I. The document provides an overview of DNA sequencing methods, including a brief history and discussion of the Sanger dideoxy method, sequencing large pieces of DNA using shotgun sequencing, and progress towards achieving the "$1,000 genome".
II. It describes the Sanger dideoxy chain termination method and how primers, templates, and reagents are used. Newer methods like pyrosequencing that can sequence many DNA molecules in parallel are also covered.
III. The document discusses how sequenced DNA can be assembled and annotated, and tools for identifying genes and predicting functions like BLAST searches of databases. Reducing the cost of genome sequencing enables more widespread applications.
The document discusses different methods of DNA and protein sequencing. It begins by mentioning Sanger sequencing and chain termination sequencing. It then provides details on the principles and processes of Sanger sequencing, including how it utilizes chain terminating ddNTPs. The document discusses how Sanger sequencing was automated and led to first generation sequencing. Finally, it introduces high throughput next generation sequencing methods that allow sequencing of large genomes in a fast and cheap manner through massively parallel reactions.
Sequencing genes and genomes in biology. 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
whole genome analysis
history
needs
steps involved
human genome data
NGS
pyrosequencing
illumina
SOLiD
Ion torrent
PacBio
applications
problems
benefits
DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCINGPuneet Kulyana
This presentation will give you a brief idea about the various DNA sequencing methods and various strategies used for genome sequencing and much more vital information related to gene expression and analysis
The document discusses various DNA and RNA sequencing methods and technologies. It begins with an overview of sequencing-based markers like DNA sequencing, RNA sequencing, SNPs, epigenetic markers, and omics. The document then provides more details on the history and development of sequencing technologies, including early methods like Sanger and Maxam-Gilbert sequencing. It discusses next generation sequencing platforms like MPSS, 454 pyrosequencing, Illumina, Ion Torrent, ABI-SOLiD, and their approaches. The document concludes with an overview of third generation long-read sequencing technologies like SMRT and nanopore sequencing.
sequencing presentation. providing deep and insightful points about Sanger sequencing, Maxam-gilbert sequencing, Illumina sequencing, and single molecule sequencing.
Gene sequencing is the technique that determines the order of nucleotide bases in DNA. It allows researchers to read genetic information and understand genes. The first genome sequenced was a bacteriophage in 1977. Major advances include sequencing the human genome in 2001. Current technologies like Illumina and Ion Torrent can generate billions of reads faster and cheaper than Sanger sequencing. Gene sequencing has applications in medicine, forensics, agriculture, and more. It is an important tool for understanding genomes and their relationship to traits and disease.
Gene sequencing is the technique that determines the order of nucleotide bases in DNA. It allows researchers to read genetic information and understand genes. The first genome sequenced was a bacteriophage in 1977. Techniques have advanced from Sanger sequencing to second-generation sequencing using platforms like Illumina and third-generation single-molecule techniques. Gene sequencing has various applications in medicine, forensics, agriculture, cancer research and more. It is an important tool for understanding genomes and their relationship to traits and disease.
The document discusses different methods of DNA and protein sequencing. It begins by mentioning Sanger sequencing and chain termination sequencing. It then provides details on the principles and processes of Sanger sequencing, including how it utilizes chain terminating ddNTPs. The document discusses how Sanger sequencing was automated and led to first generation sequencing. Finally, it introduces high throughput next generation sequencing methods that allow sequencing of large genomes in a fast and cheap manner through massively parallel reactions.
Sequencing genes and genomes in biology. 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
whole genome analysis
history
needs
steps involved
human genome data
NGS
pyrosequencing
illumina
SOLiD
Ion torrent
PacBio
applications
problems
benefits
DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCINGPuneet Kulyana
This presentation will give you a brief idea about the various DNA sequencing methods and various strategies used for genome sequencing and much more vital information related to gene expression and analysis
The document discusses various DNA and RNA sequencing methods and technologies. It begins with an overview of sequencing-based markers like DNA sequencing, RNA sequencing, SNPs, epigenetic markers, and omics. The document then provides more details on the history and development of sequencing technologies, including early methods like Sanger and Maxam-Gilbert sequencing. It discusses next generation sequencing platforms like MPSS, 454 pyrosequencing, Illumina, Ion Torrent, ABI-SOLiD, and their approaches. The document concludes with an overview of third generation long-read sequencing technologies like SMRT and nanopore sequencing.
sequencing presentation. providing deep and insightful points about Sanger sequencing, Maxam-gilbert sequencing, Illumina sequencing, and single molecule sequencing.
Gene sequencing is the technique that determines the order of nucleotide bases in DNA. It allows researchers to read genetic information and understand genes. The first genome sequenced was a bacteriophage in 1977. Major advances include sequencing the human genome in 2001. Current technologies like Illumina and Ion Torrent can generate billions of reads faster and cheaper than Sanger sequencing. Gene sequencing has applications in medicine, forensics, agriculture, and more. It is an important tool for understanding genomes and their relationship to traits and disease.
Gene sequencing is the technique that determines the order of nucleotide bases in DNA. It allows researchers to read genetic information and understand genes. The first genome sequenced was a bacteriophage in 1977. Techniques have advanced from Sanger sequencing to second-generation sequencing using platforms like Illumina and third-generation single-molecule techniques. Gene sequencing has various applications in medicine, forensics, agriculture, cancer research and more. It is an important tool for understanding genomes and their relationship to traits and disease.
This document summarizes trends in DNA sequencing methods and applications. It discusses the purpose and historical methods of DNA sequencing, including the Maxam-Gilbert and Sanger methods. Next generation sequencing methods like Roche 454, Illumina, SOLiD, Ion Torrent, and PacBio are described. Applications of sequencing include analyzing gene structure, detecting mutations, microbial identification, and whole genome sequencing. The document provides details on sequencing techniques, platforms, yields, and error rates.
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.
The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA. First described in 1985, it was made possible by the discovery of Taq polymerase. The primary reagents used in PCR are DNA nucleotides, template DNA, primers, and DNA polymerase.
NEED OF GENETIC SEQUENCING
- Understanding the particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment.
- An alteration in a DNA sequence can lead to an altered or non functional protein and hence to a harmful effect in a plant or animal.
- Simple point mutations can cause altered protein shape and function.
DNA sequencing is a laboratory technique used to determine the exact sequence of bases (A, C, G, and T) in a DNA molecule. The DNA base sequence carries the information a cell needs to assemble protein and RNA molecules. DNA sequence information is important to scientists investigating the functions of genes.
In medicine, DNA sequencing is used for a range of purposes, including diagnosis and treatment of diseases. In general, sequencing allows health care practitioners to determine if a gene or the region that regulates a gene contains changes, called variants or mutations, that are linked to a disorder.
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. Establishing the sequence of DNA is key to understanding the function of genes and other parts of the genome. There are now several different methods available for DNA sequencing, each with its own characteristics, and the development of additional methods represents an active area of genomics research.
This document discusses gene mapping and sequencing. It defines key terms like gene, genome, and gene mapping. It describes different types of gene mapping including linkage mapping and physical mapping. It also discusses various genetic markers used in mapping like RFLPs, SNPs, AFLPs, RAPDs, SSLPs, microsatellites, and minisatellites. Details are provided on techniques like RFLP analysis, RAPD, AFLP, and their advantages and limitations. The document also covers Sanger sequencing, the chain termination method, and the chemical cleavage method developed by Maxam and Gilbert.
theoretical perspectives on marriage and familyRameenIqbal1
This document provides an overview of DNA sequencing, including its definition, types, uses, and impact. It discusses the key types of sequencing, including the Sanger method and high-throughput sequencing techniques like Illumina and Roche 454. The document also outlines some common uses of sequencing in areas like diagnostics, genomics, and forensics. Overall, the document highlights how advances in sequencing technology have enabled sequencing of entire genomes and provided important insights into human biology, disease, and more.
This document discusses gene mapping and sequencing. It begins by defining genomics and genetic markers such as RFLP, SSLP, and SNP that are used to track inheritance. Gene mapping involves determining the locus and distance between genes on chromosomes, which is important for diagnosing genetic diseases. There are two main types of gene mapping: linkage mapping which measures recombination frequency to determine if genes are linked, and physical mapping which precisely locates DNA sequences on chromosomes using techniques like fluorescence in situ hybridization. The document also discusses methods for gene sequencing, including Sanger sequencing and Maxam-Gilbert sequencing, as well as newer techniques like shotgun sequencing and Illumina sequencing.
Next-generation sequencing (NGS) has various applications in livestock genetics and breeding including:
1. Whole genome sequencing to identify genetic variations within and between species and quantify introgression.
2. RNA sequencing to detect differentially expressed genes between control and infected/challenged animals and identify genes related to disease resistance.
3. Genome-wide association studies using SNPs identified through NGS to map quantitative trait loci and guide marker-assisted selection for improved traits.
This document discusses molecular genetic methods such as polymerase chain reaction (PCR), DNA sequencing, DNA fingerprinting, and single nucleotide polymorphisms. It provides details on how each method works, including how PCR amplifies DNA, the process of manual and automated DNA sequencing, using variable number tandem repeats as markers for DNA fingerprinting, and applications of these molecular genetic techniques.
Genome sequencing and comparative genomics are important tools in plant breeding. Genome sequencing determines the order of DNA nucleotides in individual genes, chromosomes, or entire genomes. Comparative genomics analyzes and compares genetic material between species to study evolution, gene function, and disease. Next generation sequencing techniques like Illumina sequencing have made genome sequencing faster, cheaper, and able to sequence thousands of sequences at once. Comparative genomics is used to understand differences between species by comparing gene location, structure, sequence similarity and other characteristics. This aids in understanding evolution and identifying genes responsible for unique traits.
The document provides an overview of gene sequencing and DNA sequencing techniques. It discusses how DNA is composed of nucleotides containing phosphate, sugar and nitrogen bases. The order of these bases determines the genetic instructions. Each sequence of bases that codes for a protein is known as a gene. It then describes several methods for DNA sequencing, including the Maxam-Gilbert and Sanger methods. The document outlines key applications of gene sequencing such as in medicine, forensics and agriculture. Recent advances in sequencing technology including Illumina, Roche 454 and solid sequencing are also summarized.
Detailed explanation about gene sequencing methods
Sequencing the gene is an important step toward understanding the gene.
A gene sequence contains some clues about where genes are.
Gene sequencing give us understanding how the genome as a whole works-how genes work together to direct the growth, development and maintenance of an entire organism.
It help scientists to study the part of genome outside the genes-regulatory regions
Genomics is the study of genomes through DNA sequencing and analysis. There are several types of genomics including structural, functional, comparative, epigenomics, and metagenomics. DNA sequencing methods have advanced from Sanger sequencing to next-generation sequencing using high-throughput technologies. Genome sequencing has provided insights into disease, evolution, and applications in medicine, agriculture, and environmental science.
Next generation sequencing (NGS) refers to modern DNA sequencing technologies that allow for high-speed, low-cost sequencing of entire genomes. NGS works by massively parallel sequencing of millions of DNA fragments. The Illumina sequencing by synthesis method is the most commonly used NGS approach. It involves library preparation, cluster generation on a flow cell, sequencing via reversible dye-terminator chemistry, and computational analysis of sequenced reads. Key advantages of NGS include its scalability, unlimited dynamic range, tunable coverage levels, and ability to multiplex many samples simultaneously in a single run.
This document discusses various microbiological techniques for isolating and culturing microorganisms, including serial dilution, pour and spread plating, using a micromanipulator to isolate single cells under a microscope, and the roll tube method for culturing obligate anaerobes by pumping inert gas through molten agar in a test tube to remove oxygen before solidification.
Isolation and screening_Class I - Copy.pptxaaaa bbb
Industrial microbiology began based on the natural process of fermentation, which ancient humans practiced but did not fully understand. The discovery of microorganisms under the microscope led to a clearer understanding of fermentation and the development of industrial microbiology. The history of industrial microbiology can be divided into five phases from alcohol fermentation pre-1900 to the current biotechnology period beginning in 1979. Microbial strains for industrial use can be isolated from natural sources like soil, water, food, and animals or obtained from commercial and educational culture collections. Desired strains are screened based on their ability to grow quickly and produce non-toxic end products while being genetically stable and resistant to microbial threats.
This document summarizes trends in DNA sequencing methods and applications. It discusses the purpose and historical methods of DNA sequencing, including the Maxam-Gilbert and Sanger methods. Next generation sequencing methods like Roche 454, Illumina, SOLiD, Ion Torrent, and PacBio are described. Applications of sequencing include analyzing gene structure, detecting mutations, microbial identification, and whole genome sequencing. The document provides details on sequencing techniques, platforms, yields, and error rates.
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.
The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA. First described in 1985, it was made possible by the discovery of Taq polymerase. The primary reagents used in PCR are DNA nucleotides, template DNA, primers, and DNA polymerase.
NEED OF GENETIC SEQUENCING
- Understanding the particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment.
- An alteration in a DNA sequence can lead to an altered or non functional protein and hence to a harmful effect in a plant or animal.
- Simple point mutations can cause altered protein shape and function.
DNA sequencing is a laboratory technique used to determine the exact sequence of bases (A, C, G, and T) in a DNA molecule. The DNA base sequence carries the information a cell needs to assemble protein and RNA molecules. DNA sequence information is important to scientists investigating the functions of genes.
In medicine, DNA sequencing is used for a range of purposes, including diagnosis and treatment of diseases. In general, sequencing allows health care practitioners to determine if a gene or the region that regulates a gene contains changes, called variants or mutations, that are linked to a disorder.
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. Establishing the sequence of DNA is key to understanding the function of genes and other parts of the genome. There are now several different methods available for DNA sequencing, each with its own characteristics, and the development of additional methods represents an active area of genomics research.
This document discusses gene mapping and sequencing. It defines key terms like gene, genome, and gene mapping. It describes different types of gene mapping including linkage mapping and physical mapping. It also discusses various genetic markers used in mapping like RFLPs, SNPs, AFLPs, RAPDs, SSLPs, microsatellites, and minisatellites. Details are provided on techniques like RFLP analysis, RAPD, AFLP, and their advantages and limitations. The document also covers Sanger sequencing, the chain termination method, and the chemical cleavage method developed by Maxam and Gilbert.
theoretical perspectives on marriage and familyRameenIqbal1
This document provides an overview of DNA sequencing, including its definition, types, uses, and impact. It discusses the key types of sequencing, including the Sanger method and high-throughput sequencing techniques like Illumina and Roche 454. The document also outlines some common uses of sequencing in areas like diagnostics, genomics, and forensics. Overall, the document highlights how advances in sequencing technology have enabled sequencing of entire genomes and provided important insights into human biology, disease, and more.
This document discusses gene mapping and sequencing. It begins by defining genomics and genetic markers such as RFLP, SSLP, and SNP that are used to track inheritance. Gene mapping involves determining the locus and distance between genes on chromosomes, which is important for diagnosing genetic diseases. There are two main types of gene mapping: linkage mapping which measures recombination frequency to determine if genes are linked, and physical mapping which precisely locates DNA sequences on chromosomes using techniques like fluorescence in situ hybridization. The document also discusses methods for gene sequencing, including Sanger sequencing and Maxam-Gilbert sequencing, as well as newer techniques like shotgun sequencing and Illumina sequencing.
Next-generation sequencing (NGS) has various applications in livestock genetics and breeding including:
1. Whole genome sequencing to identify genetic variations within and between species and quantify introgression.
2. RNA sequencing to detect differentially expressed genes between control and infected/challenged animals and identify genes related to disease resistance.
3. Genome-wide association studies using SNPs identified through NGS to map quantitative trait loci and guide marker-assisted selection for improved traits.
This document discusses molecular genetic methods such as polymerase chain reaction (PCR), DNA sequencing, DNA fingerprinting, and single nucleotide polymorphisms. It provides details on how each method works, including how PCR amplifies DNA, the process of manual and automated DNA sequencing, using variable number tandem repeats as markers for DNA fingerprinting, and applications of these molecular genetic techniques.
Genome sequencing and comparative genomics are important tools in plant breeding. Genome sequencing determines the order of DNA nucleotides in individual genes, chromosomes, or entire genomes. Comparative genomics analyzes and compares genetic material between species to study evolution, gene function, and disease. Next generation sequencing techniques like Illumina sequencing have made genome sequencing faster, cheaper, and able to sequence thousands of sequences at once. Comparative genomics is used to understand differences between species by comparing gene location, structure, sequence similarity and other characteristics. This aids in understanding evolution and identifying genes responsible for unique traits.
The document provides an overview of gene sequencing and DNA sequencing techniques. It discusses how DNA is composed of nucleotides containing phosphate, sugar and nitrogen bases. The order of these bases determines the genetic instructions. Each sequence of bases that codes for a protein is known as a gene. It then describes several methods for DNA sequencing, including the Maxam-Gilbert and Sanger methods. The document outlines key applications of gene sequencing such as in medicine, forensics and agriculture. Recent advances in sequencing technology including Illumina, Roche 454 and solid sequencing are also summarized.
Detailed explanation about gene sequencing methods
Sequencing the gene is an important step toward understanding the gene.
A gene sequence contains some clues about where genes are.
Gene sequencing give us understanding how the genome as a whole works-how genes work together to direct the growth, development and maintenance of an entire organism.
It help scientists to study the part of genome outside the genes-regulatory regions
Genomics is the study of genomes through DNA sequencing and analysis. There are several types of genomics including structural, functional, comparative, epigenomics, and metagenomics. DNA sequencing methods have advanced from Sanger sequencing to next-generation sequencing using high-throughput technologies. Genome sequencing has provided insights into disease, evolution, and applications in medicine, agriculture, and environmental science.
Next generation sequencing (NGS) refers to modern DNA sequencing technologies that allow for high-speed, low-cost sequencing of entire genomes. NGS works by massively parallel sequencing of millions of DNA fragments. The Illumina sequencing by synthesis method is the most commonly used NGS approach. It involves library preparation, cluster generation on a flow cell, sequencing via reversible dye-terminator chemistry, and computational analysis of sequenced reads. Key advantages of NGS include its scalability, unlimited dynamic range, tunable coverage levels, and ability to multiplex many samples simultaneously in a single run.
This document discusses various microbiological techniques for isolating and culturing microorganisms, including serial dilution, pour and spread plating, using a micromanipulator to isolate single cells under a microscope, and the roll tube method for culturing obligate anaerobes by pumping inert gas through molten agar in a test tube to remove oxygen before solidification.
Isolation and screening_Class I - Copy.pptxaaaa bbb
Industrial microbiology began based on the natural process of fermentation, which ancient humans practiced but did not fully understand. The discovery of microorganisms under the microscope led to a clearer understanding of fermentation and the development of industrial microbiology. The history of industrial microbiology can be divided into five phases from alcohol fermentation pre-1900 to the current biotechnology period beginning in 1979. Microbial strains for industrial use can be isolated from natural sources like soil, water, food, and animals or obtained from commercial and educational culture collections. Desired strains are screened based on their ability to grow quickly and produce non-toxic end products while being genetically stable and resistant to microbial threats.
The document summarizes biodiversity hotspots in India. It discusses four main hotspots: the Himalayas, Indo-Burma Region, Western Ghats, and Sundaland. Each hotspot contains numerous endemic plant and animal species but has lost significant habitat. The hotspots support a high percentage of the country's and world's biodiversity despite comprising only a small area. Threats like habitat loss and poaching have endangered many species.
This document discusses the potential for co-producing multiple valuable compounds from marine microalgae. Some key compounds produced include lipids, pigments, carbohydrates, and proteins. While these compounds exhibit beneficial bioactivities, their low concentrations in microalgae make production costs high. The document proposes methods for integrated extraction of compounds through similar physicochemical properties or stepwise extraction. It also discusses manipulating culture conditions like light intensity to boost production of different compounds. Overall, the document advocates developing strategies to simultaneously extract multiple high-value compounds from microalgae to improve production efficiency and lower costs.
Marine algae are promising sources of novel bioactive compounds with various biological activities useful for cancer therapy. Metabolites from algae like terpenoids, alkaloids, and polyphenols have shown antitumor, antioxidant, anti-inflammatory and other therapeutic effects. Specifically, polysaccharides, sulfated polysaccharides, fucoxanthin and fucoidan from algae downregulate anti-apoptotic proteins and induce apoptosis in cancer cells through caspase activation and intrinsic/extrinsic pathways without harming normal cells. Algal extracts contain various phytochemicals that can act as chemotherapeutic agents by selectively targeting cancer cells and driving cancers into remission.
The document discusses genomic research and sequencing technologies. It provides a history of genomic research from early sequencing methods like Sanger sequencing to modern massively parallel sequencing. It describes several next-generation sequencing platforms, their read lengths, accuracy, applications, and differences. It emphasizes that data analysis is a major challenge and recommends consulting sequencing facilities and having bioinformaticians available for analysis.
Titus Brown will be leading a workshop on shotgun metagenomics. The plan includes introductions to shotgun sequencing and metagenome assembly, binning genomes from metagenomes, functional analysis, and capstone projects. Shotgun metagenomics involves randomly sequencing all DNA in an environmental sample and computationally reconstructing genomes and identifying genes and functions without the use of primers. This provides advantages over amplicon sequencing but is more computationally challenging. Assembly of reads into contigs is discussed as a key step for binning genomes and functional analysis from metagenomes.
Adhd Medication Shortage Uk - trinexpharmacy.comreignlana06
The UK is currently facing a Adhd Medication Shortage Uk, which has left many patients and their families grappling with uncertainty and frustration. ADHD, or Attention Deficit Hyperactivity Disorder, is a chronic condition that requires consistent medication to manage effectively. This shortage has highlighted the critical role these medications play in the daily lives of those affected by ADHD. Contact : +1 (747) 209 – 3649 E-mail : sales@trinexpharmacy.com
Here is the updated list of Top Best Ayurvedic medicine for Gas and Indigestion and those are Gas-O-Go Syp for Dyspepsia | Lavizyme Syrup for Acidity | Yumzyme Hepatoprotective Capsules etc
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
Promoting Wellbeing - Applied Social Psychology - Psychology SuperNotesPsychoTech Services
A proprietary approach developed by bringing together the best of learning theories from Psychology, design principles from the world of visualization, and pedagogical methods from over a decade of training experience, that enables you to: Learn better, faster!
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptxHolistified Wellness
We’re talking about Vedic Meditation, a form of meditation that has been around for at least 5,000 years. Back then, the people who lived in the Indus Valley, now known as India and Pakistan, practised meditation as a fundamental part of daily life. This knowledge that has given us yoga and Ayurveda, was known as Veda, hence the name Vedic. And though there are some written records, the practice has been passed down verbally from generation to generation.
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
Does Over-Masturbation Contribute to Chronic Prostatitis.pptxwalterHu5
In some case, your chronic prostatitis may be related to over-masturbation. Generally, natural medicine Diuretic and Anti-inflammatory Pill can help mee get a cure.
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
Local Advanced Lung Cancer: Artificial Intelligence, Synergetics, Complex Sys...Oleg Kshivets
Overall life span (LS) was 1671.7±1721.6 days and cumulative 5YS reached 62.4%, 10 years – 50.4%, 20 years – 44.6%. 94 LCP lived more than 5 years without cancer (LS=2958.6±1723.6 days), 22 – more than 10 years (LS=5571±1841.8 days). 67 LCP died because of LC (LS=471.9±344 days). AT significantly improved 5YS (68% vs. 53.7%) (P=0.028 by log-rank test). Cox modeling displayed that 5YS of LCP significantly depended on: N0-N12, T3-4, blood cell circuit, cell ratio factors (ratio between cancer cells-CC and blood cells subpopulations), LC cell dynamics, recalcification time, heparin tolerance, prothrombin index, protein, AT, procedure type (P=0.000-0.031). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and N0-12 (rank=1), thrombocytes/CC (rank=2), segmented neutrophils/CC (3), eosinophils/CC (4), erythrocytes/CC (5), healthy cells/CC (6), lymphocytes/CC (7), stick neutrophils/CC (8), leucocytes/CC (9), monocytes/CC (10). Correct prediction of 5YS was 100% by neural networks computing (error=0.000; area under ROC curve=1.0).
Ear and its clinical correlations By Dr. Rabia Inam Gandapore.pptx
Useful.ppt
1. DNA sequencing: methods
I. Brief history of sequencing
II. Sanger dideoxy method for sequencing
III. Sequencing large pieces of DNA
VI. The “$1,000 dollar genome”
On WebCT
-- “The $1000 genome”
-- review of new sequencing techniques by George Church
2. Why sequence DNA?
• All genes available for an organism to use -- a
very important tool for biologists
• Not just sequence of genes, but also positioning
of genes and sequences of regulatory regions
• New recombinant DNA constructs must be
sequenced to verify construction or positions of
mutations
• Etc.
5. Methods of sequencing
A. Sanger dideoxy (primer extension/chain-termination)
method: most popular protocol for sequencing, very
adaptable, scalable to large sequencing projects
B. Maxam-Gilbert chemical cleavage method: DNA is
labelled and then chemically cleaved in a sequence-
dependent manner. This method is not easily scaled and
is rather tedious
C. Pyrosequencing: measuring chain extension by
pyrophosphate monitoring
6. for dideoxy sequencing you need:
1) Single stranded DNA template
2) A primer for DNA synthesis
3) DNA polymerase
4) Deoxynucleoside triphosphates and
dideoxynucleotide triphosphates
7. Primers for DNA sequencing
• Oligonucleotide primers can be synthesized by
phosphoramidite chemistry--usually designed
manually and then purchased
• Sequence of the oligo must be complimentary to
DNA flanking sequenced region
• Oligos are usually 15-30 nucleotides in length
8. DNA templates for sequencing:
• Single stranded DNA isolated from
recombinant M13 bacteriophage containing
DNA of interest
• Double-stranded DNA that has been
denatured
• Non-denatured double stranded DNA (cycle
sequencing)
9. One way for obtaining single-stranded DNA from a double
stranded source--magnets
10. Reagents for sequencing:
DNA polymerases
• Should be highly processive, and
incorporate ddNTPs efficiently
• Should lack exonuclease activity
• Thermostability required for “cycle
sequencing”
11. Single stranded DNA 5’
3’
5’ 3’
Sanger dideoxy sequencing--basic method
a) Anneal the primer
12. Sanger dideoxy sequencing: basic method
b) Extend the
primer with DNA
polymerase in the
presence of all four
dNTPs, with a
limited amount of a
dideoxy NTP
(ddNTP)
5’
3’
Direction of
DNA
polymerase
travel
13. DNA polymerase incorporates ddNTP in a template-
dependent manner, but it works best if the DNA pol
lacks 3’ to 5’ exonuclease (proofreading) activity
14. Sanger dideoxy sequencing: basic method
5’
3’
5’ 3’
T T T
T
ddA
ddA
ddA
ddA
ddATP in the
reaction: anywhere
there’s a T in the
template strand,
occasionally a ddA
will be added to the
growing strand
15. How to visualize DNA fragments?
• Radioactivity
– Radiolabeled primers (kinase with 32P)
– Radiolabelled dNTPs (gamma 35S or 32P)
• Fluorescence
– ddNTPs chemically synthesized to contain fluors
– Each ddNTP fluoresces at a different wavelength
allowing identification
16. Analysis of sequencing products:
Polyacrylamide gel electrophoresis--good
resolution of fragments differing by a single
dNTP
– Slab gels: as previously described
– Capillary gels: require only a tiny amount of
sample to be loaded, run much faster than
slab gels, best for high throughput
sequencing
17. DNA sequencing gels: old school
Analyze sequencing
products by gel
electrophoresis,
autoradiography
Different ddNTP used in
separate reactions
Radioactively labelled primer or
dNTP in sequencing reaction
18.
19. cycle sequencing: denaturation
occurs during temperature cycles
94°C:DNA denatures
45°C: primer anneals
60-72°C: thermostable DNA
pol extends primer
Repeat 25-35 times
Advantages: don’t need a lot
of template DNA
Disadvantages: DNA pol
may incorporate ddNTPs
poorly
20. Animation of cycle sequencing: see
http://www.dnai.org/
Click on:
“manipulation”
“techniques”
“sorting and sequencing”
22. Current trends in sequencing:
It is rare for labs to do their own sequencing:
--costly, perishable reagents
--time consuming
--success rate varies
Instead most labs send out for sequencing:
--You prepare the DNA (usually plasmid, M13, or PCR product),
supply the primer, company or university sequencing center
does the rest
--The sequence is recorded by an automated sequencer as an
“electropherogram”
23. ~160 kbp
~1 kbp
Assemble sequences by
matching overlaps
BAC sequence
BAC overlaps give genome sequence
BREAK UP THE GENOME,
PUT IT BACK TOGETHER
24. Sequencing large pieces of DNA:
the “shotgun” method
• Break DNA into small pieces (typically sizes of around
1000 base pairs is preferable)
• Clone pieces of DNA into M13
• Sequence enough M13 clones to ensure complete
coverage (eg. sequencing a 3 million base pair genome
would require 5x to 10x 3 million base pairs to have a
reliable representation of the genome)
• Assemble genome through overlap analysis using
computer algorithms, also “polish” sequences using
mapping information from individual clones,
characterized genes, and genetic markers
• This process is assisted by robotics
25. Sequencing done by TIGR (Maryland) and The
Sanger Institute (Cambridge, UK)
“Here we report an analysis of the genome sequence of P.
falciparum clone 3D7, including descriptions of chromosome
structure, gene content, functional classification of proteins,
metabolism and transport, and other features of parasite
biology.”
26. Sequencing strategy
A whole chromosome shotgun sequencing
strategy was used to determine the genome
sequence of P. falciparum clone 3D7. This approach
was taken because a whole genome shotgun
strategy was not feasible or cost-effective with the
technology that was available at the beginning of the
project. Also, high-quality large insert libraries of (A -
T)-rich P. falciparum DNA have never been
constructed in Escherichia coli, which ruled out a
clone-by-clone sequencing strategy. The
chromosomes were separated on pulsed field gels,
and chromosomal DNA was extracted…
27. The shotgun sequences were assembled into
contiguous DNA sequences (contigs), in some cases with
low coverage shotgun sequences of yeast artificial
chromosome (YAC) clones to assist in the ordering of
contigs for closure. Sequence tagged sites (STSs)10,
microsatellite markers11,12 and HAPPY mapping7 were
also used to place and orient contigs during the gap
closure process. The high (A /T) content of the genome
made gap closure extremely difficult7–9.
Chromosomes 1–5, 9 and 12 were closed,
whereas chromosomes 6–8, 10, 11, 13 and 14 contained
3–37 gaps (most less than 2.5 kb) per chromosome at the
beginning of genome annotation. Efforts to close the
remaining gaps are continuing.
28. Methods: Sequencing, gap closure and annotation
The techniques used at each of the three participating
centres for sequencing, closure and annotation are described in
the accompanying Letters7–9. To ensure that each centres’
annotation procedures produced roughly equivalent results, the
Wellcome Trust Sanger Institute (‘Sanger’) and the Institute for
Genomic Research (‘TIGR’) annotated the same100-kb
segment of chromosome 14. The number of genes predicted in
this sequence by the two centres was 22 and 23; the
discrepancy being due to the merging of two single genes by
one centre. Of the 74 exons predicted by the two centres, 50
(68%) were identical, 9 (2%) overlapped, 6 (8%) overlapped
and shared one boundary, and the remainder were predicted by
one centre but not the other. Thus 88% of the exons predicted
by the two centres in the 100-kb fragment were identical or
overlapped.
29. The $1000 dollar genome
Venter Foundation (2003): The first group to produce a
technology capable of a $1000 human genome will win
$500,000 …
X - Prize Foundation: no, $5 - 20 million …
National Institutes of Health (2004): $70 million grant program
to reach the $1000 genome
30. Previous sequencing techniques: one DNA molecule at a time
Needed: many DNA molecules at a time -- arrays
One of these: “pyrosequencing”
Cut a genome to DNA fragments 300 - 500 bases long
Immobilize single strands on a very small plastic bead (one
piece of DNA per bead)
Amplify the DNA on each bead to cover each bead to boost the
signal
Separate each bead on a plate with up to 1.6 million wells
31. Sequence by DNA polymerase -dependent chain extension,
one base at a time in the presence of a reporter (luciferase)
Luciferase is an enzyme that will emit a photon of light in
response to the pyrophosphate (PPi) released upon nucleotide
addition by DNA polymerase
Flashes of light and their intensity are recorded
32. Extension with individual dNTPs gives a readout
A B
A B
The readout is recorded by
a detector that measures
position of light flashes and
intensity of light flashes
33. APS = Adenosine phosphosulfate From www.454.com
25 million bases in
about 4 hours
34. Height of peak indicates the number of
dNTPs added
This sequence: TTTGGGGTTGCAGTT
35. DNA sequencing: methods
I. Brief history of sequencing
II. Sanger dideoxy method for sequencing
III. Sequencing large pieces of DNA
VI. The “$1,000 dollar genome”
On WebCT
-- “The $1000 genome”
-- review of new sequencing techniques by George Church
36. Introduction to bioinformatics
1) Making biological sense of DNA
sequences
2) Online databases: a brief survey
3) Database in depth: NCBI
4) What is BLAST?
5) Using BLAST for sequence analysis
6) “Biology workbench”, etc.
www.ncbi.nlm.nih.gov
www.tigr.org
http://workbench.sdsc.edu
37. There’s plenty of DNA to make sense of
http://www.genomesonline.org/
(2006)
38. Making sense of genome sequences:
1) Genes
a) Protein-coding
• Where are the open reading frames?
• What are the ORFs most similar to? (What is
the function/structure/evolution history?)
b) RNA
2) Non-genes
a) Regulation: promoters and factor-binding sites
b) Transactions: replication, repair, and
segregation, DNA packaging (nucleosomes)
42. ORF map 1) Where are the potential starts (ATG)
and stops (TAA, TAG, TGA)?
2) Which reading frame is correct?
= ATG
= stop
codon
Reading frame #1 appears to encode a protein
43. Cautions in ORF identification
• Not all genes initiate with ATG, particularly in certain
microbes (archaea)
• What is the shortest possible length of a real ORF? 50
amino acids? 25 amino acids? Cut-off is somewhat
arbitrary.
• In eukaryotes, ORFs can be difficult to identify because
of introns
• Are there other sequences surrounding the ORF that
indicate it might be functional?
– promoter sequences for RNA polymerase binding
– Shine-Dalgarno sequences for ribosome binding?
44. What is the function of
the sequenced gene?
Classical methods:
-- mutate gene, characterize phenotype for clues to function
(genetics)
-- purify protein product, characterize in vitro (biochemistry)
Comparison to previously characterized genes:
-- genes sequences that have high sequence similarity
usually have similar functions
-- if your gene has been previously characterized
(using classical methods) by someone else, you want
to know right away! (avoid duplication of labor)
45. NCBI
NCBI home page --Go to www.ncbi.nlm.nih.gov for the following
pages
Pubmed: search tool for literature--search by author, subject, title
words, etc.
All databases: “a retrieval system for searching several linked
databases”
BLAST: Basic Local Alignment Sequence Tool
OMIM: Online Mendelian Inheritance in Man
Books: many online textbooks available
Tax Browser: A taxonomic organization of organisms and their
genomes
Structure: Clearinghouse for solved molecular structures
46. What does BLAST do?
1) Searches chosen sequence database
and identifies sequences with similarity
to test sequence
2) Ranks similar sequences by degree of
homology (E value)
3) Illustrates alignment between test
sequence and similar sequences
47. Alignment of sequences:
The principle: two homologous sequences derived from the
same ancestral sequence will have at least some identical
(similar) amino acid residues
Fraction of identical amino acids is called “percent identity”
Similar amino acids: some amino acids have similar
physical/chemical properties, and more likely to substitute for
each other--these give specific similarity scores in
alignments
Gaps in similar/homologous sequences are rare, and are
given penalty scores
48. Homology of proteins
Homology: similarity of biological structure, physiology,
development, and evolution, based on genetic inheritance
Homologous proteins: statistically similar sequence, therefore
similar functions (often, but not always…)
Alignment of TFB and TFIIB sequences
49. High sequence similarity correlates with functional similarity
40-20% identity: fold can be predicted by similarity but precise
function cannot be predicted (the 40% rule)
enzymes
Non-enzymes
50. Programs available for BLAST searches
Protein sequence (this is the best option)
blastp--compares an amino acid query sequence against a protein
sequence database
tblastn--compares a protein query sequence against a nucleotide
sequence database translated in all reading frames
DNA sequence
blastn--compares a nucleotide query sequence against a nucleotide
sequence database
blastx--compares a nucleotide query sequence translated in all reading
frames against a protein sequence database
tblastx--compares the six-frame translations of a nucleotide query
sequence against the six-frame translations of a nucleotide sequence
database.
51. BLAST considers all possible combinations of
matches
mismatches
gaps
in any given alignment
Gives the “best” (highest scoring) alignment of sequences
Three scores
1) percent identity
2) similarity score
3) E-value--probability that two sequences will have
the similarity they have by chance (lower number, higher
probability of evolutionary homology, higher probability of
similar function)
52. What is the E-value?
The E value represents the chance that the similarity is
random and therefore insignificant. Essentially, the E value
describes the random background noise that exists for
matches between sequences. For example, an E value of 1
assigned to a hit can be interpreted as meaning that in a
database of the current size one might expect to see 1
match with a similar score simply by chance.
You can change the Expect value threshold on most main
BLAST search pages. When the Expect value is increased
from the default value of 10, a larger list with more low-
scoring hits can be reported.
53. E values (continued)
From the BLAST tutorial:
Although hits with E values much higher than 0.1 are
unlikely to reflect true sequence relatives, it is useful
to examine hits with lower significance (E values
between 0.1 and 10) for short regions of similarity. In
the absence of longer similarities, these short
regions may allow the tentative assignment of
biochemical activities to the ORF in question. The
significance of any such regions must be assessed
on a case by case basis.
54. Relationship between E-value and function
Single domain proteins
Multi-domain proteins
E value greater than 10-10, similar structure but possibly
different functions
57. BLAST against (go to genomes page):
-- Microbial genomes
-- environmental sequences (genomes)
Results:
1) Distribution of hits: query sequence and positions in
sequence that gave alignments
2) Sequences producing significant alignments
1) Accession number (this takes you to the sequence that
yielded the hit: gene or contig)
2) Name of sequence (sometimes identifies the gene)
3) Similarity score
4) E-value
3) Alignments arranged by E value, with links to gene reports
58. 2) Large percentages of
coding proteins cannot be
assigned function based
on homology
1) Homology? the function is
only inferred (NOT known)
Two problems with BLAST
59. For a current list of databases and bioinformatics
tools see: Nucleic Acids Research annual
bioinformatics issue (comes out every January).
List of all the databases described, by category:
http://www.oxfordjournals.org/nar/database/cap/
Guide to NCBI: see Webct
60. Bioinformatics:
making sense of biological sequence
• New DNA sequences are analyzed for ORFs
(Open Reading Frames: protein)
• Any DNA or protein sequence can then be
compared to all other sequences in databases,
and similar sequences identified
• There is much more -- a great diversity of
programs and databases are available
61. Massively parallel measurements of gene
expression: microarrays
• Defining the “transcriptome”
• The northern blot revisited
• Detecting expression of many genes: arrays
• A typical array experiment
• What to do with all this data?
Brown and Botstein (1999) “Exploring the new world
of the genome with DNA microarrays” Nature
Genetics 21, p. 33-37.
63. The value of DNA microarrays for
studying gene expression
1) Study all transcripts at same time
2) Transcript abundance usually correlates with level of gene
expression--much gene control is at level of transcription
3) Changes in transcription patterns often occur as a response to
changing environment--this can be detected with a microarray
64. Detection of mRNA transcripts
• Northern Blot -- immobilize mRNA on membrane,
detect specific sequence by hybridization with one
labeled probe--requires a separate blotting for
each probe
• DNA microarray -- immobilize many probes
(thousands) in an ordered array, hybridize (base
pair) with labelled mRNA or cDNA
65. Generating an array of probes
• Identify open reading frames (orfs)
1) PCR each orf (several for each orf), attach
(spot) each PCR product to a solid support in a
specific order (pioneered by Pat Brown’s lab,
Stanford)
2) Chemically synthesize orf-specific
oligonucleotide probes directly on microchip
(Affymetrix)
66. http://derisilab.ucsf.edu/microarray/
(Derisi Lab at UCSF)
The chip defines
the genes you are
measuring
The hybridization
represents the
measurement
The RNA comes
from the cells and
conditions you are
interested in
67.
68. A print head for generating arrays
of probes
Print head travels from DNA probe
source (microtiter plate) to solid
support (treated glass slide)
Small amount of DNA probe is put
on a specific spot at a specific
location
Each spot (DNA probe sequence)
has a specific “address”
Print head
Printing needles
69.
70.
71. A yeast array experiment
vegetative sporulating
Isolate mRNA
Prepare fluorescently
labeled cDNA with two
different-colored fluors
hybridize read-out
72. Example microarray data
Green: mRNA
more abundant
in vegetative
cells
Red: mRNA more
abundant in
sporulating cells
Yellow: equivalent
mRNA abundance
in vegetative and
sporulating cells
73. What to do with all that data?
Overarching patterns may become apparent
1) Organize data by hierarchical clustering,
profiling to find patterns
2) Display data graphically to allow
assimilation/comprehension
75. MIAME:
The Minimum Information About a Microarray Experiment
(#6 helps correct for variations in the quantity of
starting RNA, and for variable labelling and
detection efficiencies)
77. Analysis of the proteome: “proteomics”
• Which proteins are present and when?
• What are the proteins doing?
– What interacts with what?
• Protein-DNA interactions (chromatin
immunoprecipitation)
• Protein-protein interactions
– Functions of proteins?
Phizicky et al. (2003) “Protein analysis on a proteomic
scale” Nature 422, p. 208-215
78. Which proteins are expressed?
Classical method
– Detect presence of a specific protein
• Using antibodies or specific assay
• Measure changes in protein levels with
changing environment, in different tissues
– Very labor intensive, expensive to scale up to
proteome
79. Massively parallel detection and
identification of proteins
• 2D gel electrophoresis
– Separate proteins in a given organism or tissue type by migration in gel
electrophoresis
– Identify protein (cut out of gel, sequence or mass-spec)
– Pattern of spots like a barcode for hi-throughput studies
• Mass spectrometry
– Separate individual proteins from cell by charge and mass, individual
proteins can be identified (but need genome sequence information for
this)
• Microarrays: isolate things that bind proteins
80. 2D gel electrophoresis
1) Separate proteins on the basis of isoelectric point
This technique is usually
done on a long, narrow gel
4 10
81. 2D gel
electrophoresis
Lay gel containing
isoelectrically focused
protein on SDS page
gel, separate on the
basis of size
E.coli protein profile
From swissprot database,
www.expasy.ch
82. Mass spectrometry for identifying proteins in a
mixture
From J.R. Yates 1998 “Mass spectrometry and the age
of the proteome” J Mass Spec. 33, p 1-19
Liquid chromatography
and tandem mass
spectrometry
Software for processing
data
83. Defining protein function
• Classical methods:
– Define activity of protein, develop an assay for
activity
• Biochemistry: use assay to purify protein from
cell, characterize structure/function of protein in
vitro
• Genetics: obtain mutants with change in activity,
characterize phenotype of mutant, obtain
suppressors to identify genes that interact with
protein of interest
– Time intensive, expensive
84. Protein activity at the proteome level
• Protein-DNA interactions: identifying binding
sites for DNA-binding proteins: regulation of
gene expression
• Massively parallel screens for activity--protein
arrays
85. “chromatin immunoprecipitation” (ChIP)
1) Grow cells, add
formaldehyde to cross-link
everything to everything
(including DNA to protein)
2) Lyse cells, break up DNA
by shearing
3) Retrieve protein of interest
(and the DNA it is bound to)
using specific antibody to that
protein (immunoprecipitation)
4) Determine presence of
DNA by quantitative PCR
V. Orlando (2000) TIBS 25, p. 99
87. Protein arrays for function
Proteins immobilized,
usually by virtue of a tag
sequence (6 x his tag,
biotin, etc.)
Probe all proteins
at once for a
specific activity
88. Example of a protein microarray
Proteins fused to GST with
6 x histidine tags,
immobilized on Ni++ matrix
Anti-GST tells how much
protein is immobilized on
surface
Specific assays identify
proteins with specific
activities--calmodulin
binding, phosphoinositide
binding