After sequencing of the genome has been done, the first thing that comes to mind is "Where are the genes?". Genome annotation is the process of attaching information to the biological sequences. It is an active area of research and it would help scientists a lot to undergo with their wet lab projects once they know the coding parts of a genome.
Genome annotation is the process of analyzing genomic DNA sequences to extract biological meaning and context. It involves two main steps - structural annotation, which locates gene elements like exons and introns, and functional annotation, which predicts the functions of gene products. Computational tools are crucial given the vast amounts of sequence data. They use various approaches like identifying open reading frames, conserved sequences, statistical patterns and sequence similarities to model gene structures and infer functions. The results are then integrated into automated annotation pipelines to generate comprehensive and reliable gene annotations for genomes.
BTC 506 Gene Identification using Bioinformatic Tools-230302130331.pptxChijiokeNsofor
This document discusses several bioinformatics tools and methods for identifying genes from genomic sequences, including:
1. Obtaining sequence data through sequencing technologies and preprocessing data.
2. Using tools like Ensembl, RefSeq and UCSC Genome Browser for gene identification and annotation.
3. Using gene prediction tools like Augustus, GeneMark and Glimmer to predict gene locations and structures.
4. Validating predicted genes through comparison to known genes or experimental validation with RNA-seq or RT-PCR.
This document discusses various bioinformatics tools and methods for identifying genes from genomic sequences. It begins by defining genes and genomes, then describes reference databases like RefSeq that are important for gene identification. It outlines the general workflow for gene identification, including obtaining sequences, preprocessing, annotation, prediction, and validation. Specific tools mentioned include GENSCAN, Glimmer, and Augustus for gene prediction, and BLAST for sequence alignment. The document also discusses identifying other genomic features like promoters, repeats, and open reading frames. It emphasizes that accurate gene identification requires both computational and experimental approaches.
This is an introduction to conducting manual annotation efforts using Apollo. This webinar was offered to members of the i5K Research community on 2015-10-07.
Apollo is a web-based application that supports and enables collaborative genome curation in real time, allowing teams of curators to improve on existing automated gene models through an intuitive interface. Apollo allows researchers to break down large amounts of data into manageable portions to mobilize groups of researchers with shared interests.
The i5K, an initiative to sequence the genomes of 5,000 insect and related arthropod species, is a broad and inclusive effort that seeks to involve scientists from around the world in their genome curation process, and Apollo is serving as the platform to empower this community.
This presentation is an introduction to Apollo for the members of the i5K Pilot Project on Eurytemora affinis
This document discusses functional genomics and its approaches. It defines functional genomics as the worldwide experimental approach to access the function of genes by using information from structural genomics. The key functional genomics approaches discussed are transcriptomics, proteomics, metabolomics, interactomics, epigenetics, and nutrigenomics. Modern techniques discussed include expressed sequence tags (ESTs), serial analysis of gene expression (SAGE), and microarray analysis.
This document discusses gene prediction and promoter prediction. It begins by explaining that gene prediction involves locating protein-coding genes within sequenced genomes in order to understand their functional content. Various computational methods are used for gene prediction, including searching for signals like start/stop codons, searching coding content, and comparing sequences to find homologs. Promoter prediction involves locating DNA elements that regulate gene expression and is challenging due to diversity and short, conserved motifs. Ab initio and comparative phylogenetic footprinting methods are used to predict promoters and regulatory elements in prokaryotes and eukaryotes.
After sequencing of the genome has been done, the first thing that comes to mind is "Where are the genes?". Genome annotation is the process of attaching information to the biological sequences. It is an active area of research and it would help scientists a lot to undergo with their wet lab projects once they know the coding parts of a genome.
Genome annotation is the process of analyzing genomic DNA sequences to extract biological meaning and context. It involves two main steps - structural annotation, which locates gene elements like exons and introns, and functional annotation, which predicts the functions of gene products. Computational tools are crucial given the vast amounts of sequence data. They use various approaches like identifying open reading frames, conserved sequences, statistical patterns and sequence similarities to model gene structures and infer functions. The results are then integrated into automated annotation pipelines to generate comprehensive and reliable gene annotations for genomes.
BTC 506 Gene Identification using Bioinformatic Tools-230302130331.pptxChijiokeNsofor
This document discusses several bioinformatics tools and methods for identifying genes from genomic sequences, including:
1. Obtaining sequence data through sequencing technologies and preprocessing data.
2. Using tools like Ensembl, RefSeq and UCSC Genome Browser for gene identification and annotation.
3. Using gene prediction tools like Augustus, GeneMark and Glimmer to predict gene locations and structures.
4. Validating predicted genes through comparison to known genes or experimental validation with RNA-seq or RT-PCR.
This document discusses various bioinformatics tools and methods for identifying genes from genomic sequences. It begins by defining genes and genomes, then describes reference databases like RefSeq that are important for gene identification. It outlines the general workflow for gene identification, including obtaining sequences, preprocessing, annotation, prediction, and validation. Specific tools mentioned include GENSCAN, Glimmer, and Augustus for gene prediction, and BLAST for sequence alignment. The document also discusses identifying other genomic features like promoters, repeats, and open reading frames. It emphasizes that accurate gene identification requires both computational and experimental approaches.
This is an introduction to conducting manual annotation efforts using Apollo. This webinar was offered to members of the i5K Research community on 2015-10-07.
Apollo is a web-based application that supports and enables collaborative genome curation in real time, allowing teams of curators to improve on existing automated gene models through an intuitive interface. Apollo allows researchers to break down large amounts of data into manageable portions to mobilize groups of researchers with shared interests.
The i5K, an initiative to sequence the genomes of 5,000 insect and related arthropod species, is a broad and inclusive effort that seeks to involve scientists from around the world in their genome curation process, and Apollo is serving as the platform to empower this community.
This presentation is an introduction to Apollo for the members of the i5K Pilot Project on Eurytemora affinis
This document discusses functional genomics and its approaches. It defines functional genomics as the worldwide experimental approach to access the function of genes by using information from structural genomics. The key functional genomics approaches discussed are transcriptomics, proteomics, metabolomics, interactomics, epigenetics, and nutrigenomics. Modern techniques discussed include expressed sequence tags (ESTs), serial analysis of gene expression (SAGE), and microarray analysis.
This document discusses gene prediction and promoter prediction. It begins by explaining that gene prediction involves locating protein-coding genes within sequenced genomes in order to understand their functional content. Various computational methods are used for gene prediction, including searching for signals like start/stop codons, searching coding content, and comparing sequences to find homologs. Promoter prediction involves locating DNA elements that regulate gene expression and is challenging due to diversity and short, conserved motifs. Ab initio and comparative phylogenetic footprinting methods are used to predict promoters and regulatory elements in prokaryotes and eukaryotes.
This document provides an introduction and overview of manual genome annotation using the Apollo genome annotation tool. It begins with an outline of the webinar topics, which include an introduction to manual annotation and its necessity, an overview of the Apollo tool and its functionality for collaborative curation, and examples and demonstrations. The document then covers key concepts for manual annotation such as the definition of a gene, genome curation steps, transcription and translation including reading frames, splice sites, and phase. The goal of the webinar is to help participants better understand genome curation and manual annotation using Apollo to identify and modify gene models.
The document discusses genomics and comparative genomics. It defines genomics as the study of genomes and notes that comparative genomics compares two or more genomes to discover similarities and differences. Comparative genomics can provide insights into evolutionary biology, drug discovery, gene function prediction, and identification of genes and regulatory elements. The document outlines different levels of genome comparison including nucleotide statistics, genome structure at the DNA and gene levels, and describes various methods used in comparative genomic analyses.
Introduction to Apollo: A webinar for the i5K Research CommunityMonica Munoz-Torres
This document provides an introduction and outline for a webinar on using the Apollo genome annotation editing tool. It was presented by Monica Munoz-Torres of BBOP to the i5K Research Community. The webinar aimed to help participants better understand genome curation in the context of automated and manual annotation. It also aimed to familiarize participants with Apollo's functionality and how to identify homologs of known genes, corroborate gene models using evidence, and modify automated annotations in Apollo. The document includes sections on genome sequencing projects, the objectives and uses of genome annotation, and a biological refresher on concepts relevant to manual annotation like genes, transcription, translation, and genome curation steps.
Apollo: A workshop for the Manakin Research Coordination NetworkMonica Munoz-Torres
Apollo is a web-based, collaborative genomic annotation editing platform. We need annotation editing tools to modify and refine precise location and structure of the genome elements that predictive algorithms cannot yet resolve automatically.
This presentation is an introduction to how the manual annotation process takes place using Apollo. It is addressed to the members of the Manakin Genomics research community.
Bioinformatics uses computers to store, organize, and analyze biological data, particularly DNA and protein sequences. Key data types include DNA, RNA, and protein sequences, as well as data from experiments like transcriptomics and proteomics. Common analyses include sequence comparisons and searches for coding regions. DNA contains genetic information encoded as sequences of nucleotides that are read from 5' to 3'. It is double-stranded and antiparallel. Genes encode proteins through transcription of DNA to mRNA and translation of mRNA to protein.
The document provides information about various bioinformatics tools for DNA sequence analysis. It describes tools for finding protein coding regions like GeneMark and GENSCAN. It discusses tools for predicting promoters like SoftBerry Promoter and Promoter 2.0. It outlines how Tandem Repeat Finder can detect tandem repeats and how RepeatMasker can mask interspersed repeats in a sequence. It also discusses UTRScan for finding UTR locations and CpG Islands for detecting CpG islands. For each tool, it provides the procedure and interpretation of sample results.
Comparative genome analysis requires high quality annotations of all genomic elements. Today’s sequencing projects face numerous challenges including lower coverage, more frequent assembly errors, and the lack of closely related species with well-annotated genomes. Precise elucidation of the many different biological features encoded in any genome requires careful examination and review. We need genome annotation editing tools to modify and refine the location and structure of the genome elements that predictive algorithms cannot yet resolve automatically. During the manual annotation process, curators identify elements that best represent the underlying biology and eliminate elements that reflect systemic errors of automated analyses.
Apollo is a web-based application that supports and enables collaborative genome curation in real time, analogous to Google Docs, allowing teams of curators to improve on existing automated gene models through an intuitive interface. Researchers from nearly one hundred institutions worldwide are currently using Apollo for distributed curation efforts in over sixty genome projects across the tree of life: from plants to arthropods, to fungi, to species of fish and other vertebrates including human, cattle (bovine), and dog.
Apollo - A webinar for the Phascolarctos cinereus research communityMonica Munoz-Torres
This document provides an overview of a webinar introducing the Apollo genome annotation tool. The webinar aims to help the koala genome research community better understand genome curation processes involving automated annotation and manual curation using Apollo. It outlines the webinar topics which will explain gene prediction, the Apollo interface for collaborative curation, and demonstrations of identifying gene homologs and modifying automated annotations. The webinar aims to familiarize participants with genome curation concepts and the Apollo tool.
1) The document discusses a study analyzing the impact of gene length on detecting differentially expressed genes using RNA-seq technology.
2) The study will first test the reproducibility of RNA-seq and the effect of normalization. It will then compare different statistical tests for identifying differentially expressed genes.
3) Finally, the study will specifically test how gene length impacts the likelihood of a gene being identified as differentially expressed, as longer genes are easier to map with short reads.
description of functional genomics and structural genomics and the techniques involved in it and also decribing the models of forward genetics and techniques involved in it and reverse genetics and techniques involved in it
annotation is nothing but the extra informations. the genome annotation is the extra informations about the DNA sequence of the organism. without annotation the squence doesnt make any sense of the sequencing.The current gene prediction methods can be classified into two major categories, abinitio–based and homology-based approaches.
The ab initio–based approach predicts genes based on the given sequence alone.
The homology-based approach predicts a gene using the alignment of the protein or RNA sequence/ gene models in evolutionary related species.
The analysis of global gene expression and transcription factor regulation, global approaches to alternative splicing and its regulation, long noncoding RNAs, gene expression models of signalling pathways, from gene expression to disease phenotypes, introduction to isoform sequencing, systematic and integrative analysis of gene expression to identify feature genes underlying human diseases.
This document discusses gene identification and genome annotation. It describes how gene finding in eukaryotes is difficult due to smaller percentages of genes in genomes like humans, and larger intron sizes. It covers open reading frames, complications with introns, and the use of six-frame translation to find protein coding sequences. Software tools for structural and functional annotation are outlined, including identifying genes through homology searching and ab initio prediction using hidden Markov models. The accuracy challenges of ab initio prediction are also summarized.
International Journal of Engineering Research and DevelopmentIJERD Editor
This document discusses a study that uses the ke-REM (ke-Rule Extraction Method) classifier to predict promoter regions in DNA sequences. The study evaluates the performance of ke-REM compared to existing promoter prediction techniques. ke-REM constructs rules based on attribute-value pairs from a dataset of 106 E. coli DNA sequences, each containing 57 nucleotides. The results show that ke-REM competes well with existing methods for identifying promoter regions in DNA.
1. Bioinformatics is the science of using computer hardware and software to analyze biological data such as DNA sequences, protein sequences, and gene expression data.
2. It has three main branches - genomics which analyzes genome sequences, transcriptomics which analyzes gene expression data, and proteomics which analyzes protein sequences and structures.
3. The goals of bioinformatics include acquiring biological data, developing tools and databases, analyzing the data, and integrating different types of biological data to gain new biological insights.
Comparative genome analysis requires high quality annotations of all genomic elements. Today’s sequencing projects face numerous challenges including lower coverage, more frequent assembly errors, and the lack of closely related species with well-annotated genomes. Precise elucidation of the many different biological features encoded in any genome requires careful examination and review. We need genome annotation editing tools to modify and refine the location and structure of the genome elements that predictive algorithms cannot yet resolve automatically. During the manual annotation process, curators identify elements that best represent the underlying biology and eliminate elements that reflect systemic errors of automated analyses.
Apollo is a web-based application that supports and enables collaborative genome curation in real time, analogous to Google Docs, allowing teams of curators to improve on existing automated gene models through an intuitive interface. Researchers from nearly one hundred institutions worldwide are currently using Apollo for distributed curation efforts in over sixty genome projects across the tree of life: from plants to arthropods, to fungi, to species of fish and other vertebrates including human, cattle (bovine), and dog.
This document discusses using hidden Markov models (HMMs) for gene prediction and analysis of unknown DNA sequences. It explains that HMMs allow for probabilistic modeling of sequences that accounts for insertions and deletions, and can be used to identify coding regions, splice sites, repeats and other features in genomic sequences. The document provides examples of using HMMs to represent proteins and DNA as probabilistic state machines, and describes how HMMs can incorporate profile data to enable database searching and gene prediction.
1.introduction to genetic engineering and restriction enzymesGetachew Birhanu
An introduction to Genetic engineering
A short background and history of Genetic Engineering
Classification of DNA manipulating Enzymes, nomenclature
Restriction recognition sequences, the anatomy of a gene and the flow of genetic information
More emphasis is given for the essential DNA Manipulating Enzymes
Finally Restriction mapping (analysis)
This document provides an introduction and overview of manual genome annotation using the Apollo genome annotation tool. It begins with an outline of the webinar topics, which include an introduction to manual annotation and its necessity, an overview of the Apollo tool and its functionality for collaborative curation, and examples and demonstrations. The document then covers key concepts for manual annotation such as the definition of a gene, genome curation steps, transcription and translation including reading frames, splice sites, and phase. The goal of the webinar is to help participants better understand genome curation and manual annotation using Apollo to identify and modify gene models.
The document discusses genomics and comparative genomics. It defines genomics as the study of genomes and notes that comparative genomics compares two or more genomes to discover similarities and differences. Comparative genomics can provide insights into evolutionary biology, drug discovery, gene function prediction, and identification of genes and regulatory elements. The document outlines different levels of genome comparison including nucleotide statistics, genome structure at the DNA and gene levels, and describes various methods used in comparative genomic analyses.
Introduction to Apollo: A webinar for the i5K Research CommunityMonica Munoz-Torres
This document provides an introduction and outline for a webinar on using the Apollo genome annotation editing tool. It was presented by Monica Munoz-Torres of BBOP to the i5K Research Community. The webinar aimed to help participants better understand genome curation in the context of automated and manual annotation. It also aimed to familiarize participants with Apollo's functionality and how to identify homologs of known genes, corroborate gene models using evidence, and modify automated annotations in Apollo. The document includes sections on genome sequencing projects, the objectives and uses of genome annotation, and a biological refresher on concepts relevant to manual annotation like genes, transcription, translation, and genome curation steps.
Apollo: A workshop for the Manakin Research Coordination NetworkMonica Munoz-Torres
Apollo is a web-based, collaborative genomic annotation editing platform. We need annotation editing tools to modify and refine precise location and structure of the genome elements that predictive algorithms cannot yet resolve automatically.
This presentation is an introduction to how the manual annotation process takes place using Apollo. It is addressed to the members of the Manakin Genomics research community.
Bioinformatics uses computers to store, organize, and analyze biological data, particularly DNA and protein sequences. Key data types include DNA, RNA, and protein sequences, as well as data from experiments like transcriptomics and proteomics. Common analyses include sequence comparisons and searches for coding regions. DNA contains genetic information encoded as sequences of nucleotides that are read from 5' to 3'. It is double-stranded and antiparallel. Genes encode proteins through transcription of DNA to mRNA and translation of mRNA to protein.
The document provides information about various bioinformatics tools for DNA sequence analysis. It describes tools for finding protein coding regions like GeneMark and GENSCAN. It discusses tools for predicting promoters like SoftBerry Promoter and Promoter 2.0. It outlines how Tandem Repeat Finder can detect tandem repeats and how RepeatMasker can mask interspersed repeats in a sequence. It also discusses UTRScan for finding UTR locations and CpG Islands for detecting CpG islands. For each tool, it provides the procedure and interpretation of sample results.
Comparative genome analysis requires high quality annotations of all genomic elements. Today’s sequencing projects face numerous challenges including lower coverage, more frequent assembly errors, and the lack of closely related species with well-annotated genomes. Precise elucidation of the many different biological features encoded in any genome requires careful examination and review. We need genome annotation editing tools to modify and refine the location and structure of the genome elements that predictive algorithms cannot yet resolve automatically. During the manual annotation process, curators identify elements that best represent the underlying biology and eliminate elements that reflect systemic errors of automated analyses.
Apollo is a web-based application that supports and enables collaborative genome curation in real time, analogous to Google Docs, allowing teams of curators to improve on existing automated gene models through an intuitive interface. Researchers from nearly one hundred institutions worldwide are currently using Apollo for distributed curation efforts in over sixty genome projects across the tree of life: from plants to arthropods, to fungi, to species of fish and other vertebrates including human, cattle (bovine), and dog.
Apollo - A webinar for the Phascolarctos cinereus research communityMonica Munoz-Torres
This document provides an overview of a webinar introducing the Apollo genome annotation tool. The webinar aims to help the koala genome research community better understand genome curation processes involving automated annotation and manual curation using Apollo. It outlines the webinar topics which will explain gene prediction, the Apollo interface for collaborative curation, and demonstrations of identifying gene homologs and modifying automated annotations. The webinar aims to familiarize participants with genome curation concepts and the Apollo tool.
1) The document discusses a study analyzing the impact of gene length on detecting differentially expressed genes using RNA-seq technology.
2) The study will first test the reproducibility of RNA-seq and the effect of normalization. It will then compare different statistical tests for identifying differentially expressed genes.
3) Finally, the study will specifically test how gene length impacts the likelihood of a gene being identified as differentially expressed, as longer genes are easier to map with short reads.
description of functional genomics and structural genomics and the techniques involved in it and also decribing the models of forward genetics and techniques involved in it and reverse genetics and techniques involved in it
annotation is nothing but the extra informations. the genome annotation is the extra informations about the DNA sequence of the organism. without annotation the squence doesnt make any sense of the sequencing.The current gene prediction methods can be classified into two major categories, abinitio–based and homology-based approaches.
The ab initio–based approach predicts genes based on the given sequence alone.
The homology-based approach predicts a gene using the alignment of the protein or RNA sequence/ gene models in evolutionary related species.
The analysis of global gene expression and transcription factor regulation, global approaches to alternative splicing and its regulation, long noncoding RNAs, gene expression models of signalling pathways, from gene expression to disease phenotypes, introduction to isoform sequencing, systematic and integrative analysis of gene expression to identify feature genes underlying human diseases.
This document discusses gene identification and genome annotation. It describes how gene finding in eukaryotes is difficult due to smaller percentages of genes in genomes like humans, and larger intron sizes. It covers open reading frames, complications with introns, and the use of six-frame translation to find protein coding sequences. Software tools for structural and functional annotation are outlined, including identifying genes through homology searching and ab initio prediction using hidden Markov models. The accuracy challenges of ab initio prediction are also summarized.
International Journal of Engineering Research and DevelopmentIJERD Editor
This document discusses a study that uses the ke-REM (ke-Rule Extraction Method) classifier to predict promoter regions in DNA sequences. The study evaluates the performance of ke-REM compared to existing promoter prediction techniques. ke-REM constructs rules based on attribute-value pairs from a dataset of 106 E. coli DNA sequences, each containing 57 nucleotides. The results show that ke-REM competes well with existing methods for identifying promoter regions in DNA.
1. Bioinformatics is the science of using computer hardware and software to analyze biological data such as DNA sequences, protein sequences, and gene expression data.
2. It has three main branches - genomics which analyzes genome sequences, transcriptomics which analyzes gene expression data, and proteomics which analyzes protein sequences and structures.
3. The goals of bioinformatics include acquiring biological data, developing tools and databases, analyzing the data, and integrating different types of biological data to gain new biological insights.
Comparative genome analysis requires high quality annotations of all genomic elements. Today’s sequencing projects face numerous challenges including lower coverage, more frequent assembly errors, and the lack of closely related species with well-annotated genomes. Precise elucidation of the many different biological features encoded in any genome requires careful examination and review. We need genome annotation editing tools to modify and refine the location and structure of the genome elements that predictive algorithms cannot yet resolve automatically. During the manual annotation process, curators identify elements that best represent the underlying biology and eliminate elements that reflect systemic errors of automated analyses.
Apollo is a web-based application that supports and enables collaborative genome curation in real time, analogous to Google Docs, allowing teams of curators to improve on existing automated gene models through an intuitive interface. Researchers from nearly one hundred institutions worldwide are currently using Apollo for distributed curation efforts in over sixty genome projects across the tree of life: from plants to arthropods, to fungi, to species of fish and other vertebrates including human, cattle (bovine), and dog.
This document discusses using hidden Markov models (HMMs) for gene prediction and analysis of unknown DNA sequences. It explains that HMMs allow for probabilistic modeling of sequences that accounts for insertions and deletions, and can be used to identify coding regions, splice sites, repeats and other features in genomic sequences. The document provides examples of using HMMs to represent proteins and DNA as probabilistic state machines, and describes how HMMs can incorporate profile data to enable database searching and gene prediction.
1.introduction to genetic engineering and restriction enzymesGetachew Birhanu
An introduction to Genetic engineering
A short background and history of Genetic Engineering
Classification of DNA manipulating Enzymes, nomenclature
Restriction recognition sequences, the anatomy of a gene and the flow of genetic information
More emphasis is given for the essential DNA Manipulating Enzymes
Finally Restriction mapping (analysis)
Similar to Structural annotation................pptx (20)
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...Leonel Morgado
Current descriptions of immersive learning cases are often difficult or impossible to compare. This is due to a myriad of different options on what details to include, which aspects are relevant, and on the descriptive approaches employed. Also, these aspects often combine very specific details with more general guidelines or indicate intents and rationales without clarifying their implementation. In this paper we provide a method to describe immersive learning cases that is structured to enable comparisons, yet flexible enough to allow researchers and practitioners to decide which aspects to include. This method leverages a taxonomy that classifies educational aspects at three levels (uses, practices, and strategies) and then utilizes two frameworks, the Immersive Learning Brain and the Immersion Cube, to enable a structured description and interpretation of immersive learning cases. The method is then demonstrated on a published immersive learning case on training for wind turbine maintenance using virtual reality. Applying the method results in a structured artifact, the Immersive Learning Case Sheet, that tags the case with its proximal uses, practices, and strategies, and refines the free text case description to ensure that matching details are included. This contribution is thus a case description method in support of future comparative research of immersive learning cases. We then discuss how the resulting description and interpretation can be leveraged to change immersion learning cases, by enriching them (considering low-effort changes or additions) or innovating (exploring more challenging avenues of transformation). The method holds significant promise to support better-grounded research in immersive learning.
The binding of cosmological structures by massless topological defectsSérgio Sacani
Assuming spherical symmetry and weak field, it is shown that if one solves the Poisson equation or the Einstein field
equations sourced by a topological defect, i.e. a singularity of a very specific form, the result is a localized gravitational
field capable of driving flat rotation (i.e. Keplerian circular orbits at a constant speed for all radii) of test masses on a thin
spherical shell without any underlying mass. Moreover, a large-scale structure which exploits this solution by assembling
concentrically a number of such topological defects can establish a flat stellar or galactic rotation curve, and can also deflect
light in the same manner as an equipotential (isothermal) sphere. Thus, the need for dark matter or modified gravity theory is
mitigated, at least in part.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
Immersive Learning That Works: Research Grounding and Paths ForwardLeonel Morgado
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
2. INTRODUCTION
• The scope of genome annotation has expanded since the first complete annotation
of the Haemophilus influenzae genome in 1995( Fleishmann et al., 1995).
• Once a DNA sequence has been obtained, whether it is the sequence of a single
cloned fragment or of an entire chromosome, then various methods can be
employed to locate the genes that are present.
• These methods can be divided into those that involve simply inspecting the
sequence, by eye or more frequently by computer, to look for the special sequence
features associated with genes, and those methods that locate genes by
experimental analysis of the DNA sequence. The computer methods form part of the
methodology called bioinformatics.
• The first software used to analyze sequencing reads is the ‘Staden Package’
created by Rodger Staden in 1977( Staden, 1977).
3. STRUCTURAL ANNOTATION
• Finding features of DNA—exons, introns, promoters, transposons, etc.—is known as
structural annotation. Structural annotation attempts to find genes in a genomic sequence.
• A gene can be defined as "a sequence region necessary for generating functional
products" . Functional products of genes are proteins and RNAs. Genes that lead to the
production of proteins are called protein-coding genes.
• Other genes that do not code proteins, but instead functional RNA molecules, are called
noncoding genes. Noncoding RNA genes include genes for ribosomal RNA (rRNA),
transfer RNA (tRNA), microRNA (miRNA), small nuclear RNA and nucleolar RNA (snRNA
and snoRNA, respectively) and long noncoding RNA (lncRNA).
• Structural annotations also identify pseudogenes. They were initially considered to be
functionless and evolutionary dead-ends. We now know that they sometimes participate in
gene regulation. Hence, their prediction improves our understanding of genomes.
4. • Sequence inspection can be used to locate genes because genes are not random series of nucleotides but
instead have distinctive features.
• At present we do not fully understand the nature of all of these specific features, and sequence inspection is
therefore not a foolproof way of locating genes, but it is still a powerful tool and is usually the first method that
is applied to analysis of a new genome sequence.
GENE LOCATION BY SEQUENCE INSPECTION
5. The coding regions of genes are open reading frames
• Genes that code for proteins comprise open reading frames
(ORFs) consisting of a series of codons that specify the amino
acid sequence of the protein that the gene codes for.
• The ORF begins with an initiation codon usually (but not
always) ATG-and ends with a termination codon: TAA, TAG, or
TGA . Searching a DNA sequence for ORFs that begin with an
ATG and end with a termination triplet is therefore one way of
looking for genes.
• The analysis is complicated by the fact that each DNA
sequence has six reading frames, three in one direction and
three in the reverse direction on the complementary strand , but
computers are quite capable of scanning all six reading frames
for ORFs.
A PROTEIN-CODING GENE IS AN OPEN READING FRAME
OF TRIPLET CODONS
6. • With bacterial genomes, simple ORF scanning is an effective way of locating most of the genes in a
DNA sequence.
• With bacteria the analysis is further simplified by the fact that the genes are very closely spaced and
hence there is relatively little intergenic DNA in the genome (only 11% for E. coli).
• If we assume that the real genes do not overlap, which is true for most bacterial genes, then it is only
in the intergenic regions that there is a possibility of mistaking a short, spurious ORF for a real gene.
• So if the intergenic component of a genome is small, then there is a reduced chance of making
mistakes in interpreting the results of a simple ORF scan.
7. Repeats
• The first step in structural annotation involves repeat masking. DNA repeats occur in both
prokaryotic and eukaryotic organisms.
• The repeats account for 0% to over 42% of the prokaryotic genome . Similarly, eukaryotic
genomes can harbor millions of repeats.
• For instance, repeats account for two-thirds of the human genome . Repeat sequences can be
localized in tandem, i.e., adjacent to one other, and are typically found in the centromere .
Alternatively, they can be interspersed in different forms of transposable elements, e.g., in long
and short interspersed nuclear elements (LINEs and SINEs), DNA transposons, etc. .
• Repeat masking tools rely on databases with lists of already identified repeats. RepeatMasker is
a good example of such tool.
• Aligning transcript and protein evidence after masking is the second step of structural annotation
before gene identification, although it is not mandatory. BLAST or BLAT can be used to align the
transcript and protein evidence.
• Further, RNA-seq evidence can be aligned using TopHat or HISAT .
8. Predictions of Gene and Different Features
• Identifying protein-coding genes and other regulatory elements takes center stage in gene annotation. Gene
prediction is a complex process, especially for eukaryotic DNA .
• The varying sizes of introns(noncoding sequences) in-between exons and alternative splice variants make
gene structure prediction difficult.
• Many gene prediction programs exist. They can be categorized into three groups: ab initio methods,
homology-based methods, and combined methods.
• Approaches for gene prediction based on nucleotide sequence are called ab initio methods. Ab initio
approaches rely on statistical models, such as the hidden Markov model (HMM), to identify promoters, coding
or noncoding regions, and intron–exon junctions in the genome sequence.
• The second approach aligns the sequence with expressed sequence tags (EST), complementary DNA (cDNA),
or protein evidence, and uses detected similarities for gene prediction.
9. • The other group comprises programs that combine ab initio and evidence- or homology-based
approaches for gene prediction.
• In addition, gene prediction programs should be able to predict alternative splicing sites because
alternative splicing is a major actor in the regulation of gene expression, and transcriptome and proteome
diversity .
• Accordingly, gene prediction programs use various models to predict splice sites. Since approximately
99% of the introns in sequenced genomes begin with GT and end with AG, these features are denoted as
mandatory by most gene prediction systems for splice site detection.
• In addition, incorporation of a strong splice donor consensus, such as the GC–AG splice site, improves
the accuracy of gene prediction programs.
13. Databases for Structural Annotation
• Annotations require supporting data that can be used or presented as evidence of predicted
assignments. Currently, homology-based methods play a central role in genome annotation because
of the huge amount of EST and cDNA sequences available .
• Homology-based methods depend on DNA, RNA, or protein sequence alignment data, which can
easily be retrieved from biological databases. Ab initio annotations, on the other hand, identify genes
and their structures using mathematical models.
• Nonetheless, the ab initio gene predictors have to be trained using high-quality gene models or
organism-specific genome traits, such as codon frequency and intron–exon length distribution .
Further, ab initio models require ESTs, RNA-seq data, and proteins to improve prediction accuracy.
• Databases readily provide such data. Nucleotide and protein sequence or structure can easily be
found in comprehensive public-domain databases, e.g., the GenBank , European Nucleotide Archive
(ENA) , and DNA Databank of Japan (DDBJ). UniProt , which is a protein sequence database that
combines UniProtKB/Swiss-Prot (over 560,000 manually curated sequences) and
UniProtKB/TrEMBL (180 million automatically annotated sequences), provides the scientific
community with high-quality and freely accessible protein sequences with the associated functional
information.
14. Comparative Annotation Methods
• Genome annotation achieved by comparison of genes and genomes across species can be a reliable
information source for understanding genome evolution. Comparative annotation allows annotations of a
well-studied genome to be projected onto an evolutionarily close species. It often focuses on the coding
genes.
• Valuable information for comparative annotation can be found from genome alignment. A well-aligned
genome will yield sound data for comparative annotation .
• Approaches to comparative annotation of genomes can be categorized into ab initio methods and
homology-based methods, considering the input information used for annotation, i.e., either a statistical
model of genes, or protein sequence, EST, and cDNA, accordingly.
• Ab initio approaches are preferred for genes that are weakly or not at all represented in RNA-seq library and
have insufficient similarity to any known protein and lack other evidence.
15. • Related species have genomes that share similarities inherited from their common ancestor,
over- laid with species-specific differences that have arisen since the species began to evolve
independently. Because of natural selection, the sequence similarities between related genomes
are greatest within the genes and lowest in the intergenic regions.
• Therefore, when related genomes are compared, homologous genes are easily identified
because they have high sequence similarity, and any ORF that does not have a clear homolog in
the second genome can be discounted as almost certainly being a chance sequence and not a
genuine gene. This type of analysis-called comparative genomics
16. Homology-Based Annotation
• For predict and annotate genes by identifying significant matches from a well annotated genome
sequence by employing alignment tools such as BLAST.
• Homology-based annotations use the coding sequences (CDS), usually protein sequences and
sometimes transcripts in the form of mRNA, cDNA, or EST to predict genes, assuming similar sequence
regions encode homologous proteins.
• Tools like Exonerate and DIALIGN can be used for sequence alignment; GenomeThreader and
AGenDA are used for gene predictions. Increased evolutionary distance between the input protein and
the target protein reduces the accuracy of homology-based gene finding. This happens because of
heavy reliance on the alignment and information derived from the already known genes, which creates a
challenge in identifying genes whose properties are different from those of referenced genes.
• However, newer comparative approaches solve this issue by relying to a greater degree on sequence
conservation, which enables them to identify genes with new features and different statistical
composition.
• TWINSCAN and SGP2 are examples of tools in which gene prediction uses the analysis of sequence
conservation patterns between genomic sequences of evolutionarily related organisms.
17. Ab Initio Annotation
• Ab initio annotation relies on ab initio gene predictors, which in turn rely on training data to construct an
algorithm or model.
• Prediction is done based on the genomic sequence in question, using statistical analysis and other gene
signals such as k-mer statistics and frame length.
• Some popular ab initio gene predictors are discussed below. AUGUSTUS defines the probability distributions
for eukaryotic genome sequences based on GHMM. AUGUSTUS is re-trainable and it can predict alterative
splicing, and the 50UTR and 30UTR, including introns. AUGUSTUS is one of the most accurate ab initio
gene prediction programs for the species it has been trained for .
• FGENESH is an HMM-based, very fast, and accurate ab initio gene structure prediction program for
humans, Drosophila, plants, yeasts, and nematodes.
• This renders it the fastest tool among HMM-based gene finding programs. GENSCAN is another HMM-
based ab initio tool for predicting locations and exon–intron structures of genes in genomic sequences of a
variety of organisms.
18. • Vertebrate and invertebrate versions of GENSCAN are available. The accuracy of the latter is lower
because the original tool was primarily designed for the detection of genes in human and vertebrate
genomic sequences.
• It is becoming a common practice to use ab initio annotation methods in combine a sequences
transcriptome information such as that provided by RNA-seq.
• This can be viewed as an evidence-based or extrinsic approach. For example, a newer version of
AUGUSTUS can incorporate information from EST and protein alignments.
• In addition, a variant of FGENESH called FGENESH-C uses HMM and cDNA for predictions, while
GenomeScan (an extension of GENSCAN) uses extrinsic information of protein BLAST alignments for
gene structure prediction.
19. Ab initio and Homology based annotation tools summary.
20. Annotation Pipelines
• Analysis of large amounts of data generated by the sequencing requires multiple computationally-intensive
steps . Sets of algorithms that process sequence data and are executed in a predefined order are called a
bioinformatic pipelines.
• Pipelines process massive amounts of sequence data and the associated metadata using multiple
software components, databases, and environments.
• They are comprehensive, holistic packages that try to exploit relevant information provided by both ab
initio and similarity-based gene predictors.
21. Structural Pipelines
• MAKER2 is a multi-threaded, parallelized genome annotation and data management application, which
builds up on MAKER.
• Ab initio gene prediction tools SNAP, AUGUSTUS, and GenMark-ES are integrated in MAKER2. Novel
genomes with limited training data available can be annotated with MAKER2. The tool can also be used
to improve annotation quality by integrating mRNA-seq data.
• NCBI Eukaryotic Annotation Pipeline is an automated pipeline for eukaryotes, in which coding and
noncoding genes, transcripts, and proteins in both finished and draft genomes can be annotated. This
pipeline uses Splign and ProSplign for alignment. It also has its own gene prediction tool called
GNOMON which combines HMM-based ab initio models and homology search information extracted
from experimental evidence.
• Comparative Annotation Toolkit (CAT) is a fully open-source software toolkit for end-to-end annotation.
CAT uses Progressive Cactus for multiple alignments. It’s output, together with previously annotated
genomes, is used to project annotations using TransMAP .
22. • CAT uses AUGUSTUS for gene prediction both from transMap projections and for ab initio gene
prediction.
• CAT wan developed by the GENCODE, and was utilized for the annotation of genomes of laboratory
mouse strains and great apes .
• BRAKER1 is a fully automated and highly accurate unsupervised RNA-seq–based genome
annotation pipeline for eukaryotic genomes.
23. Annotation Visualization
• File Formats
Most bioinformatic tools use the FASTA format as a standard for sequence data sharing. The FASTA
format is used for searching sequence databases, evaluating similarity scores, and identification of
periodic similarity scores.
Other standard file formats exisformat can accommodate additional information and can be used by
different programs, and interpreformat human users. It format genomic features in a standard text file
format.
• Genome Browsers
• Researchers and users utilize genome browsers to integrate various types of information, as well as
analyze and visualize data related to annotation.
• Genome browsers are usually used to efficiently and conveniently browse, search, retrieve, and
examine genomic sequence and annotation data, via a graphical interface. The UCSC Genome
Browser is the most commonly used genome browser; many visualization tools are modeled based on
this tool.
• The Ensembl genome browser is another widely used genome browser for vertebrate genomes, which
supports comparative genomics, sequence variation analysis, and transcriptional regulation analysis.
• Generic Model Organism Database (GMOD) is a collection of interconnected open-source software
tools and databases for managing, visualizing, storing, and sharing genetic and genomic information.
24. Re-Annotation
• We have seen that as a result of the increasing volume of data from genome sequencing projects,
computational analysis methods have become a considerable element of genome annotations. However, this
has led to high levels of misannotation in public databases .
• Re-annotation benefits the end-user by providing the latest resources. Updating a previously annotated
genome can be seen as re-annotation . Automated annotations save time and resources, but manual
annotations, although time-consuming, are better than automated annotations.
• Re-annotation can be used to create large complete genomes, and indeed, there are tools that can be used
for this purpose. Restauro-G is rapid bacterial genome re-annotation software that utilizes a BLAST-like
alignment tool for re-annotation.
• MAKER2 incorporates an external annotation pass-through mechanism that accepts pre-existing genome
annotations.
• Wiki-based sites have been proven successful in providing accurate, useful, and updated information,
despite the fear of being filled with unreliable and inaccurate data. Currently, new information emerges from
different corners of bioinformatic fields, which impacts gene annotation, rendering re-annotation a never-
ending process, to some degree.
25. EXPERIMENTAL TECHNIQUES FOR GENE LOCATION
• Most experimental methods for gene location are not based on direct examination of
DNA molecules but instead rely on detection of the RNA molecules that are
transcribed from genes.
• All genes are transcribed into RNA, and if the gene is discontinuous then the primary
transcript is subsequently processed to remove the introns and link up the exons .
• Techniques that map the positions of transcribed sequences in a DNA fragment can
therefore be used to locate exons and entire genes. The only problem to be kept in
mind is that the transcript is usually longer than the coding part of the gene because it
begins several tens of nucleotides upstream of the initiation codon and continues
several tens or hundreds of nucleotides downstream of the termination codon.
26. Hybridization tests can determine if a fragment contains transcribed sequences
• The simplest procedures for studying transcribed sequences are based on hybridization analysis.
• RNA molecules can be separated by specialized forms of agarose gel electrophoresis, transferred to a
nitrocellulose or nylon membrane, and examined by the process called northern hybridization.
• This differs from Southern hybridization only in the precise conditions under which the transfer is carried
out, and the fact that it was not invented by a Dr Northern and so does not have a capital "N."
• If a northern blot of cellular RNA is probed with a labeled fragment of the genome, then RNAs
transcribed from genes within that fragment will be detected. Northern hybridization is therefore,
theoretically, a means of determining the number of genes present in a DNA fragment and the size of
each coding region.
27. • An RNA is electrophoresed under denaturing
conditions in an agarose gel.
• After ethidium bromide staining, two bands are seen.
These are the two largest rRNA molecules , which are
abundant in most cells.
• The smaller rRNAs, which are also abundant, are not
seen because they are so short that they run out the
bottom of the gel and, in most cells, none of the
mRNAs are enough to form a band visible after
ethidium bromide staining.
• The gel is blotted onto a nylon membrane and, in this
example, probed with a radioactively labeled DNA
fragment.
• A single band is visible on the autoradiograph, showing
that the DNA fragment used as the probe contains part
or all of one transcribed sequence. northern hybridization
28. zoo-blotting
• A second type of hybridization analysis avoids the problems with
poorly expressed and tissue-specific genes by searching not for
RNAs but for related sequences in the DNAs of other organisms.
• This approach, like homology searching, is based on the fact that
homologous genes in related organisms have similar sequences,
whereas the intergenic DNA is usually quite different. a DNA
from one species is used to probe a Southern transfer of DNAs
from related species, and one or more hybridization signals are
obtained, then it is likely that the probe contains one or more
genes. This is called zoo-blotting.
zoo-blotting.
29. cDNA sequencing enables genes to be mapped within DNA fragments
• Northern hybridization and zoo-blotting enable the presence or absence of genes in a DNA fragment to be
determined, but give no positional information relating to the location of those genes in the DNA sequence.
The easiest way to obtain this information is to sequence the relevant cDNAs.
• A cDNA is a copy of an mRNA and so corresponds to the coding region of a gene, plus any leader or trailer
sequences that are also transcribed. Comparing a cDNA sequence with a genomic DNA sequence
therefore delineates the position of the relevant gene and reveals the exon-intron boundaries.
• In order to obtain an individual cDNA, a cDNA library must first be prepared from all of the mRNA in the
tissue being studied. Once the library has been prepared, the success of cDNA sequencing as a means of
gene location depends on two factors.
30. • The first concerns the frequency of the desired cDNAs in the library. As with northern hybridization,
the problem relates to the different expression levels of different genes. If the DNA fragment being
studied contains one or more poorly expressed genes, then the relevant cDNAs will be rare in the
library and it might be necessary to screen many clones before the desired one is identified.
• To get around this problem, various methods of cDNA capture or cDNA selection have been
devised, in which the DNA fragment being studied is repeatedly hybridized to the pool of cDNAs in
order to enrich the pool for the desired clones.
• Because the cDNA pool contains so many different sequences, it is generally not possible to
discard all the irrelevant clones by these repeated hybridizations, but it is possible to increase
significantly the frequency of those clones that specifically hybridize to the DNA fragment. This
reduces the size of the library that must subsequently be screened under stringent conditions to
identify the desired clones.
31. • A second factor that determines success or failure is the completeness of the individual cDNA
molecules. Usually, cDNAs are made by copying RNA molecules into single-stranded DNA with
reverse transcriptase and then converting the single-stranded DNA into double-stranded DNA with a
DNA polymerase..
• There is always a chance that one or other of the strand synthesis reactions will not proceed to
completion, resulting in a truncated cDNA. The presence of intramolecular base pairs in the RNA can
also lead to incomplete copying. Truncated cDNAs may lack some of the information needed to locate
the start and end points of a gene and all its exon-intron boundaries.
32. Methods are available for precise mapping of the ends of transcripts – (RACE)
• The problems with incomplete cDNAs mean that more robust methods are needed for locating the
precise start and end points of gene transcripts.
• One possibility is a special type of PCR that uses RNA rather than DNA as the starting material. The
first step in this type of PCR is to convert the RNA into cDNA with reverse transcriptase, after which
the cDNA is amplified with Taq polymerase in the same way as in a normal PCR. These methods go
under the collective name of reverse transcriptase PCR (RT-PCR) but the particular version that
interests us at present is rapid amplification of cDNA ends (RACE).
33. • In the simplest form of this method, one of the primers is specific for an internal region close to the
beginning of the gene being studied. This primer attaches to the mRNA for the gene and directs the
first reverse transcriptase-catalyzed stage of the process, during which a cDNA corresponding to the
start of the mRNA is made.
• Because only a small segment of the mRNA is being copied, the expectation is that the cDNA
synthesis will not terminate prematurely, so one end of the cDNA will correspond exactly with the start
of the mRNA.
• Once the cDNA has been made, a short poly(A) tail is attached to its 3' end. The second primer
anneals to this poly(A) sequence and, during the first round of the normal PCR, converts the single-
stranded cDNA into a double-stranded molecule, which is subsequently amplified as the PCR
proceeds. The sequence of this amplified molecule will reveal the precise position of the start of the
transcript.
34. RACE – Rapid Amplification of cDNA Ends.
• The RNA being studied is converted into a partial cDNA by
extension of a DNA primer that anneals at an internal
position not too distant from the 5' end of the molecule.
• The 3' end of the cDNA is further extended by treatment with
terminal deoxynucleotidyl transferase in the presence of
dNTP, which results in a series of As being added to the
cDNA.
• This series of As acts as the annealing site for the anchor
primer. Extension of the anchor primer leads to a double-
stranded DNA molecule, which can now be amplified by a
standard PCR.
• This is 5'-RACE, so-called because it results in amplification
of the 5' end of the starting RNA. A similar method- 3'-RACE-
can be used if the 3' end- sequence is desired.
35. • Other methods for precise transcript mapping involve heteroduplex analysis. If the DNA region being
studied is cloned as a restriction fragment in an M13 vector then it can be obtained as single-stranded
DNA. When mixed with an appropriate RNA preparation, the transcribed sequence in the cloned DNA
hybridizes with the equivalent mRNA, forming a double-stranded heteroduplex.
• The start of this mRNA lies within the cloned restriction fragment, so some of the cloned fragment
participates in the heteroduplex, but the rest does not. The single-stranded regions can be digested
by treatment with a single-strand-specific nuclease such as S1.
• The size of the heteroduplex is determined by degrading the RNA component with alkali and
electrophoresing the resulting single-stranded DNA in an agarose gel.
• This size measurement is then used to position the start of the transcript relative to the restriction site
at the end of the cloned fragment. Heteroduplex analysis can also be used to locate exon-intron
boundaries.
Heteroduplex analysis.
36. Exon-intron boundaries can also be located with precision
• A second method for finding exons in a genome sequence is called exon trapping.
• This requires a special type of vector that contains a minigene consisting of two exons flanking an
intron sequence, the first exon being preceded by the sequence signals needed to initiate transcription
in a eukaryotic cell.
• To use the vector, the piece of DNA to be studied is inserted into a restriction site located within the
vector's intron region.
• The vector is then introduced into a suitable eukaryotic cell line, where it is transcribed and the RNA
produced from it is spliced.
• The result is that any exon contained in the genomic fragment becomes attached between the
upstream and downstream exons from the minigene.
• RT-PCR with primers annealing within the two minigene exons is now used to amplify a DNA fragment,
which is sequenced. As the mini- gene sequence is already known, the nucleotide positions at which
the insertedexon starts and ends can be determined, precisely delineating this exon.