is a DNA sequencing technique based on detecting pyrophosphate release upon nucleotide incorporation, unlike Sanger sequencing which uses chain termination.
2. In pyrosequencing, only one of the four possible nucleotides is added at a time so light emission determines which nucleotide is incorporated on the template.
3. Next generation sequencing includes 454 pyrosequencing which was the first commercially successful technique and works by parallelizing pyrosequencing on many DNA fragments attached to beads in a picotiter plate.
This document discusses several types of PCR techniques and their applications. It begins by explaining standard PCR and its development. It then describes several specialized PCR techniques including allele-specific PCR, asymmetric PCR, assembly PCR, hot-start PCR, helicase-dependent amplification, in situ PCR, inverse PCR, ligation-mediated PCR, and multiplex ligation-dependent probe amplification. Each technique is explained and examples of its uses and applications are provided.
DNA is made up of a sugar-phosphate backbone and four nucleobases - adenine, guanine, cytosine, and thymine. The bases pair up (A with T, C with G) and the paired bases stack to form the double helix structure of DNA. Early DNA sequencing methods, like Sanger sequencing, determined sequences by chain termination with dideoxynucleotides. Next generation sequencing uses reversible terminators and sequencing by synthesis to sequence millions of DNA fragments in parallel. Third generation sequencers can now sequence single DNA molecules in real time.
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.
This presentation summarizes three topics: polymerase chain reaction (PCR), antisense therapy, and the complement system. PCR is an in vitro technique used to amplify a specific DNA sequence and was invented by Kary Mullis in 1983. It involves DNA denaturation, annealing of primers, and extension of the DNA sequence. Applications include genome mapping, disease diagnosis, and forensics. Antisense therapy inhibits gene expression by using single-stranded oligonucleotides to block transcription. It is used to treat various cancers and infections. The complement system consists of serum proteins that help activate the innate immune response. It can be activated via the classical or alternative pathway and acts through mechanisms like opsonization, inflammation, and
The chain-termination method developed by Frederick Sanger and coworkers in 1977. This method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers.
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.
The next generation sequencing platform of roche 454creativebiogene1
454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
DNA sequencing determines the order of nucleotides in a DNA molecule. The Sanger dideoxy chain termination method is commonly used and involves DNA polymerase, dNTPs, and chain-terminating ddNTPs. This generates DNA fragments of different lengths that can be separated by gel electrophoresis and used to determine the DNA sequence. Modern sequencing uses fluorescent dye-labeled ddNTPs and capillary electrophoresis for higher throughput automated sequencing. DNA sequencing is important for understanding genetic disorders and developing treatments.
Automated DNA sequencing is now commonly used and allows for rapid and accurate sequencing of up to 100,000 nucleotides per day at low cost. It works by incorporating fluorescent tags into terminating DNA strands during sequencing reactions, then separating the strands via electrophoresis and detecting them by their fluorescence. DNA fingerprinting compares restriction fragment length polymorphisms between crime scene DNA samples and suspect samples. Variable number tandem repeats are commonly used as probes, since copy number varies greatly between individuals, allowing identification. A match between crime scene and suspect samples can provide evidence the suspect was present.
Automated DNA sequencing ; Protein sequencingRima Joseph
This document discusses several methods for DNA and protein sequencing. It describes automated DNA sequencing which is based on the Sanger method but uses fluorescent labels and allows direct computer storage of sequence data. It then discusses various methods for protein sequencing including purification, amino acid composition analysis, N-terminal sequencing using Edman degradation or other methods, C-terminal sequencing, breaking disulfide bonds, cleaving the protein into peptides, ordering peptides by overlap, and locating disulfide bonds. Newer methods discussed are using genomic data and mass spectrometry techniques.
1) DNA sequencing refers to determining the order of nucleotide bases (A, G, C, T) in a DNA molecule. This provides essential genetic information for growth and development.
2) Two major early methods for DNA sequencing were the chemical cleavage method developed by Maxam and Gilbert in 1977 and the chain termination method developed by Sanger. Sanger's method became more popular due to fewer toxic chemicals.
3) Modern DNA sequencing often uses fluorescent dye-labeled chain terminators and capillary electrophoresis. Each dye fluoresces at a different wavelength, allowing all four reactions to occur in one tube. This high-throughput automated approach has accelerated genomic research.
Next generation DNA sequencing refers to modern massively parallel DNA sequencing technologies that can generate millions of sequences simultaneously. It involves sequencing small DNA fragments in parallel and then bioinformatics to assemble the sequences. There are several NGS platforms including pyrosequencing, Illumina sequencing, and Ion Torrent sequencing which use different chemistries to sequence DNA in a massively parallel fashion, enabling large-scale genome and transcriptome sequencing. These technologies have significantly reduced the cost of DNA sequencing.
Pyrosequencing is a sequencing by synthesis technique that uses a luciferase enzyme system to monitor DNA synthesis. It works by adding DNA polymerase and a single nucleotide to the DNA fragments, generating pyrophosphate that is converted to light. The light is detected and identifies the nucleotide incorporated. Pyrosequencing has applications in cDNA analysis, mutation detection, re-sequencing of disease genes, and identifying single nucleotide polymorphisms and typing bacteria and viruses.
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
The Sanger technique is a method for DNA sequencing developed in 1977 that uses chain-terminating dideoxynucleotides. It relies on the fact that DNA synthesis will stop if a dideoxynucleotide is incorporated instead of a normal deoxynucleotide. The technique involves incubating a single-stranded DNA with the four dideoxynucleotides separately, which results in DNA fragments of different lengths that can be separated via gel electrophoresis to determine the sequence.
DNA sequencing is the process of determining the sequence of nucleotides (A, T, G, and C) in the DNA. It includes method or technology that is used to determine the order of the four bases: adenine, thymine, guanine and cytosine.
This document discusses next generation sequencing technologies. It provides details on several massively parallel sequencing platforms and describes their advantages over traditional Sanger sequencing such as higher throughput, lower costs, and ability to process millions of reads in parallel. It then outlines several applications of next generation sequencing like mutation discovery, transcriptome analysis, metagenomics, epigenetics research and discovery of non-coding RNAs.
1. DNA sequencing is used in basic biological research and applied fields like diagnostics, biotechnology, forensics, and systematics to determine the sequence of nucleotides in DNA.
2. Frederick Sanger developed the chain termination method of DNA sequencing, winning two Nobel Prizes for his work. The method involves using DNA polymerase, dNTPs, fluorescently labeled ddNTPs, and thermal cycling to generate terminated fragments of varying lengths that can then be separated by size.
3. Automated DNA sequencing further developed this method, using capillary electrophoresis to separate the terminated fragments by size before detecting the different fluorescent dyes with a laser and CCD, generating an electropherogram that software then uses
This Is the enzymatic method of of DNA sequencing developed by senger et. al.
but on this server animation is not working , plz beware of server errors.
This document summarizes the chemical synthesis of DNA through the phosphoramidite method. It describes the evolution of DNA synthesis from early phosphodiester and phosphotriester methods to the current phosphoramidite approach. The key steps of the phosphoramidite method are detritylation, activation and coupling of nucleotides, capping, and oxidation. Automated DNA synthesizers utilize this method to programmatically assemble DNA strands from nucleotide building blocks. Chemically synthesized DNA oligonucleotides have many applications, including gene cloning, sequencing, and engineering genes with novel properties.
A detailed description about the basic steps involved in the - PCR - Polymerase Chain Reaction, its applications,its limitations and steps to overcome it.
Polymerase chain reaction is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence
PCR allows for the amplification of small amounts of DNA, generating millions of copies. It involves three steps - denaturation of the DNA template, annealing of primers, and extension of the primers by DNA polymerase. Repeating this process results in exponential growth in the number of DNA copies. DNA sequencing, such as the Sanger method, is used to determine the exact order of nucleotides in a DNA fragment and can be used to map genomes and detect genetic variations.
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.
This presentation summarizes the usage of the verbs "make" and "do" by providing examples of how each are used in context. It lists 20 examples for "make" and 15 examples for "do" to illustrate their meanings and typical uses. The document aims to clarify the differences between these commonly confused verbs.
Pyrosequencing is a DNA sequencing method that detects DNA polymerase activity through the release of pyrophosphate, which generates a light signal. It works by sequentially adding individual nucleotides and detecting incorporation in real time. For massively parallel pyrosequencing, DNA fragments are attached to beads and amplified in individual emulsion droplets, then sequenced in separate wells to obtain hundreds of thousands of sequences simultaneously. This allows for rapid, high-throughput genome sequencing.
The next generation sequencing platform of roche 454creativebiogene1
454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
DNA sequencing determines the order of nucleotides in a DNA molecule. The Sanger dideoxy chain termination method is commonly used and involves DNA polymerase, dNTPs, and chain-terminating ddNTPs. This generates DNA fragments of different lengths that can be separated by gel electrophoresis and used to determine the DNA sequence. Modern sequencing uses fluorescent dye-labeled ddNTPs and capillary electrophoresis for higher throughput automated sequencing. DNA sequencing is important for understanding genetic disorders and developing treatments.
Automated DNA sequencing is now commonly used and allows for rapid and accurate sequencing of up to 100,000 nucleotides per day at low cost. It works by incorporating fluorescent tags into terminating DNA strands during sequencing reactions, then separating the strands via electrophoresis and detecting them by their fluorescence. DNA fingerprinting compares restriction fragment length polymorphisms between crime scene DNA samples and suspect samples. Variable number tandem repeats are commonly used as probes, since copy number varies greatly between individuals, allowing identification. A match between crime scene and suspect samples can provide evidence the suspect was present.
Automated DNA sequencing ; Protein sequencingRima Joseph
This document discusses several methods for DNA and protein sequencing. It describes automated DNA sequencing which is based on the Sanger method but uses fluorescent labels and allows direct computer storage of sequence data. It then discusses various methods for protein sequencing including purification, amino acid composition analysis, N-terminal sequencing using Edman degradation or other methods, C-terminal sequencing, breaking disulfide bonds, cleaving the protein into peptides, ordering peptides by overlap, and locating disulfide bonds. Newer methods discussed are using genomic data and mass spectrometry techniques.
1) DNA sequencing refers to determining the order of nucleotide bases (A, G, C, T) in a DNA molecule. This provides essential genetic information for growth and development.
2) Two major early methods for DNA sequencing were the chemical cleavage method developed by Maxam and Gilbert in 1977 and the chain termination method developed by Sanger. Sanger's method became more popular due to fewer toxic chemicals.
3) Modern DNA sequencing often uses fluorescent dye-labeled chain terminators and capillary electrophoresis. Each dye fluoresces at a different wavelength, allowing all four reactions to occur in one tube. This high-throughput automated approach has accelerated genomic research.
Next generation DNA sequencing refers to modern massively parallel DNA sequencing technologies that can generate millions of sequences simultaneously. It involves sequencing small DNA fragments in parallel and then bioinformatics to assemble the sequences. There are several NGS platforms including pyrosequencing, Illumina sequencing, and Ion Torrent sequencing which use different chemistries to sequence DNA in a massively parallel fashion, enabling large-scale genome and transcriptome sequencing. These technologies have significantly reduced the cost of DNA sequencing.
Pyrosequencing is a sequencing by synthesis technique that uses a luciferase enzyme system to monitor DNA synthesis. It works by adding DNA polymerase and a single nucleotide to the DNA fragments, generating pyrophosphate that is converted to light. The light is detected and identifies the nucleotide incorporated. Pyrosequencing has applications in cDNA analysis, mutation detection, re-sequencing of disease genes, and identifying single nucleotide polymorphisms and typing bacteria and viruses.
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
The Sanger technique is a method for DNA sequencing developed in 1977 that uses chain-terminating dideoxynucleotides. It relies on the fact that DNA synthesis will stop if a dideoxynucleotide is incorporated instead of a normal deoxynucleotide. The technique involves incubating a single-stranded DNA with the four dideoxynucleotides separately, which results in DNA fragments of different lengths that can be separated via gel electrophoresis to determine the sequence.
DNA sequencing is the process of determining the sequence of nucleotides (A, T, G, and C) in the DNA. It includes method or technology that is used to determine the order of the four bases: adenine, thymine, guanine and cytosine.
This document discusses next generation sequencing technologies. It provides details on several massively parallel sequencing platforms and describes their advantages over traditional Sanger sequencing such as higher throughput, lower costs, and ability to process millions of reads in parallel. It then outlines several applications of next generation sequencing like mutation discovery, transcriptome analysis, metagenomics, epigenetics research and discovery of non-coding RNAs.
1. DNA sequencing is used in basic biological research and applied fields like diagnostics, biotechnology, forensics, and systematics to determine the sequence of nucleotides in DNA.
2. Frederick Sanger developed the chain termination method of DNA sequencing, winning two Nobel Prizes for his work. The method involves using DNA polymerase, dNTPs, fluorescently labeled ddNTPs, and thermal cycling to generate terminated fragments of varying lengths that can then be separated by size.
3. Automated DNA sequencing further developed this method, using capillary electrophoresis to separate the terminated fragments by size before detecting the different fluorescent dyes with a laser and CCD, generating an electropherogram that software then uses
This Is the enzymatic method of of DNA sequencing developed by senger et. al.
but on this server animation is not working , plz beware of server errors.
This document summarizes the chemical synthesis of DNA through the phosphoramidite method. It describes the evolution of DNA synthesis from early phosphodiester and phosphotriester methods to the current phosphoramidite approach. The key steps of the phosphoramidite method are detritylation, activation and coupling of nucleotides, capping, and oxidation. Automated DNA synthesizers utilize this method to programmatically assemble DNA strands from nucleotide building blocks. Chemically synthesized DNA oligonucleotides have many applications, including gene cloning, sequencing, and engineering genes with novel properties.
A detailed description about the basic steps involved in the - PCR - Polymerase Chain Reaction, its applications,its limitations and steps to overcome it.
Polymerase chain reaction is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence
PCR allows for the amplification of small amounts of DNA, generating millions of copies. It involves three steps - denaturation of the DNA template, annealing of primers, and extension of the primers by DNA polymerase. Repeating this process results in exponential growth in the number of DNA copies. DNA sequencing, such as the Sanger method, is used to determine the exact order of nucleotides in a DNA fragment and can be used to map genomes and detect genetic variations.
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.
This presentation summarizes the usage of the verbs "make" and "do" by providing examples of how each are used in context. It lists 20 examples for "make" and 15 examples for "do" to illustrate their meanings and typical uses. The document aims to clarify the differences between these commonly confused verbs.
Pyrosequencing is a DNA sequencing method that detects DNA polymerase activity through the release of pyrophosphate, which generates a light signal. It works by sequentially adding individual nucleotides and detecting incorporation in real time. For massively parallel pyrosequencing, DNA fragments are attached to beads and amplified in individual emulsion droplets, then sequenced in separate wells to obtain hundreds of thousands of sequences simultaneously. This allows for rapid, high-throughput genome sequencing.
Qingdao Unionchem Co., Ltd. produces polyacrylamide polymer, which is a white powder or granular substance that is soluble in cold or hot water. It can disperse in water and efficiently flocculate suspended particles, making it useful for applications like waste water treatment, paper making, mining, and civil engineering. The polymer comes in anionic, non-ionic, and cationic forms and is usually packaged in 25kg paper bags.
The document discusses various topics related to protein structure and function. It defines different types of bonds in proteins including peptide bonds, disulfide bonds, and hydrogen bonds. It describes the 20 common amino acids that make up proteins and different secondary structures such as alpha helices and beta sheets. It discusses the four levels of protein structure - primary, secondary, tertiary, and quaternary structure. It also covers protein folding driven by hydrophobic interactions and hydrogen bonding, as well as denaturation of proteins.
This document discusses polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It defines gels and describes different types including organogels, xerogels, and hydrogels. It then focuses on polyacrylamide gels, explaining how they are made from acrylamide and bis-acrylamide monomers and can be used for electrophoresis. The document outlines the SDS-PAGE procedure, including how SDS treats proteins uniformly, and its applications in proteomics and ichthyotaxonomy.
Acrylics are a family of transparent plastics that include polymethyl methacrylate (PMMA). PMMA was first synthesized in 1877 and commercialized in the 1930s for uses like aircraft canopies. It is produced through radical polymerization of methyl methacrylate. PMMA has good clarity, weatherability, and scratch resistance but limited chemical resistance. It finds wide use in glazing, lighting, medical devices, and coatings. Other acrylics include polyacrylamide, used as a flocculant and soil conditioner, and sodium polyacrylate, a super absorbent polymer used in diapers and water-retention products.
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.
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.
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
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.
There are two main methods of DNA sequencing: the chain termination method (Sanger sequencing) and fluorescent sequencing. Sanger sequencing uses dideoxynucleotides that terminate DNA synthesis, producing fragments of different lengths that can be resolved on a gel. Fluorescent sequencing labels each dideoxynucleotide with a different colored dye, then uses software to analyze electrophoresed fragments by color and size. Next-generation sequencing allows high-throughput parallel sequencing of multiple DNA segments. It can be used for whole genome sequencing, targeted exome sequencing, or custom panels. Metagenomics applies next-generation sequencing to study the genomes of multiple organisms within an environmental sample.
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
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 refers to determining the order of nucleotide bases in a DNA molecule. Frederick Sanger developed the chain termination/dideoxy method in 1977, which uses DNA polymerase and dideoxynucleotides to generate fragmented DNA strands of different lengths. Pyrosequencing was later developed, detecting light pulses from nucleotide incorporation without electrophoresis. Both methods have advanced biological research and applications in medicine, biotechnology and forensics.
DNA sequencing is a process to determine the order of nucleotides in a DNA molecule. It was discovered in the 1970s by scientists like Frederick Sanger who developed the chain termination method. This method involves DNA replication with modified nucleotides that cause the growing DNA strand to terminate at that point. The fragments are then separated by size to reveal the sequence. Automated sequencing now uses fluorescent dyes and capillary electrophoresis for faster and higher throughput sequencing. DNA sequencing has applications in medicine, forensics, and agriculture.
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.
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 consists of a linear string of nucleotides, or bases, for simplicity, referred to by the first letters of their chemical names--A, T, C and G. The process of deducing the order of nucleotides in DNA is called DNA sequencing. Since the DNA sequence confers information that the cell uses to make RNA molecules and proteins, establishing the sequence of DNA is key for understanding how genomes work. The technology for DNA sequencing was made faster and less expensive as a part of the Human Genome Project. And recent developments have profoundly increased the efficiency of DNA sequencing even further.
The Helicos Genetic Analysis System was the first commercial NGS platform to use single molecule fluorescent sequencing. It sequences DNA fragments hybridized to a flow cell one nucleotide at a time. Fluorescent nucleotides are added and imaged individually to determine each base. Read lengths of around 30 nucleotides were achieved. Advantages included simpler sample prep avoiding PCR bias, but disadvantages included high error rates from imaging noise and slower sequencing speed.
The document summarizes a presentation on DNA sequencing techniques. It describes DNA and its functions of storing genetic information, self-duplication, and expressing genetic messages. It then explains the historical Maxam-Gilbert and Sanger DNA sequencing methods and how modern sequencing uses variations of the Sanger technique. The document provides details on the chemical degradation approach of Maxam-Gilbert and the chain termination approach of Sanger sequencing before polymerase chain reaction and gel electrophoresis. It compares the two methods and discusses applications and future directions of DNA sequencing.
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.
dna sequencing is the one of the most important technique in today's biotech field and in this ppt I cover up the most imporatant techniques of DNA sequencing methods
3. Sequencing Genes and Genomes
The most important technique
available to the molecular biologist
is DNA sequencing, by which
the precise order of nucleotides in
a piece of DNA can be
determined.
DNA sequencing methods have
been around for 40 years, and
since the mid-1970s rapid and
efficient sequencing has been
possible.
4. The methodology for DNA sequencing
There are several procedures for DNA
sequencing, the most popular being the chain
termination method first devised by Fred
Sanger and colleagues in the mid-1970s.
Chain termination sequencing has gained
preeminence for several reasons, not least being
the relative ease with which the technique can be
automated.
5. The methodology for DNA sequencing
In order to sequence an entire genome a huge
number of individual sequencing experiments
must be carried out, and it would take many
years to perform all of these by hand.
Automated sequencing techniques are
therefore essential if a genome project is to be
completed in a reasonable time span.
6. The methodology for DNA sequencing
Partof the automation strategy is to design
systems that enable many individual sequencing
experiments to be carried out at once.
With the chain termination method, up to 96
sequences can be obtained simultaneously in a
single run of a sequencing machine.
7. The methodology for DNA sequencing
Thisis still not enough to fully satisfy the demands
of genome sequencing, and during the last few
years an alternative method called
pyrosequencing has become popular.
Pyrosequencing, which was invented in 1998,
forms the basis to a massively parallel strategy
that enables hundreds of thousands of short
sequences to be generated at the same time.
8. Chain termination DNA sequencing
http://highered.mcgraw-
hill.com/sites/0072556781/student_view0/chapter15/anim
ation_quiz_1.html
9. Chain termination DNA sequencing
Chain termination DNA
sequencing is based on the
principle that single-
stranded DNA molecules
that differ in length by just a
single nucleotide can be
separated from one another
by polyacrylamide gel
electrophoresis.
10. Chain termination DNA sequencing
This means that it is
possible to resolve a family
of molecules, representing
all lengths from 10 to 1500
nucleotides, into a series of
bands in a slab or capillary
gel
11. What do we need for Chain termination
method?
1. The starting material for a chain termination
sequencing experiment is a preparation of identical
single-stranded DNA molecules.
2. The first step is to anneal a short oligonucleotide
to the same position on each molecule.
This oligonucleotide subsequently acting as the
primer for synthesis of a new DNA strand that is
complementary to the template
12.
13. What do we need for Chain termination
method?
3. The strand synthesis reaction, which is catalyzed by a
DNA polymerase enzyme and requires the four
deoxyribonucleotide triphosphates:
(dNTPs— dATP; Deoxyadenosine triphosphate,
dCTP; Deoxycytidine triphosphate, dGTP;
Deoxyguanosine triphosphate, and dTTP;
Deoxythymidine triphosphate) as substrates, would
normally continue until several thousand nucleotides
had been polymerized.
14.
15. What do we need for Chain termination
method?
4. Because,
as well as the four deoxynucleotides, a
small amount of each of four
dideoxynucleotides (ddNTPs—ddATP, ddCTP,
ddGTP, and ddTTP) is added to the reaction.
Each of these dideoxynucleotides is labeled with a
different fluorescent marker.
16. What do we need for Chain termination
method?
The polymerase enzyme does
not discriminate between
deoxy- and
dideoxynucleotides.
But once incorporated, a
dideoxynucleotide blocks
further elongation because it
lacks the 3′-hydroxyl group
needed to form a connection
with the next nucleotide.
17. What happens in Chain termination
sequencing method?
1. A single strand of DNA to be sequenced (yellow) is
hybridized to a 5’ end labeled synthetic
deoxynucleotide primer(Brown).
18. What happens in Chain termination
sequencing method?
2. The primer is elongated using DNA polymerase
in four separate reaction mixtures containing
four normal deoxynucleotide triphosphates
(dNTPs) plus one of dideoxynucleotide
triphosphate (ddNTPs) in a ratio of 100 :1.
19. What happens in Chain termination
sequencing method?
3. In each tube, the primer enlongation is
terminated by the incorporation of a
dideoxynucleotide triphosphate into the newly
synthesized chain.
20.
21. What happens in Chain termination
sequencing method?
4. The synthesized DNA chains can then be
separated by polyacrylamide gel electrophoresis.
Using this gel analysis of fragments, the
sequence of the template DNA chain be
determined.
22.
23. What happens in Chain termination
sequencing method?
Why do we use Polyacrylamide gel, not
agarose gel?
Have smaller pores than agarose can separate
DNA fragments which range in size from 10-500 bp.
DNA fragments which differ in size by one nucleotide
can be separated from each other.
NOTE: Polyacrylamide gel electrophoresis is also
used to separate protein molecules.
24.
25. What happens in Chain termination
sequencing method?
Because the normal deoxynucleotides are also
present, in larger amounts than the
dideoxynucleotides, the strand synthesis does not
always terminate close to the prime.
In fact, several hundred nucleotides may be
polymerized before a dideoxynucleotide is eventually
incorporated.
26. What happens in Chain termination
sequencing method?
The result is a set of new molecules, all of different
lengths, and each ending in a dideoxynucleotide
whose identity indicates the nucleotide—A, C, G, or
T—that is present at the equivalent position in the
template DNA
27. What happens in Chain termination
sequencing method?
The mixture is loaded into a well of a polyacrylamide
gel and electrophoresis carried out to separate the
molecules according to their lengths.
After separation, the molecules are run past a
fluorescent detector capable of discriminating the
labels attached to the dideoxynucleotides
29. What happens in Chain termination
?sequencing method
The detector therefore
determines if each molecule
ends in an A, C, G, or T.
The sequence can be printed
out for examination by the
operator.
Or entered directly into a
storage device for future
analysis.
30. Not all DNA polymerases can be used for
sequencing
Any DNA polymerase is capable of extending a
primer that has been annealed to a single- stranded
DNA molecule, but not all polymerases can be
used for DNA sequencing.
This is because many DNA polymerases have a
mixed enzymatic activity, being able to degrade as
well as synthesize DNA.
31. Not all DNA polymerases can be used for
sequencing
Degradation can occur in either the 5′→3′ or 3 ′→5′
direction.
And both activities are detrimental to accurate
chain termination sequencing.
32. Not all DNA polymerases can be used for
sequencing
33. Not all DNA polymerases can be used for
sequencing
The 5′→ 3′ exonuclease activity enables the
polymerase to remove nucleotides from the 5′ ends of
the newly-synthesized strands, changing the lengths of
these strands so that they no longer run through the
polyacrylamide gel in the appropriate order.
The 3′→ 5′ activity could have the same effect, but
more importantly, will remove a dideoxynucleotide
that has just been added at the 3′ end, preventing chain
termination from occurring.
34. Not all DNA polymerases can be used for
sequencing
In the original method for chain termination
sequencing, the Klenow polymerase was used as
the sequencing enzyme.
This is a modified version of the DNA
polymerase I enzyme from E. coli, the
modification removing the 5′→3′ exonuclease activity
of the standard enzyme.
35. Not all DNA polymerases can be used for
sequencing
However, the Klenow polymerase has low
processivity, meaning that it can only synthesize a
relatively short DNA strand before dissociating from
the template.
This limits the length of sequence that can be
obtained from a single experiment to about 250 bp.
36. Not all DNA polymerases can be used for
sequencing
Toavoid this problem, most sequencing today makes use
of a more specialized enzyme, such as Sequenase, a
modified version of the DNA polymerase encoded by
bacteriophage T7.
Sequenase has high processivity and no exonuclease
activity and so is ideal for chain termination sequencing,
enabling sequences of up to 750bp to be obtained in a
single experiment.
37. Chain termination sequencing requires a
single-stranded DNA template
The template for a chain termination experiment is
a single-stranded version of the DNA
molecule to be sequenced.
One way of obtaining single-stranded DNA is to
use an M13 vector, but the M13 system, although
designed specifically to provide DNA for chain
termination sequencing, is not ideal for this
purpose.
38. Chain termination sequencing requires a
single-stranded DNA template
The problem is that cloned DNA fragments that
are longer than about 3 kb are unstable in an M13
vector and can undergo deletions and
rearrangements.
Thismeans that M13 cloning can only be
used with short pieces of DNA.
39. Chain termination sequencing requires a
single-stranded DNA template
Plasmid vectors, which do not suffer instability
problems, are therefore more popular.
But some means is needed of converting the
double-stranded plasmid into a single-stranded
form.
40. Chain termination sequencing requires a
single-stranded DNA template
There are two possibilities for using the plasmid
vectors:
1. Double-stranded plasmid DNA can be converted into
single-stranded DNA by denaturation with alkali or by
boiling.
2. The DNA can be cloned in a phagemid, a plasmid
vector that contains an M13 origin of replication and
which can therefore be obtained as both double- and
single-stranded DNA versions
41. Chain termination sequencing requires a
single-stranded DNA template
Phagemids avoid the
instabilities of M13
cloning and can be used
with fragments up to 10
kb or more.
3. The need for single-
stranded DNA can also
be sidestepped by using a
thermostable DNA
polymerase as the
sequencing enzyme.
42. Chain termination sequencing requires a
single-stranded DNA template
This method, called
thermal cycle
sequencing, is carried
out in a similar way to
PCR, but just one
primer is used and the
reaction mixture
includes the four
dideoxynucleotides
43. The primer determines the region of the
template DNA that will be sequenced
In the first stage of a chain
termination sequencing
experiment, an oligonucleotide
primer is annealed onto the
template DNA.
The main function of the
primer is to provide the short
double-stranded region that is
needed in order for the DNA
polymerase to initiate DNA
synthesis.
44. The primer determines the region of the
template DNA that will be sequenced
The primer also plays a
second critical role in
determining the region of
the template molecule that
will be sequenced.
45. The primer determines the region of the
template DNA that will be sequenced
For most sequencing
experiments a
universal primer is
used.
This being one that is
complementary to
the part of the vector
DNA immediately
adjacent to the point
into which new DNA
is ligated.
46. The primer determines the region of the
template DNA that will be sequenced
The 3′ end of the
primer points toward
the inserted DNA, so
the sequence that is
obtained starts with a
short stretch of the
vector and then
progresses into the
cloned DNA
fragment.
47. The primer determines the region of the
template DNA that will be sequenced
If the DNA is cloned in a plasmid vector, then both
forward and reverse universal primers can be used,
enabling sequences to be obtained from both ends
of the insert.
This is an advantage if the cloned DNA is more
than 750 bp and hence too long to be sequenced
completely in one experiment.