Dna sequencing AND ANALYSIS
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Compare and contrast the chemical (Maxam/Gilbert) and chain termination (Sanger) sequencing methods. List the components and molecular reactions that occur in chain termination sequencing. Discuss the advantages of dye primer and dye terminator sequencing. Derive a text DNA sequence from raw sequencing data. Describe examples of alternative sequencing methods, such as bisulfite sequencing and pyrosequencing. Maxam/Gilbert chemical sequencing Sanger chain termination sequencing Pyrosequencing Array sequencing With addition of enzyme (DNA polymerase), the primer is extended until a ddNTP is encountered. The chain will end with the incorporation of the ddNTP. With the proper dNTP:ddNTP ratio, the chain will terminate throughout the length of the template. All terminated chains will end in the ddNTP added to that reaction. The collection of fragments is a sequencing ladder. The resulting terminated chains are resolved by electrophoresis. Fragments from each of the four tubes are placed in four separate gel lanes. Cycle sequencing is chain termination sequencing performed in a thermal cycler. Cycle sequencing requires a heat-stable DNA polymerase. Fluorescent dyes are multicyclic molecules that absorb and emit fluorescent light at specific wavelengths. Examples are fluorescein and rhodamine derivatives. For sequencing applications, these molecules can be covalently attached to nucleotides. In dye primer sequencing, the primer contains fluorescent dye–conjugated nucleotides, labeling the sequencing ladder at the 5′ ends of the chains. In dye terminator sequencing, the fluorescent dye molecules are covalently attached to the dideoxynucleotides, labeling the sequencing ladder at the 3′ ends of the chains.
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Loop Mediated Isothermal Amplification (LAMP)
Diseases are the primary constrains for the development of any living forms i.e., both plants and animals including humans.
Diagnosis is the critical step for effective treatment and control of infectious diseases. PCR (Polymerase Chain Reaction) has revolutionized the earlier detection of infection, tumour etc.,
However, LAMP has several pros over PCR. LAMP is used in rapid diagnosis of viral, bacterial and parasitic diseases. 2. It helps in the identification of genus and species-specific parasites
ABBREVATION: LOOP MEDIATED ISOTHERMAL AMPLIFICATION
First reported by Notomi et al in 2000 of EIKEN Chemical Co. Ltd., Japan.
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Determining Amino Acid Composition
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Mass spectrometry
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Type of Nucleic Acid Sequencing
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Method of DNA Sequencing
Application of DNA Sequencing
DNA Sequencing Institutes
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Reference
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This document discusses loop-mediated isothermal amplification (LAMP), a DNA amplification technique. LAMP uses 4-6 specially designed primers to amplify DNA under isothermal conditions. It has advantages over PCR such as faster amplification time (30-60 minutes), constant reaction temperature, and simpler reaction setup. LAMP can detect as few as 6 copies of DNA and has been used to detect various pathogens. The document compares LAMP to PCR and other techniques and discusses LAMP primer design, reaction principles, visualization methods, advantages, and limitations.
Loop Mediated Isothermal Amplification
The document summarizes Loop-mediated Isothermal Amplification (LAMP), a sensitive and efficient nucleic acid amplification technique. LAMP uses 4-6 primers that recognize 6 distinct regions on the target gene. The reaction proceeds at a constant temperature without thermal cycling. It produces billions of copies of target DNA in less than an hour. LAMP has applications in rapid diagnosis of diseases and identification of parasites due to its low cost, simplicity and speed compared to PCR.
Prabhakar singh ii sem-paper-dna sequencing
Prabhakar singh ii sem-paper-dna sequencingDepartment of Biochemistry, Veer Bahadur Singh Purvanchal Univarsity, Jaunpur
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.LAMP - Loop-mediated isothermal amplificaton of DNA
Loop-mediated isothermal amplification (LAMP) is a DNA amplification method that was developed by Eiken Chemical Co. in 2000. It allows for rapid, cost-effective, and simple amplification of DNA with high specificity under isothermal conditions without the need for thermocycling. LAMP uses 4-6 designed primers that recognize 6 distinct regions on the target gene to initiate self-sustained amplification. The process results in the production of stem-loop DNA structures with inverted repeats and ca be detected through turbidity or fluorescence without gel electrophoresis. LAMP has various applications including disease diagnosis, food inspection, and environmental testing.
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The document discusses loop-mediated isothermal amplification (LAMP) as a simple, rapid diagnostic tool for detecting microbial diseases. LAMP can amplify DNA under isothermal conditions in less than an hour using a single-tube reaction. It uses a set of six special primers that recognize eight distinct sequences on the target DNA to achieve high specificity. By utilizing a polymerase with strand displacement activity, LAMP amplifies DNA without the need for thermal cycling, producing large amounts of target DNA. This allows for visual detection of results without gel electrophoresis.
Cryptosporidium LAMP
The document describes the development of a LAMP method for detecting the parasite Cryptosporidium. Key points:
- LAMP amplifies DNA at a single temperature using multiple primers, allowing for simpler detection than PCR. The researcher developed LAMP primers to detect Cryptosporidium.
- Initial results found the isothermal amplification buffer was better than ThermoPol buffer at detecting a range of Cryptosporidium strains.
- Further work is needed to establish minimum detection levels and field-safe visualization methods like a portable spectrophotometer for use in detecting Cryptosporidium outside the lab.
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Diseases are the primary constrains for the development of any living forms i.e., both plants and animals including humans.
Diagnosis is the critical step for effective treatment and control of infectious diseases. PCR (Polymerase Chain Reaction) has revolutionized the earlier detection of infection, tumour etc.,
However, LAMP has several pros over PCR. LAMP is used in rapid diagnosis of viral, bacterial and parasitic diseases. 2. It helps in the identification of genus and species-specific parasites
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First reported by Notomi et al in 2000 of EIKEN Chemical Co. Ltd., Japan.
It is a very sensitive, easy and time efficient method. The LAMP reaction proceeds at a constant temperature using a strand displacement reaction.
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Introduction
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History of Protein Sequencing
Determining Amino Acid Composition
N-terminal amino acid analysis
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The Edman degradation reaction
Limitations of the Edman degradation
Mass spectrometry
Nucleic Acid Sequencing
Introduction
Nucleic Acid Sequencing
Type of Nucleic Acid Sequencing
DNA Sequencing
Method of DNA Sequencing
Application of DNA Sequencing
DNA Sequencing Institutes
Conclusion
Reference
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This document discusses loop-mediated isothermal amplification (LAMP), a DNA amplification technique. LAMP uses 4-6 specially designed primers to amplify DNA under isothermal conditions. It has advantages over PCR such as faster amplification time (30-60 minutes), constant reaction temperature, and simpler reaction setup. LAMP can detect as few as 6 copies of DNA and has been used to detect various pathogens. The document compares LAMP to PCR and other techniques and discusses LAMP primer design, reaction principles, visualization methods, advantages, and limitations.
Loop Mediated Isothermal Amplification
The document summarizes Loop-mediated Isothermal Amplification (LAMP), a sensitive and efficient nucleic acid amplification technique. LAMP uses 4-6 primers that recognize 6 distinct regions on the target gene. The reaction proceeds at a constant temperature without thermal cycling. It produces billions of copies of target DNA in less than an hour. LAMP has applications in rapid diagnosis of diseases and identification of parasites due to its low cost, simplicity and speed compared to PCR.
Prabhakar singh ii sem-paper-dna sequencing
Prabhakar singh ii sem-paper-dna sequencingDepartment of Biochemistry, Veer Bahadur Singh Purvanchal Univarsity, Jaunpur
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.LAMP - Loop-mediated isothermal amplificaton of DNA
Loop-mediated isothermal amplification (LAMP) is a DNA amplification method that was developed by Eiken Chemical Co. in 2000. It allows for rapid, cost-effective, and simple amplification of DNA with high specificity under isothermal conditions without the need for thermocycling. LAMP uses 4-6 designed primers that recognize 6 distinct regions on the target gene to initiate self-sustained amplification. The process results in the production of stem-loop DNA structures with inverted repeats and ca be detected through turbidity or fluorescence without gel electrophoresis. LAMP has various applications including disease diagnosis, food inspection, and environmental testing.
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The document discusses loop-mediated isothermal amplification (LAMP) as a simple, rapid diagnostic tool for detecting microbial diseases. LAMP can amplify DNA under isothermal conditions in less than an hour using a single-tube reaction. It uses a set of six special primers that recognize eight distinct sequences on the target DNA to achieve high specificity. By utilizing a polymerase with strand displacement activity, LAMP amplifies DNA without the need for thermal cycling, producing large amounts of target DNA. This allows for visual detection of results without gel electrophoresis.
Cryptosporidium LAMP
The document describes the development of a LAMP method for detecting the parasite Cryptosporidium. Key points:
- LAMP amplifies DNA at a single temperature using multiple primers, allowing for simpler detection than PCR. The researcher developed LAMP primers to detect Cryptosporidium.
- Initial results found the isothermal amplification buffer was better than ThermoPol buffer at detecting a range of Cryptosporidium strains.
- Further work is needed to establish minimum detection levels and field-safe visualization methods like a portable spectrophotometer for use in detecting Cryptosporidium outside the lab.
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LAMP PCR is a nucleic acid amplification technique that uses a DNA polymerase with strand displacement activity. It requires 4-6 designed primers that recognize 6 distinct regions on the target gene. Amplification occurs isothermally without temperature cycling. It produces stem-loop DNA structures with dumbbell ends that are amplified quickly using loop primers. LAMP has advantages over PCR like higher specificity, greater detection limit, simpler operation without expensive equipment, and ability to use crude samples. It is a sensitive, specific and cost-effective technique used for diagnosing infectious diseases.
Journal club 11 06-12
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This document provides an overview of Loop Mediated Isothermal Amplification (LAMP), a gene amplification technique that allows for rapid detection of pathogens. LAMP uses a unique set of six primers that recognize eight distinct sequences on the target gene for high specificity amplification under isothermal conditions. It has advantages over PCR as amplification can be completed in less than an hour without a thermal cycler. LAMP products can be visually detected with the naked eye through turbidity or fluorescence and has applications for point-of-care diagnosis of various infectious diseases.
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This document discusses LAMP (loop-mediated isothermal amplification), a diagnostic technique for visual virus detection. LAMP offers advantages over PCR like higher sensitivity, specificity, speed, and simplicity. It can detect as little as 10 femtograms of DNA under isothermal conditions in 60 minutes. The document reviews the ideal properties of diagnostic tests, different isothermal amplification techniques, LAMP primers and enzymes, result interpretation methods, LAMP stability and applications for detecting various animal viruses. While sensitive, LAMP is prone to product cross-contamination; solutions to address this like closed-tube reactions and pre-made mixtures are proposed to make LAMP suitable for field-level pathogen diagnosis.
Loop Mediated Isothermal Amplification (LAMP)
Loop Mediated Isothermal Amplification (LAMP) is a kinds of PCR reaction. This technology is most reliable and convenient than conventional PCR procedure. We can call it updated version of PCR. Rapid, Easy detectable and cheap to accomplish the process.
Pyrosequencing
This document discusses two approaches to pyrosequencing technology - solid phase and liquid phase. The solid phase approach utilizes streptavidin coated beads to immobilize biotin-labeled DNA templates. It involves sequential addition of nucleotides followed by washing steps to remove unincoporated nucleotides. The liquid phase approach introduced an enzyme called apyrase that degrades unincorporated nucleotides, eliminating the need for washing steps. It involves a cascade of four enzymes - DNA polymerase, ATP sulfurylase, luciferase, and apyrase to continuously degrade unincorporated nucleotides and determine DNA sequences from light signals.
Lecture on DNA sequencing
The document discusses different methods of DNA sequencing including the Maxam-Gilbert and Sanger chain termination methods as well as newer next generation sequencing techniques. It describes the principles, steps, and significance of the Maxam-Gilbert and Sanger methods and how next generation sequencing improved DNA sequencing by allowing millions of DNA molecules to be sequenced simultaneously in an automated process.
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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.
Polymerase Chain Reaction
Polymerase chain reaction or PCR is a laboratory technique that has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing.
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.
Transcription Prokaryotes
ATP transcription in prokaryotes involves RNA polymerase or transcriptase binding to DNA and initiating transcription. RNA polymerase moves along the DNA strand and synthesizes a complementary RNA molecule using one DNA strand as a template. The newly synthesized RNA molecule is then released from the DNA template and can be further processed.
Cloning pcr
This document discusses PCR cloning and various applications of PCR. It provides details on:
1. PCR cloning allows amplification of DNA fragments and vectors without restriction enzymes, requiring only small amounts of DNA.
2. PCR has numerous applications including gene cloning, paternity testing, and mutation detection for inherited diseases.
3. Key aspects of PCR include its three main stages - denaturation, annealing of primers, and extension. Proofreading enzymes play an important role by identifying and removing mismatched bases to ensure high fidelity DNA replication.
Cloning pcr amirtham
This document discusses various applications of PCR cloning including its advantages over traditional cloning methods. It provides details on:
1) PCR cloning allows cloning of DNA fragments without restriction enzymes and works well for projects requiring high throughput. It can clone DNA fragments that are not available in large amounts.
2) Common types of PCR like real-time PCR, reverse transcriptase PCR, and applications like paternity testing, mutation detection, gene recombination through addition, deletion, and site-specific mutagenesis.
3) The role of proofreading enzymes in ensuring faithful DNA replication by identifying and removing mismatched bases.
Pcr molecular diagnosis
This document discusses several molecular diagnosis techniques, including single-strand conformation polymorphism (SSCP) analysis and chemical cleavage of mismatch (CCM). It describes the CCM method, which uses specific reagents to modify and cleave DNA at the site of mutations, allowing detection of point mutations and insertions/deletions. PCR heteroduplexes are treated with reagents that modify unpaired bases before cleavage and electrophoresis identifies the location and type of mutation. The CCM method has higher diagnostic sensitivity than other techniques and can analyze longer DNA fragments of up to 2 kb.
Polymerase Chain Reaction(PCR)
Polymerase Chain Reaction (PCR) is a technique used to amplify small amounts of DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to denature and replicate the target DNA. Each cycle doubles the amount of target DNA, exponentially increasing its quantity for analysis. PCR uses primers, DNA polymerase, and dNTPs to selectively amplify the target DNA sequence. It has revolutionized molecular biology and is widely used for DNA cloning, detection of genetic diseases and mutations, forensic analysis, and more.
T/A CLONING
TA cloning is a subcloning technique that relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize and ligate together without using restriction enzymes. PCR products are amplified with Taq DNA polymerase, which adds an adenine to the 3' end. These inserts are cloned into vectors that are linearized and given complementary 3' thymine overhangs. The process is simpler and faster than traditional cloning as it does not require restriction enzymes, with commercial kits expediting the workflow, though the gene has a 50% chance of inserting in the reverse direction.
Dna sequencing
DNA sequencing is the process of determining the order of nucleotides in DNA. There are several methods, including classical sequencing techniques like Sanger sequencing and Maxam-Gilbert sequencing, as well as next-generation sequencing techniques like pyrosequencing. Sanger sequencing uses chain termination with dideoxynucleotides to generate DNA fragments of different lengths that can then be separated and read. Pyrosequencing detects nucleotide incorporation in real-time by detecting pyrophosphate release using a luciferase reaction that produces light. DNA sequencing is important for understanding genes, detecting diseases, personalized medicine, forensics, and evolutionary studies.
Lecture2 nucleic acid (1)
DNA replication is semiconservative and involves unwinding of the DNA double helix by helicase, stabilization by SSB proteins, and use of RNA primers by primase. DNA polymerase extends the primers using free 3'OH groups and dNTPs. Replication is continuous on the leading strand but discontinuous on the lagging strand, producing Okazaki fragments. RNA primers are replaced by DNA and gaps sealed by ligase. DNA sequencing uses dideoxynucleotides and DNA polymerase to differentially terminate DNA strand extension, producing fragments of different lengths that can be resolved by gel electrophoresis to determine the DNA sequence.
Sengar method of dna sequencing
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.
PCR
A detailed description about the basic steps involved in the - PCR - Polymerase Chain Reaction, its applications,its limitations and steps to overcome it.
DNA sequencing
DNA sequencing refers to determining the order of nucleotides in a DNA molecule. The first DNA sequence was obtained in the 1970s using chromatography. Modern methods use dye-based sequencing and automation. The two main historical methods are the Maxam-Gilbert chemical degradation method and the Sanger dideoxy chain termination method. Next generation sequencing now allows millions of DNA molecules to be sequenced in parallel through massively parallel sequencing technologies.
GENE SEQUENCING
Gene sequencing is the process of determining the order of nucleotides in a gene or genome. It allows scientists to understand how alterations in DNA sequences can cause genetic conditions or affect protein function. There are several methods for gene sequencing, including Sanger sequencing which uses chain termination with dideoxynucleotides, and next generation sequencing techniques like pyrosequencing. Gene sequencing provides insight into genetic variations and metabolic pathways that can aid disease research and treatment development.
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LAMP PCR
LAMP PCR is a nucleic acid amplification technique that uses a DNA polymerase with strand displacement activity. It requires 4-6 designed primers that recognize 6 distinct regions on the target gene. Amplification occurs isothermally without temperature cycling. It produces stem-loop DNA structures with dumbbell ends that are amplified quickly using loop primers. LAMP has advantages over PCR like higher specificity, greater detection limit, simpler operation without expensive equipment, and ability to use crude samples. It is a sensitive, specific and cost-effective technique used for diagnosing infectious diseases.
Journal club 11 06-12
Loop-mediated isothermal amplification (LAMP) is a technique that amplifies DNA under isothermal conditions for rapid and accurate diagnosis of infectious diseases. LAMP uses 4-6 primers that recognize 6 distinct regions on the target DNA to initiate self-sustained amplification at 60-65°C. The resulting products are numerous copies of stem-loop DNA structures with various lengths and cauliflower-like structures. LAMP has advantages over PCR as it does not require thermal cycling and can produce 10^9-10^10 copies of target in less than an hour, making it suitable for resource-limited settings.
Loop mediated isothermal amplification by dr.pavulraj.s
This document provides an overview of Loop Mediated Isothermal Amplification (LAMP), a gene amplification technique that allows for rapid detection of pathogens. LAMP uses a unique set of six primers that recognize eight distinct sequences on the target gene for high specificity amplification under isothermal conditions. It has advantages over PCR as amplification can be completed in less than an hour without a thermal cycler. LAMP products can be visually detected with the naked eye through turbidity or fluorescence and has applications for point-of-care diagnosis of various infectious diseases.
LAMP- Daignostic assay
This document discusses LAMP (loop-mediated isothermal amplification), a diagnostic technique for visual virus detection. LAMP offers advantages over PCR like higher sensitivity, specificity, speed, and simplicity. It can detect as little as 10 femtograms of DNA under isothermal conditions in 60 minutes. The document reviews the ideal properties of diagnostic tests, different isothermal amplification techniques, LAMP primers and enzymes, result interpretation methods, LAMP stability and applications for detecting various animal viruses. While sensitive, LAMP is prone to product cross-contamination; solutions to address this like closed-tube reactions and pre-made mixtures are proposed to make LAMP suitable for field-level pathogen diagnosis.
Loop Mediated Isothermal Amplification (LAMP)
Loop Mediated Isothermal Amplification (LAMP) is a kinds of PCR reaction. This technology is most reliable and convenient than conventional PCR procedure. We can call it updated version of PCR. Rapid, Easy detectable and cheap to accomplish the process.
Pyrosequencing
This document discusses two approaches to pyrosequencing technology - solid phase and liquid phase. The solid phase approach utilizes streptavidin coated beads to immobilize biotin-labeled DNA templates. It involves sequential addition of nucleotides followed by washing steps to remove unincoporated nucleotides. The liquid phase approach introduced an enzyme called apyrase that degrades unincorporated nucleotides, eliminating the need for washing steps. It involves a cascade of four enzymes - DNA polymerase, ATP sulfurylase, luciferase, and apyrase to continuously degrade unincorporated nucleotides and determine DNA sequences from light signals.
Lecture on DNA sequencing
The document discusses different methods of DNA sequencing including the Maxam-Gilbert and Sanger chain termination methods as well as newer next generation sequencing techniques. It describes the principles, steps, and significance of the Maxam-Gilbert and Sanger methods and how next generation sequencing improved DNA sequencing by allowing millions of DNA molecules to be sequenced simultaneously in an automated process.
Automated DNA sequencing ; Protein sequencing
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.
Polymerase Chain Reaction
Polymerase chain reaction or PCR is a laboratory technique that has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing.
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.
Transcription Prokaryotes
ATP transcription in prokaryotes involves RNA polymerase or transcriptase binding to DNA and initiating transcription. RNA polymerase moves along the DNA strand and synthesizes a complementary RNA molecule using one DNA strand as a template. The newly synthesized RNA molecule is then released from the DNA template and can be further processed.
Cloning pcr
This document discusses PCR cloning and various applications of PCR. It provides details on:
1. PCR cloning allows amplification of DNA fragments and vectors without restriction enzymes, requiring only small amounts of DNA.
2. PCR has numerous applications including gene cloning, paternity testing, and mutation detection for inherited diseases.
3. Key aspects of PCR include its three main stages - denaturation, annealing of primers, and extension. Proofreading enzymes play an important role by identifying and removing mismatched bases to ensure high fidelity DNA replication.
Cloning pcr amirtham
This document discusses various applications of PCR cloning including its advantages over traditional cloning methods. It provides details on:
1) PCR cloning allows cloning of DNA fragments without restriction enzymes and works well for projects requiring high throughput. It can clone DNA fragments that are not available in large amounts.
2) Common types of PCR like real-time PCR, reverse transcriptase PCR, and applications like paternity testing, mutation detection, gene recombination through addition, deletion, and site-specific mutagenesis.
3) The role of proofreading enzymes in ensuring faithful DNA replication by identifying and removing mismatched bases.
Pcr molecular diagnosis
This document discusses several molecular diagnosis techniques, including single-strand conformation polymorphism (SSCP) analysis and chemical cleavage of mismatch (CCM). It describes the CCM method, which uses specific reagents to modify and cleave DNA at the site of mutations, allowing detection of point mutations and insertions/deletions. PCR heteroduplexes are treated with reagents that modify unpaired bases before cleavage and electrophoresis identifies the location and type of mutation. The CCM method has higher diagnostic sensitivity than other techniques and can analyze longer DNA fragments of up to 2 kb.
Polymerase Chain Reaction(PCR)
Polymerase Chain Reaction (PCR) is a technique used to amplify small amounts of DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to denature and replicate the target DNA. Each cycle doubles the amount of target DNA, exponentially increasing its quantity for analysis. PCR uses primers, DNA polymerase, and dNTPs to selectively amplify the target DNA sequence. It has revolutionized molecular biology and is widely used for DNA cloning, detection of genetic diseases and mutations, forensic analysis, and more.
T/A CLONING
TA cloning is a subcloning technique that relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize and ligate together without using restriction enzymes. PCR products are amplified with Taq DNA polymerase, which adds an adenine to the 3' end. These inserts are cloned into vectors that are linearized and given complementary 3' thymine overhangs. The process is simpler and faster than traditional cloning as it does not require restriction enzymes, with commercial kits expediting the workflow, though the gene has a 50% chance of inserting in the reverse direction.
Dna sequencing
DNA sequencing is the process of determining the order of nucleotides in DNA. There are several methods, including classical sequencing techniques like Sanger sequencing and Maxam-Gilbert sequencing, as well as next-generation sequencing techniques like pyrosequencing. Sanger sequencing uses chain termination with dideoxynucleotides to generate DNA fragments of different lengths that can then be separated and read. Pyrosequencing detects nucleotide incorporation in real-time by detecting pyrophosphate release using a luciferase reaction that produces light. DNA sequencing is important for understanding genes, detecting diseases, personalized medicine, forensics, and evolutionary studies.
Lecture2 nucleic acid (1)
DNA replication is semiconservative and involves unwinding of the DNA double helix by helicase, stabilization by SSB proteins, and use of RNA primers by primase. DNA polymerase extends the primers using free 3'OH groups and dNTPs. Replication is continuous on the leading strand but discontinuous on the lagging strand, producing Okazaki fragments. RNA primers are replaced by DNA and gaps sealed by ligase. DNA sequencing uses dideoxynucleotides and DNA polymerase to differentially terminate DNA strand extension, producing fragments of different lengths that can be resolved by gel electrophoresis to determine the DNA sequence.
Sengar method of dna sequencing
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.
PCR
A detailed description about the basic steps involved in the - PCR - Polymerase Chain Reaction, its applications,its limitations and steps to overcome it.
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Loop mediated isothermal amplification by dr.pavulraj.s
Loop mediated isothermal amplification by dr.pavulraj.s
Similar to Dna sequencing AND ANALYSIS
DNA sequencing
DNA sequencing refers to determining the order of nucleotides in a DNA molecule. The first DNA sequence was obtained in the 1970s using chromatography. Modern methods use dye-based sequencing and automation. The two main historical methods are the Maxam-Gilbert chemical degradation method and the Sanger dideoxy chain termination method. Next generation sequencing now allows millions of DNA molecules to be sequenced in parallel through massively parallel sequencing technologies.
GENE SEQUENCING
Gene sequencing is the process of determining the order of nucleotides in a gene or genome. It allows scientists to understand how alterations in DNA sequences can cause genetic conditions or affect protein function. There are several methods for gene sequencing, including Sanger sequencing which uses chain termination with dideoxynucleotides, and next generation sequencing techniques like pyrosequencing. Gene sequencing provides insight into genetic variations and metabolic pathways that can aid disease research and treatment development.
Dna sequencing
DNA sequencing determines the order of nucleotide bases in a DNA sample. The Sanger method, also known as dideoxy or chain termination method, was the first widely used DNA sequencing technique. It involves DNA polymerase, dNTPs, a primer, and ddNTPs to terminate DNA strand extension. The fragments are separated by gel electrophoresis and the sequence is read. Next-generation sequencing methods like pyrosequencing do not require electrophoresis or fragment separation. They detect incorporated nucleotides in real-time. Nanopore sequencing detects changes in electrical current as single-stranded DNA passes through a nanopore protein.
Dna sequencing
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.
DNA sequencing
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.
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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.
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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
Lecture 2 final sols 2019
The document provides an overview of DNA replication through several key experiments and findings:
1. The Meselson-Stahl experiment in 1958 supported the semiconservative model of DNA replication proposed by Watson and Crick. They found that E. coli DNA replicated semiconservatively after growing bacteria in media containing different nitrogen isotopes.
2. DNA replication requires unwinding of the DNA double helix by helicase, stabilization by SSB proteins, and relaxation of supercoiling by topoisomerase.
3. RNA primers are added by primase and extended by DNA polymerase, which can only extend from a 3' OH group. Replication is continuous on the leading strand but discontinuous via Ok
DNA polymerases (DNA manupliation Enzymes).pdf
the Secrets of DNA Manipulation: A Comprehensive Exploration of DNA Polymerase and Enzymes
In this PDF presentation entitled "Enzymes that Manipulate DNA, Specially DNA Polymerase," we delve deep into the mechanisms and functions of these remarkable enzymes that play a pivotal role in the realm of molecular biology.
🧬 Key Highlights:
Introduction to DNA Polymerase:
Uncover the fundamental aspects of DNA polymerase, a key player in DNA replication and repair. Explore its structure, functions, and the indispensable role it plays in maintaining the genetic integrity of living organisms.
Types of DNA Polymerases:
Delve into the diverse landscape of DNA polymerases, ranging from prokaryotic to eukaryotic systems. Understand how different types of DNA polymerases contribute to the precision and efficiency of DNA synthesis.
Examples of polymerases:
•DNA polymerase 1
•klenow fragment
•sequenase
•Taq polymerase
•Reverse Transcriptase
DNA Replication
Take a closer look at the intricate dance of enzymes during DNA replication. Follow the step-by-step process, and gain insights into how DNA polymerase ensures the accurate transmission of genetic information from one generation to the next.
Technological Applications:
Unleash the potential of DNA polymerase in various biotechnological applications. From PCR (Polymerase Chain Reaction) to DNA sequencing, discover how these enzymes have revolutionized molecular biology and genetic research.
Emerging Trends and Future Prospects:
Stay ahead of the curve by exploring the latest advancements and emerging trends in DNA manipulation. Witness the ongoing research that promises to unlock new possibilities in the field.
🎓 Who Should Explore This Presentation?
Students and researchers in molecular biology and genetics
Biotechnologists and professionals in the field of genetic engineering
Enthusiasts curious about the molecular machinery behind DNA manipulation
PCR
PCR (polymerase chain reaction) is a technique used to amplify a specific region of DNA. It involves repeated cycles of heating and cooling of the DNA sample to denature the DNA strands, allow primers to anneal to the target region, and extend new strands using a DNA polymerase. Each cycle doubles the number of copies of the target region, allowing millions of copies to be produced from a single DNA molecule. PCR was invented in 1985 and has many applications, including detecting DNA sequences and quantifying gene expression levels.
DNA sequencing
DNA sequencing methods determine the order of nucleotides in a DNA molecule. Maxam-Gilbert sequencing uses specific chemical agents to randomly cleave DNA fragments at individual nucleotide positions, generating labeled fragments that are separated by gel electrophoresis. Sanger sequencing uses DNA polymerase and modified nucleotides called dideoxynucleotides to terminate DNA replication in a sequence-specific manner, producing a collection of labeled fragments of different lengths that define the DNA sequence. Automated DNA sequencing now translates gel images into nucleotide sequences using fluorescent tags and detection of signal intensities.
Next Generation Sequencing
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.
Dna sequencing and its types
This power point gives you very good explanations about conventional sequencing and Next generation sequencing methods
DNA sequencing.pptx
Sanger sequencing, also known as chain termination sequencing, was developed in 1977 and involves DNA polymerase extending a primer until a dideoxynucleotide is incorporated, terminating DNA synthesis. The resulting fragments of different lengths are separated by gel electrophoresis and read to determine the DNA sequence. Maxam-Gilbert sequencing uses chemical reactions that selectively modify bases followed by piperidine cleavage, generating labeled fragments that are resolved on gels to reveal the DNA sequence. Both methods were crucial for determining DNA sequences in the Human Genome Project.
Dhanu
DNA sequencing determines the order of bases in a DNA sample. Two major early methods are the Maxam-Gilbert chemical cleavage method using double-stranded DNA and the Sanger chain termination method using single-stranded DNA. The Sanger method involves primer annealing, complementary strand synthesis using labeled chain terminators, and resolution on a polyacrylamide gel to read the sequence. Bacteria protect their DNA from foreign DNA through modification and restriction. Modification involves host-specific methylation while restriction enzymes cut foreign DNA at specific recognition sequences. There are four classes of restriction enzymes that differ in subunit composition, cofactor requirements, and cleavage position relative to the recognition site.
Dna sequencing.
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.
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.
Similar to Dna sequencing AND ANALYSIS (20)
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Ramsar Wetlands of pakistan
23rd November 1976: The convention on wetlands came into force for Pakistan
1976: Total wetland surface area 7,800 sq km with 9 wetlands of global significance
2001: Number raised to 16
2013:Among 1,888 Ramsar sites, 19 sites of Pakistan bear global importance
Total surface area of Pakistan Ramsar sites is 1,343,627 hectares
Pakistan’s 19 sites Internationally recognized by Ramsar Convention (RC) Bureau
2PK009
Astola (Haft Talar) Island, Balochistan
Russel’s viper (Echis carinatus astolae), is an endemic species and a highly poisonous snake, which is reported only from this Island
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The amazing biological facts
The amazing biological facts
What are facts
Difference between facts and reality
Difference between facts and opinion
Humans mysterious facts
Scary plants facts
Biology of Quran
Astonishing emotional facts
References
The reality, state of affairs, or relations that make statements true. “It’s a fact that 1+1=2,” or “it’s a fact that john has a headache. If the statements are true, then corresponding facts exist that make them true. There are no facts that don’t exist. If something is a fact, then it exists.
Saccadic masking:
Also known as (visual) saccadic suppression, is the phenomenon in visual perception where the brain selectively blocks visual processing during eye movements in such a way that neither the motion of the eye nor the gap in visual perception is noticeable to the viewer.
Saccadic masking” specifically refers to the fact that our visual system is capable of “blanking out” or masking the perception of the saccadic motion of the eyes; in simpler terms, you’re not aware of seeing the motion blur or other clues that your eyes are constantly making very rapid small movements, called saccades.
The term DNA is an abbreviation of the genetic material in living things. The beginning of the science of genetics dates back to genetic laws drawn up by the scientist, the Austrian monk Johann Gregor Mendel in 1865. The date, a turning point in the history of science, is referred to in verse 65 of Surat al-Kahf, or verse 18:65.
In Surat al-Kahf, which refers to DNA and the year 1865 when the science of genetics began, DNA is repeated 7 times, as is RNA (the Arabic letters Ra-Nun-Alif).
The letters D-N-A appear side by side three times in this verse, in a most incomparable manner. In no other verse of the Qur’an do the letters “DNA” appear consecutively so often. The number of this exceptional verse in which the term DNA appears so strikingly is 18:65.
Snapdragons Make Cute Flowers, But When They Die… They Turn Into Creepy Skulls
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The dried seed pods clearly resemble tiny skulls
These skulls are oddly human-looking…
Ancient cultures once thought snapdragons held supernatural powers
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Darwins concept
Darwins concept
He was born February 12th 1809
Darwin developed the biological theory of evolution that explains how modern organisms evolved over long periods of time through descent from common ancestors.
In 1831, he began a 5 year voyage on the HMS Beagle that would change his life.
Darwin thought about the patterns he’d seen on his voyage
He realized that there were many similarities between the animals he’d seen
There was evidence that suggested that species were not fixed and that they could change by some natural process
Proposed that the use or disuse of organs caused organisms to gain or lose traits over time.
These new characteristics could be passed on to the next generation.
Lamarck’s hypotheses were incorrect in many ways
However, Lamarck was one of the first to:
Suggest that species are not fixed
Explain that evolution uses natural processes
Recognize that there is a link between an organism’s environment and its body structures
Lamarck’s work paved the way for later biologists, including Darwin
In 1798, Thomas Malthus noticed that people were being born faster than people were dying.
How this helped Darwin
He reasoned that what Malthus proposed for human populations also applied to all living things.
Struggle for Existence
From Malthus’ theory of supply and demand, Darwin reasoned that if more individuals are produced than can survive, they will have to compete for food, living space and other necessities of life
Darwin described this as the struggle for existence
Individuals have natural variations among their inheritable traits
Fast predators capture prey more efficiently
Prey that are faster, better camouflaged or better protected avoid being caught.
Any heritable characteristic that increases an organisms ability to survive and reproduce in its environment is called an adaptation’’
Ability to survive and reproduce in a specific environment is called FITNESS
Fitness is a result of adaptation
Good adaptations allow organisms to survive and are passed on to their offspring.
Good fitness: reproduce
Low fitness: few offspring/extinction
Chromosomal basis of inheritance
Chromosomal Basis of Inheritance
Be familiar with patterns of inheritance for autosomal and sex linked genes
Understand the concept of “Linked Genes”
Understand how traits affected by incomplete dominance and codominance differ from autosomal dominant and autosomal recessive traits
Understand how nondisjunction of chromosomes can lead to disorders.
Linked genes: are those that reside on the same chromosome and tend to be inherited together
Humans have 23 pairs of chromosomes
Autosomal genes reside on the autosomal chromosomes (pairs 1-22)
Sex-linked genes are found on the sex chromosomes
(pair 23, usually on the X)
Autosomal genes are usually represented by a pair of alleles
The phenotype of the gene reflects the dominant or recessive relationship of the alleles.
Most autosomal genetic diseases are autosomal recessive meaning the individual need to be homozygous recessive to exhibit the condition
(example: cystic fibrosis) Production of abnormmaly thick mucus. Leading to the blockage of panreatic duct, intestines and respiratory infection.
Huntington’s disease is an autosomal dominant disorder meaning that is a single Huntingtons allele is inherited, the individual will have the disease.
Some alleles do not show a dominance hierarchy
Incomplete dominance: the phenotype of a heterozygous genotype is intermediate in appearance
Codominance: each allele in the genotype for a particular gene will be expressed in the phenotype
Males and females differ in their sex chromosome combination
(females XX; males XY)
Because the X contains genes and the Y “does not”, inheritance patterns of sex-linked genes vary between the sexes
recessive traits more prevalent in males
Genetic disorders can also occur due to errors in the number of inherited chromosomes
This condition arises through a problem that occurs during meiosis
Although female mammals, including humans, inherit two X chromosomes, one X chromosome in each cell becomes almost completely inactivated during embryonic development.
Barr body
Nondisjunction:
Leads to aneuploidy:
Aneuploidy: is the condition of having less than or more than the normal diploid number of chromosomes, and is the most frequently observed type of cytogenetic abnormality.
Mendelian inheritance has its physical basis in the behavior of chromosomes during sexual life cycles.
Morgan traced a gene to a specific chromosome.
Sex-linked genes have unique patterns of inheritance.
Alterations of chromosome numbers or structure cause some genetic disorders.
Linked genes tend to be inherited together because they are located on the same chromosome.
Independent assortment of chromosomes and crossing over produce genetic variation (recombinants)
Geneticists can use recombination data to map a chromosomes genetic loci.
Chromosomal basis for sex is dependent upon the organism.
Chromosomes
Chromosomes are structures that carry genetic information in the form of DNA. They are found in the nucleus of human cells and determine characteristics like eye color and blood type. Each human cell normally contains 23 pairs of chromosomes, half inherited from each parent, for a total of 46 chromosomes in nearly every cell.
Beta oxidation
Beta-oxidation is the catabolic process by which fatty acid molecules are broken down in the cytosol in prokaryotes and in the mitochondria in eukaryotes to generate acetyl-coa, which enters the citric acid cycle, and NADH and FADH2, which are co-enzymes used in the electron transport .
Tryglycerides having one glycerol and three fatty acids .
Glycerol and fatty acids are separated with the help of enzyme lipase .
Free fatty acids can enter into the blood stream for metabolism .
There are two tissues in the body which can not metabolise it are RBCs and Brain(nervous tissue) which do not contain mitochondria.
Step 1:
Once the triglycerides are broken down into glycerol and fatty acids they must be activated before they can enter into the mitochondria and proceed on with beta-oxidation. This is done by Acyl-CoA synthetase to yield fatty acyl-CoA.
After the fatty acid has been acylated it is now ready to enter into the mitochondria.
Step 3:
There are two carrier proteins (Carnitine acyltransferase I and II),
Step 4:
Once inside the mitochondria the fatty acyl-CoA can enter into beta-oxidation
Oxidation: A fatty acyl-CoA is oxidized by Acyl-CoA dehydrogenase to yield a trans alkene. This is done with the aid of an [FAD] prosthetic group.
Hydration:
The trans alkene is then hydrated with the help of Enoyl-CoA hydratase.
The alcohol of the hydroxyacly-CoA is then oxidized by NAD+ to a carbonyl with the help of Hydroxyacyl-CoA dehydrogenase.
Finally acetyl-CoA is cleaved off with the help of Thiolase to yield an Acyl-CoA that is two carbons shorter than before. The cleaved acetyl-CoA can then enter into the TCA and ETC because it is already within the mitochondria.
Conclusions :
Fatty acid β-oxidation is major metabolic pathway that is responsible for the mitochondrial breakdown of long-chain acyl-CoA to acetyl-CoA. This process involves many steps that are regulated at the transcriptional and post-transcriptional level
Structure of muscle contraction
Muscle contraction is the activation of tension-generating sites within muscle fibers.
Muscles are composed of two major protein filaments: a thick filament composed of the protein myosin and a thin filament composed of the protein actin. Muscle contraction occurs when these filaments slide over one another in a series of repetitive events.
Components of muscle contraction:
Sarcomere
Actin
Myosin
Z-bands
I-bands
A-band
H-zone
The thick filaments and the thin filaments within myofibrils overlap in a structured way, forming units called sarcomeres
The 'H zone' is at the centre of the A band of each sarcomere. As shown below, this is the region in which there are only thick filaments, and no thin filaments
Sarcomere Bands :
A-band
A band' is a relatively darker area within the sarcomere that extends along the total length of the thick filaments
The 'I band' is the region between adjacent A bands, in which there are only thin filaments, and no thick filaments.
From all the above disscussion we can say that muscle contraction have some major components like mayosin and actin. Which form the bands on the sarcomere by interdigitating..
Parental care in amphibians and reptiles
Any form of parental behavior that appears likely to increase the fitness of the parent’s offspring.
Parents of some animals go to great lengths to increase their offspring’s survival prospects by protecting them from predators, food shortages, desiccation, and a range of other environmental hazards.
First off all they decide the oviposition site.
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Rheobatrachus females brooded tadpoles in their stomachs, and the young emerged from the females
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Dna sequencing AND ANALYSIS
- 2. Objectives Compare and contrast the chemical (Maxam/Gilbert) and chain termination (Sanger) sequencing methods. List the components and molecular reactions that occur in chain termination sequencing. Discuss the advantages of dye primer and dye terminator sequencing. Derive a text DNA sequence from raw sequencing data. Describe examples of alternative sequencing methods, such as bisulfite sequencing and pyrosequencing.
- 3. Sequencing Methods Maxam/Gilbert chemical sequencing Sanger chain termination sequencing Pyrosequencing Array sequencing
- 4. Maxam-Gilbert sequencing is performed by chain breakage at specific nucleotides. DMS G G G G FA G A G G A G A A H C T T C T C C T H+S C C C C Maxam-Gilbert Sequencing
- 5. Sequencing gels are read from bottom to top (5′ to 3′). G G+A T+C C 3′ A A G C A A C G T G C A G 5′ Longer fragments Shortest fragments G A Maxam-Gilbert Sequencing
- 6. Chain Termination (Sanger) Sequencing A modified DNA replication reaction. Growing chains are terminated by dideoxynucleotides.
- 7. Chain terminates at ddG Chain Termination (Sanger) Sequencing The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTPs.
- 8. Template area to be sequenced -3′ OH TCGACGGGC… 5′OP- Primer Template Chain Termination (Sanger) Sequencing A sequencing reaction mix includes labeled primer and template. Dideoxynucleotides are added separately to each of the four tubes.
- 9. ddATP + ddA four dNTPs dAdGdCdTdGdCdCdCdG ddCTP + dAdGddC four dNTPs dAdGdCdTdGddC dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC ddGTP + dAddG four dNTPs dAdGdCdTddG dAdGdCdTdGdCdCdCddG ddTTP + dAdGdCddT four dNTPs dAdGdCdTdGdCdCdCdG A C G T Chain Termination (Sanger) Sequencing
- 10. Chain Termination (Sanger) Sequencing With addition of enzyme (DNA polymerase), the primer is extended until a ddNTP is encountered. The chain will end with the incorporation of the ddNTP. With the proper dNTP:ddNTP ratio, the chain will terminate throughout the length of the template. All terminated chains will end in the ddNTP added to that reaction.
- 11. Chain Termination (Sanger) Sequencing The collection of fragments is a sequencing ladder. The resulting terminated chains are resolved by electrophoresis. Fragments from each of the four tubes are placed in four separate gel lanes.
- 12. Sequencing gels are read from bottom to top (5′ to 3′). G A T C 3′ G G T A A A T C A T G 5′ Longer fragments Shorter fragments ddG ddG Chain Termination (Sanger) Sequencing
- 13. Cycle Sequencing Cycle sequencing is chain termination sequencing performed in a thermal cycler. Cycle sequencing requires a heat-stable DNA polymerase.
- 14. Fluorescent Dyes Fluorescent dyes are multicyclic molecules that absorb and emit fluorescent light at specific wavelengths. Examples are fluorescein and rhodamine derivatives. For sequencing applications, these molecules can be covalently attached to nucleotides.
- 15. Fluorescent Dyes In dye primer sequencing, the primer contains fluorescent dye–conjugated nucleotides, labeling the sequencing ladder at the 5′ ends of the chains. In dye terminator sequencing, the fluorescent dye molecules are covalently attached to the dideoxynucleotides, labeling the sequencing ladder at the 3′ ends of the chains.
- 16. AC GT The fragments are distinguished by size and “color.” Dye Terminator Sequencing A distinct dye or “color” is used for each of the four ddNTP. Since the terminating nucleotides can be distinguished by color, all four reactions can be performed in a single tube. A T G T
- 17. Capillary G T C T G A Slab gel GA TCG A T C Dye Terminator Sequencing The DNA ladder is resolved in one gel lane or in a capillary.
- 18. Dye Terminator Sequencing The DNA ladder is read on an electropherogram. CapillarySlab gel 5′ AGTCTG Electropherogram
- 19. 5′ AGTCTG 5′ AG(T/A)CTG 5′ AGACTG T/T T/A A/A Automated Sequencing Dye primer or dye terminator sequencing on capillary instruments. Sequence analysis software provides analyzed sequence in text and electropherogram form. Peak patterns reflect mutations or sequence changes.
- 20. Alternative Sequencing Methods: Pyrosequencing Pyrosequencing is based on the generation of light signal through release of pyrophosphate (PPi) on nucleotide addition. DNAn + dNTP DNAn+1 + PPI PPi is used to generate ATP from adenosine phosphosulfate (APS). APS + PPI ATP ATP and luciferase generate light by conversion of luciferin to oxyluciferin.
- 21. Alternative Sequencing Methods: Pyrosequencing Each nucleotide is added in turn. Only one of four will generate a light signal. The remaining nucleotides are removed enzymatically. The light signal is recorded on a pyrogram.
- 22. Alternative Sequencing Methods: Bisulfite Sequencing Bisulfite sequencing is used to detect methylation in DNA. Bisulfite deaminates cytosine, making uracil. Methylated cytosine is not changed by bisulfite treatment. The bisulfite-treated template is then sequenced.
- 23. Alternative Sequencing Methods: Bisulfite Sequencing The sequence of treated and untreated templates is compared. GTC Methylated sequence: GTC Me GGC Me GATCTATC Me GTGCA… Treated sequence: Me GGC Me GATUTATC Me GTGUA… DNA Sequence: (Untreated) reference: ...GTCGGCGATCTATCGTGCA… Treated sequence: ...GTCGGCGATUTATCGTGUA… This sequence indicates that these Cs are methylated.
- 24. Summary Genetic information is stored in the order or sequence of nucleotides in DNA. Chain termination sequencing is the standard method for the determination of nucleotide sequence. Dideoxy-chain termination sequencing has been facilitated by the development of cycle sequencing and the use of fluorescent dye detection. Alternative methods are used for special applications, such as pyrosequencing (for resequencing and polymorphism detection) or bisulfite sequencing (to analyze methylated DNA).