Whole genome shotgun sequencing involves randomly breaking genomic DNA into small fragments, sequencing the fragments, and then reassembling the sequences using overlapping regions. The document outlines the history and procedure of shotgun sequencing. Genomic DNA is first fragmented, end-repaired, and size-selected into small, medium, and large fragments. Libraries are created for each size fragment and sequenced. A base caller filters poor calls and an assembler finds overlaps to generate continuous nucleotide sequences or contigs of the whole genome.
An open reading frame (ORF) is a part of a reading frame that contains no stop codons. ORFs are used as evidence to identify potential protein-coding genes in DNA sequences. The presence of a long ORF with codon usage matching the organism is used by some gene prediction algorithms to identify candidate protein-coding regions, but an ORF alone is not conclusive proof that a gene exists. Tools like ORF Finder, ORF Investigator, and ORF Predictor can be used to locate ORFs in DNA sequences.
This document discusses yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs). YACs are engineered chromosomes derived from yeast DNA that can clone very large DNA sequences in yeast cells of up to 1 megabase. BACs are cloning vectors derived from bacterial DNA that can clone DNA fragments of up to 300 kilobases in E. coli. Both systems allow cloning and propagation of large DNA fragments, but YACs can hold more DNA while BACs are more stable and better for functional analysis in mammalian cells.
Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.
The document discusses various computational methods for predicting the three-dimensional structure of proteins from their amino acid sequences. It describes homology modeling, which predicts structures based on known protein structural templates that share sequence homology. It also covers threading/fold recognition and ab initio modeling, which predict structures without templates by using physicochemical principles or energy minimization approaches. Key steps and programs used in each method are outlined.
This document discusses different types of DNA libraries and methods for screening libraries to identify clones containing genes of interest. It describes genomic and cDNA libraries, noting that genomic libraries contain all DNA fragments from an organism's genome while cDNA libraries contain only coding sequences. The key screening methods discussed are colony/plaque hybridization using radiolabeled probes, expression screening using antibodies, and PCR screening using gene-specific primers.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Sequence alig Sequence Alignment Pairwise alignment:-naveed ul mushtaq
Sequence Alignment Pairwise alignment:- Global Alignment and Local AlignmentTwo types of alignment Progressive Programs for multiple sequence alignment BLOSUM Point accepted mutation (PAM)PAM VS BLOSUM
Whole genome shotgun sequencing involves randomly breaking genomic DNA into small fragments, sequencing the fragments, and then reassembling the sequences using overlapping regions. The document outlines the history and procedure of shotgun sequencing. Genomic DNA is first fragmented, end-repaired, and size-selected into small, medium, and large fragments. Libraries are created for each size fragment and sequenced. A base caller filters poor calls and an assembler finds overlaps to generate continuous nucleotide sequences or contigs of the whole genome.
An open reading frame (ORF) is a part of a reading frame that contains no stop codons. ORFs are used as evidence to identify potential protein-coding genes in DNA sequences. The presence of a long ORF with codon usage matching the organism is used by some gene prediction algorithms to identify candidate protein-coding regions, but an ORF alone is not conclusive proof that a gene exists. Tools like ORF Finder, ORF Investigator, and ORF Predictor can be used to locate ORFs in DNA sequences.
This document discusses yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs). YACs are engineered chromosomes derived from yeast DNA that can clone very large DNA sequences in yeast cells of up to 1 megabase. BACs are cloning vectors derived from bacterial DNA that can clone DNA fragments of up to 300 kilobases in E. coli. Both systems allow cloning and propagation of large DNA fragments, but YACs can hold more DNA while BACs are more stable and better for functional analysis in mammalian cells.
Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.
The document discusses various computational methods for predicting the three-dimensional structure of proteins from their amino acid sequences. It describes homology modeling, which predicts structures based on known protein structural templates that share sequence homology. It also covers threading/fold recognition and ab initio modeling, which predict structures without templates by using physicochemical principles or energy minimization approaches. Key steps and programs used in each method are outlined.
This document discusses different types of DNA libraries and methods for screening libraries to identify clones containing genes of interest. It describes genomic and cDNA libraries, noting that genomic libraries contain all DNA fragments from an organism's genome while cDNA libraries contain only coding sequences. The key screening methods discussed are colony/plaque hybridization using radiolabeled probes, expression screening using antibodies, and PCR screening using gene-specific primers.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Sequence alig Sequence Alignment Pairwise alignment:-naveed ul mushtaq
Sequence Alignment Pairwise alignment:- Global Alignment and Local AlignmentTwo types of alignment Progressive Programs for multiple sequence alignment BLOSUM Point accepted mutation (PAM)PAM VS BLOSUM
Site-directed mutagenesis is a technique used to generate specific mutations in DNA at predetermined locations. It involves using a synthetic oligonucleotide primer containing the desired mutation to introduce changes into the DNA sequence during in vitro DNA replication or PCR. This allows researchers to study the effects of mutations and engineer proteins with improved or customized properties. Common methods for site-directed mutagenesis include using single or double primers, cassette mutagenesis by replacing DNA fragments, and PCR-based mutagenesis. The technique has various applications in investigating protein function and developing proteins for commercial uses.
ESTs are short sequences of DNA that represent genes expressed in certain tissues or organisms. They provide a quick and inexpensive way for scientists to discover new genes and map their positions in genomes. ESTs represent a snapshot of genes expressed in a tissue at a given time. Sequencing the beginning or end of cDNA clones produces 5' and 3' ESTs, which can help identify genes and study gene expression and regulation.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Automated sequencing of genomes require automated gene assignment
Includes detection of open reading frames (ORFs)
Identification of the introns and exons
Gene prediction a very difficult problem in pattern recognition
Coding regions generally do not have conserved sequences
Much progress made with prokaryotic gene prediction
Eukaryotic genes more difficult to predict correctly
DNA microarrays allow analysis of gene expression across thousands of genes simultaneously. They consist of DNA probes attached to a solid surface in an organized grid pattern, with each spot representing a single gene. Samples are labeled with fluorescent dyes and hybridized to the chip. Complementary sequences pair via hydrogen bonds, while non-specific sequences are washed away. The signal intensity at each spot indicates the amount of target sequence present and thus gene expression levels. DNA microarrays have applications in clinical diagnosis, drug discovery, and other fields by profiling gene expression patterns.
Genomic and cDNA libraries are constructed to isolate genes of interest from organisms. Genomic libraries contain total chromosomal DNA while cDNA libraries contain mRNA from specific cell types. DNA is digested and ligated into vectors to clone fragments. Libraries are screened using probes and PCR to identify clones containing genes of interest. cDNA libraries are useful for studying eukaryotic gene expression as they contain mRNA from specific cells. Thousands of clones may need to be screened to have high probability of isolating a particular gene fragment.
Artificial chromosomes are synthetic chromosomes introduced into host cells to propagate and transfect DNA fragments larger than plasmids can hold. Yeast artificial chromosomes (YACs) specifically are engineered chromosomes derived from yeast DNA ligated into bacterial plasmids, allowing insertion of 100-1000kb DNA fragments. YACs contain elements for yeast and bacterial replication and selection, and are useful for cloning large genomic fragments like whole human genes for mapping the genome.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
This document discusses sequence alignment methods. It describes global and local alignment, and algorithms used for alignment including dot matrix analysis, dynamic programming, and word/k-tuple methods as implemented in FASTA and BLAST programs. BLAST and FASTA are described as popular tools for sequence database searches that use heuristic methods and word matching to quickly identify regions of local similarity.
Microarray technology allows researchers to analyze the expression levels of thousands of genes simultaneously using DNA probes attached to a solid surface. There are two main types of microarrays: glass cDNA microarrays which involve spotting pre-fabricated cDNA fragments on glass slides; and high-density oligonucleotide arrays which involve the in situ synthesis of oligonucleotides on a chip. The key steps in a microarray experiment are sample preparation and labeling, hybridization of labeled cDNA to the probes, washing, and image analysis to quantify gene expression levels. Microarrays have numerous applications including gene expression profiling, comparative genomics, disease diagnosis, drug discovery, and toxicology research.
protein structure prediction methods. homology modelling, fold recognition, threading, ab initio methods. in short and easy form slides. after one time read you can easily understand methods for protein structure prediction.
TrEMBL is a computer-annotated protein sequence database created by Rolf Apweiler that contains translations of coding sequences from nucleotide databases like EMBL and GenBank as well as protein sequences from literature or submitted directly. The database provides automated classification and annotation to enrich the protein sequences.
This document discusses the use of 16S ribosomal RNA (rRNA) gene sequencing for bacterial identification and phylogenetic analysis. It explains that the 16S rRNA gene is highly conserved, making it useful for comparing distantly related organisms. The document outlines the process of 16S rRNA gene sequencing, including PCR amplification using conserved primer regions and sequencing of variable regions. It also discusses various methods that have been developed using 16S rRNA, such as TRFLP profiling and ribotyping, to study microbial communities.
Shotgun metagenomics sequencing allows researchers to comprehensively sample all genes in organisms present in a complex sample without culturing. This provides insights into bacterial diversity, abundance, and uncultured microbes. Bioinformatics pipelines guide analysis including quality filtering, assembly, binning, gene finding, fingerprinting, and phylogeny/diversity modeling to understand communities. Metagenomics has applications in antibiotic/drug discovery, bioremediation, agriculture, human microbiome mapping, and more. Tools like QIIME, Mothur, MEGAN, and MG-RAST facilitate large-scale metagenomic analysis.
The DNA Data Bank of Japan (DDBJ) is a biological database located in Japan that collects and stores nucleotide sequence data. It began operations in 1986 and exchanges data daily with the European Nucleotide Archive and GenBank to form the International Nucleotide Sequence Database Collaboration (INSDC). DDBJ accepts sequence submissions from researchers worldwide and assigns unique identification numbers to published sequences to recognize intellectual property rights. It also provides search and analysis tools and supercomputing resources to support genomic research.
Immunoprecipitation is a technique used to isolate a protein of interest from a complex protein mixture using an antibody that specifically binds to that protein. The key steps involve lysing cells, incubating the sample with the target antibody, precipitating the antibody-protein complex, washing away non-specific bindings, and then analyzing the isolated proteins. Immunoprecipitation can be used to study protein-protein interactions, identify proteins in complexes, and enrich low abundance proteins for further analysis.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
This document discusses various methods for ligating DNA fragments, including blunt end ligation, sticky end ligation using linkers or adaptors, and homopolymeric tailing. Blunt end ligation is less efficient than sticky end ligation. Linkers and adaptors are oligonucleotides used to create sticky ends for ligation, while homopolymeric tailing uses terminal transferase to add homopolymer tails to blunt ends before ligation. The goal is to efficiently join vector and insert DNA fragments for recombinant DNA construction.
SAGE (Serial analysis of Gene Expression)talhakhat
SAGE (Serial Analysis of Gene Expression) is a technique that allows for the rapid and comprehensive analysis of gene expression patterns in a given cell population. It works by isolating mRNA, synthesizing cDNA, ligating short sequence tags to the cDNA, and then counting the number of times each tag is observed to quantify gene expression levels. The tags are concatenated and sequenced to generate vast amounts of data that must be analyzed computationally to identify which genes particular tags correspond to and to compare expression profiles between cell types. SAGE provides an overview of a cell's complete transcriptional activity and has been applied to study differences in cancer vs normal cells and to identify targets of oncogenes and tumor suppressor genes.
Design of fragment screening libraries (Feb 2010 version)Peter Kenny
I have lectured on design of fragment screening libraries a number of times and, to be honest, my material is getting a bit dated. This presentation is from Feb 2010 when I was visiting CSIRO and the photo in the title slide was taken in Tierra del Fuego.
Site-directed mutagenesis is a technique used to generate specific mutations in DNA at predetermined locations. It involves using a synthetic oligonucleotide primer containing the desired mutation to introduce changes into the DNA sequence during in vitro DNA replication or PCR. This allows researchers to study the effects of mutations and engineer proteins with improved or customized properties. Common methods for site-directed mutagenesis include using single or double primers, cassette mutagenesis by replacing DNA fragments, and PCR-based mutagenesis. The technique has various applications in investigating protein function and developing proteins for commercial uses.
ESTs are short sequences of DNA that represent genes expressed in certain tissues or organisms. They provide a quick and inexpensive way for scientists to discover new genes and map their positions in genomes. ESTs represent a snapshot of genes expressed in a tissue at a given time. Sequencing the beginning or end of cDNA clones produces 5' and 3' ESTs, which can help identify genes and study gene expression and regulation.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Automated sequencing of genomes require automated gene assignment
Includes detection of open reading frames (ORFs)
Identification of the introns and exons
Gene prediction a very difficult problem in pattern recognition
Coding regions generally do not have conserved sequences
Much progress made with prokaryotic gene prediction
Eukaryotic genes more difficult to predict correctly
DNA microarrays allow analysis of gene expression across thousands of genes simultaneously. They consist of DNA probes attached to a solid surface in an organized grid pattern, with each spot representing a single gene. Samples are labeled with fluorescent dyes and hybridized to the chip. Complementary sequences pair via hydrogen bonds, while non-specific sequences are washed away. The signal intensity at each spot indicates the amount of target sequence present and thus gene expression levels. DNA microarrays have applications in clinical diagnosis, drug discovery, and other fields by profiling gene expression patterns.
Genomic and cDNA libraries are constructed to isolate genes of interest from organisms. Genomic libraries contain total chromosomal DNA while cDNA libraries contain mRNA from specific cell types. DNA is digested and ligated into vectors to clone fragments. Libraries are screened using probes and PCR to identify clones containing genes of interest. cDNA libraries are useful for studying eukaryotic gene expression as they contain mRNA from specific cells. Thousands of clones may need to be screened to have high probability of isolating a particular gene fragment.
Artificial chromosomes are synthetic chromosomes introduced into host cells to propagate and transfect DNA fragments larger than plasmids can hold. Yeast artificial chromosomes (YACs) specifically are engineered chromosomes derived from yeast DNA ligated into bacterial plasmids, allowing insertion of 100-1000kb DNA fragments. YACs contain elements for yeast and bacterial replication and selection, and are useful for cloning large genomic fragments like whole human genes for mapping the genome.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
This document discusses sequence alignment methods. It describes global and local alignment, and algorithms used for alignment including dot matrix analysis, dynamic programming, and word/k-tuple methods as implemented in FASTA and BLAST programs. BLAST and FASTA are described as popular tools for sequence database searches that use heuristic methods and word matching to quickly identify regions of local similarity.
Microarray technology allows researchers to analyze the expression levels of thousands of genes simultaneously using DNA probes attached to a solid surface. There are two main types of microarrays: glass cDNA microarrays which involve spotting pre-fabricated cDNA fragments on glass slides; and high-density oligonucleotide arrays which involve the in situ synthesis of oligonucleotides on a chip. The key steps in a microarray experiment are sample preparation and labeling, hybridization of labeled cDNA to the probes, washing, and image analysis to quantify gene expression levels. Microarrays have numerous applications including gene expression profiling, comparative genomics, disease diagnosis, drug discovery, and toxicology research.
protein structure prediction methods. homology modelling, fold recognition, threading, ab initio methods. in short and easy form slides. after one time read you can easily understand methods for protein structure prediction.
TrEMBL is a computer-annotated protein sequence database created by Rolf Apweiler that contains translations of coding sequences from nucleotide databases like EMBL and GenBank as well as protein sequences from literature or submitted directly. The database provides automated classification and annotation to enrich the protein sequences.
This document discusses the use of 16S ribosomal RNA (rRNA) gene sequencing for bacterial identification and phylogenetic analysis. It explains that the 16S rRNA gene is highly conserved, making it useful for comparing distantly related organisms. The document outlines the process of 16S rRNA gene sequencing, including PCR amplification using conserved primer regions and sequencing of variable regions. It also discusses various methods that have been developed using 16S rRNA, such as TRFLP profiling and ribotyping, to study microbial communities.
Shotgun metagenomics sequencing allows researchers to comprehensively sample all genes in organisms present in a complex sample without culturing. This provides insights into bacterial diversity, abundance, and uncultured microbes. Bioinformatics pipelines guide analysis including quality filtering, assembly, binning, gene finding, fingerprinting, and phylogeny/diversity modeling to understand communities. Metagenomics has applications in antibiotic/drug discovery, bioremediation, agriculture, human microbiome mapping, and more. Tools like QIIME, Mothur, MEGAN, and MG-RAST facilitate large-scale metagenomic analysis.
The DNA Data Bank of Japan (DDBJ) is a biological database located in Japan that collects and stores nucleotide sequence data. It began operations in 1986 and exchanges data daily with the European Nucleotide Archive and GenBank to form the International Nucleotide Sequence Database Collaboration (INSDC). DDBJ accepts sequence submissions from researchers worldwide and assigns unique identification numbers to published sequences to recognize intellectual property rights. It also provides search and analysis tools and supercomputing resources to support genomic research.
Immunoprecipitation is a technique used to isolate a protein of interest from a complex protein mixture using an antibody that specifically binds to that protein. The key steps involve lysing cells, incubating the sample with the target antibody, precipitating the antibody-protein complex, washing away non-specific bindings, and then analyzing the isolated proteins. Immunoprecipitation can be used to study protein-protein interactions, identify proteins in complexes, and enrich low abundance proteins for further analysis.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
This document discusses various methods for ligating DNA fragments, including blunt end ligation, sticky end ligation using linkers or adaptors, and homopolymeric tailing. Blunt end ligation is less efficient than sticky end ligation. Linkers and adaptors are oligonucleotides used to create sticky ends for ligation, while homopolymeric tailing uses terminal transferase to add homopolymer tails to blunt ends before ligation. The goal is to efficiently join vector and insert DNA fragments for recombinant DNA construction.
SAGE (Serial analysis of Gene Expression)talhakhat
SAGE (Serial Analysis of Gene Expression) is a technique that allows for the rapid and comprehensive analysis of gene expression patterns in a given cell population. It works by isolating mRNA, synthesizing cDNA, ligating short sequence tags to the cDNA, and then counting the number of times each tag is observed to quantify gene expression levels. The tags are concatenated and sequenced to generate vast amounts of data that must be analyzed computationally to identify which genes particular tags correspond to and to compare expression profiles between cell types. SAGE provides an overview of a cell's complete transcriptional activity and has been applied to study differences in cancer vs normal cells and to identify targets of oncogenes and tumor suppressor genes.
Design of fragment screening libraries (Feb 2010 version)Peter Kenny
I have lectured on design of fragment screening libraries a number of times and, to be honest, my material is getting a bit dated. This presentation is from Feb 2010 when I was visiting CSIRO and the photo in the title slide was taken in Tierra del Fuego.
Genomic DNA libraries contain representative copies of all DNA fragments in an organism's genome, including both expressed and non-expressed sequences. They are constructed by isolating genomic DNA, fragmenting it, and cloning the fragments into suitable vectors like lambda phage or BACs. cDNA libraries contain only expressed sequences, as they are constructed by isolating mRNA from tissues, reverse transcribing it to cDNA, and cloning the cDNA fragments. Both library types are useful for gene discovery, sequencing, mapping genomes, and studying regulatory sequences.
DNA Fingerprinting (AFLP, RFLP, RAPD) & Its advntages and applicationSwaatiSharma2
This document discusses three different DNA fingerprinting techniques: AFLP, RAPD, and RFLP. AFLP involves digesting genomic DNA with restriction enzymes, ligating adaptors, selective amplification of fragments, and analysis via electrophoresis. RAPD uses random primers in PCR to amplify random DNA fragments. RFLP involves restriction enzyme digestion of DNA, gel electrophoresis, Southern blotting, and detection using radioactive probes.
AFLP is a DNA fingerprinting technique that detects polymorphisms across the entire genome. It combines restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) methods. The AFLP process involves digesting genomic DNA with restriction enzymes, ligating adapters to the fragments, selectively amplifying subsets of fragments via PCR using adapter-specific primers with attached selective nucleotides, and separating the amplified fragments on a gel. This technique generates 50-100 restriction fragments per sample and is highly reproducible, sensitive, and applicable to any organism, making it widely used for developing polymorphic markers and genetic maps.
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.
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
This document discusses methods for constructing antibody scFv fragment libraries displayed on phage (phage display scFv libraries) for stability selection. It describes several strategies for PCR amplification and assembly of variable heavy (VH) and light (VL) chain domains connected by a linker to produce single-chain Fv fragments (scFvs). These include normal PCR, pull-through PCR, overlap extension PCR, and RACE PCR strategies. It then provides a basic protocol for constructing naive scFv libraries using peripheral blood mononuclear cells, including steps for VH and VL repertoire construction and assembly into a phagemid vector for phage display.
This document provides an overview of RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). It describes that RNA-Seq is used to profile transcriptomes and determine gene expression levels, while ChIP-Seq identifies the binding sites of DNA-associated proteins. The key steps of RNA-Seq are RNA preparation, library preparation, sequencing, and analysis to map reads, detect isoforms and expression levels. ChIP-Seq combines chromatin immunoprecipitation with sequencing to precisely map global binding sites of proteins of interest to understand gene regulation. Both techniques provide high-quality, genome-wide data with low input requirements compared to previous methods.
The document summarizes the steps taken in a cloning simulation project to clone the erythropoietin gene. Key steps included obtaining the gene sequence from GenBank, analyzing it to find the ORF and restriction sites, designing primers for PCR, cloning the gene into a TOPO vector, transforming E. coli cells, screening clones by PCR, and purifying the expressed protein using nickel affinity chromatography and SDS-PAGE.
This pdf is about the DNA Libraries / Genomic DNA vs cDNA.
For more details visit on YouTube; @SELF-EXPLANATORY; https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos Thanks...!
- Molecular markers are segments of DNA that represent genetic differences and can be used for genetic analysis, though they may not correlate with observable traits.
- An ideal molecular marker technique should be polymorphic, provide adequate genetic resolution, generate multiple independent markers simply and inexpensively using small amounts of DNA, and be linked to distinct phenotypes.
- Molecular marker techniques can be categorized as non-PCR based like RFLP analysis or PCR-based like AFLP, RAPD, and SNP analysis, which have been widely used in plant and animal research.
Next generation sequencing techniques were discussed including an overview of various sequencing platforms, their output, and common analysis workflows. Mapping short reads to reference genomes using alignment programs is a key first step for most applications. Formats like FASTQ, SAM, and BAM are commonly used to store sequencing reads and mapping results.
ChIP-seq is a technique to identify where proteins bind to DNA in the genome. It involves cross-linking proteins to DNA in cells, fragmenting the DNA, immunoprecipitating the protein-DNA complexes using an antibody for the protein of interest, and then sequencing the retrieved DNA. This allows mapping of the genomic binding sites for the protein. The document discusses experimental design considerations for ChIP-seq, such as antibody choice and controls. It also reviews data analysis steps including read mapping, peak calling to identify enriched regions, and downstream analyses like motif finding. Higher resolution techniques like ChIP-exo are also introduced that can identify protein binding sites at base pair level.
This document provides an improved protocol for preparing Nextera Mate Pair libraries with the following key modifications:
1) Optimizing tagmentation and Covaris shearing conditions to increase the yield of DNA fragments in the targeted size range.
2) Reducing volumes in library preparation steps to decrease usage of costly reagents while maintaining performance.
3) Recommending sequencing strategies like read lengths that maximize the proportion of read pairs containing junction adaptors, important for scaffolding.
This document provides an overview and introduction to RNA-seq analysis using Next Generation Sequencing. It discusses the RNA-seq workflow including mapping reads with TopHat2, transcript assembly with Cufflinks, and differential expression analysis. Key points covered include the advantages of RNA-seq over microarrays, the exponential drop in sequencing costs, mapping strategies for junction reads including TopHat, and running TopHat from the command line.
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Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
RECOMBINATION MOLECULAR BIOLOGY PPT UPDATED new.pptxSabahat Ali
This ppt is about recombination and where it occurs. Types of recombination and models of recombination along with many factors in prokaryotic and eukaryotic recombination
Good laboratory practices in a pharmaceutical lab 1Sabahat Ali
This document discusses good laboratory practices in a pharmaceutical lab. It outlines the members of a group project on this topic and provides an introduction to pharmaceutical lab testing. It then covers topics like GMP, GLP, quality control, quality assurance, reducing human errors, and the scope of QA and QC in a pharmaceutical lab. Key points include that pharmaceutical labs test raw materials, finished products, and conduct validation, stability, and analytical method development testing. GMP and GLP aim to minimize risks and ensure consistent quality production. QA and QC work to guarantee drug quality and safety at all stages from development to sales.
Degradation of PLA at Mesophillic and thermophillic conditionsSabahat Ali
This document summarizes research on the degradation of polylactic acid (PLA) under mesophilic and thermophilic conditions. Key findings include:
1) Mesophilic bacteria like Pseudomonas geniculata and Streptomyces pavanii were found to degrade PLA films at 25-40°C, with S. pavanii showing higher degradation.
2) PLA degradation was higher under thermophilic (41-122°C) conditions compared to mesophilic (20-45°C) due to PLA-degrading enzymes working best at high temperatures. Up to 90% of PLA weight loss was observed at thermophilic temperatures within 12 days of
Life cycle Assesment and waste stratigies of PLASabahat Ali
Group 2 presented on strategies for polylactic acid (PLA) waste, including recycling and biodegradation. There are three main routes for producing PLA: polymerization of lactic acid monomers, condensation of lactic acid, and fermentation. PLA can be chemically recycled through hydrolytic or alcoholytic depolymerization. An innovative process called the Zeus Waste PLA Depolymerization Process uses solvents like chloroform and alcohols like methanol at low temperatures to break PLA down into its original lactic acid monomers. PLA biodegrades through hydrolysis of ester bonds, thermal degradation, and photodegradation when exposed to sunlight.
Environmental biodegradation of PLA by Biotic and Abiotic factorsSabahat Ali
PLA is a biodegradable polymer that can degrade through both biotic and abiotic factors in the environment. Biotic degradation occurs through the action of microorganisms like bacteria and fungi that produce enzymes to break down PLA. Specific bacteria identified to degrade PLA include species of Pseudomonas and Streptomyces. Fungal degradation is also possible, with Phanerochaete chrysosporium shown to effectively degrade PLA. Abiotic degradation happens through hydrolysis when water breaks the ester bonds of PLA, which is accelerated at higher temperatures and pH levels.
The document discusses energy expenditure and basal metabolic rate (BMR). It defines energy expenditure as the amount of energy needed for bodily functions like breathing and circulation, while BMR is the minimum energy required for essential physiological processes when at rest. The document outlines several factors that affect BMR, such as age, gender, weight, and thyroid function. Maintaining caloric balance between intake and expenditure through diet and exercise can prevent weight gain.
Agriculture applications of nanobiotechnologySabahat Ali
This document discusses the potential applications of nanobiotechnology in agriculture. It begins by introducing how nanoparticles can interact with agricultural hosts and tissues. It then discusses several specific applications, including using nanoparticles for plant disease management and diagnostics, as well as for delivering pesticides, nutrients, and plant hormones. The document also notes potential applications in areas like recycling agricultural waste, soil improvement, water purification, and plant breeding. It acknowledges both the promise and challenges of nanotechnology for modernizing agriculture to address issues like increasing food supply to support population growth amid changing environmental conditions.
Macronutrients provide energy and are essential for growth and maintenance of the body. The document discusses the three main macronutrients - carbohydrates, proteins, and fats. Carbohydrates are divided into simple and complex categories, with simple carbs like sugars providing quick energy and complex carbs like whole grains being more filling and nutritious. Proteins are essential building blocks and energy sources, with animal products providing complete proteins and plant sources providing complementary proteins when combined. Fats serve various functions in the body and are classified based on their structure.
The document discusses methods to enhance the biodegradation of polylactic acid (PLA). It analyzes modifications to PLA's physical properties and amending the environment with various factors like stimulants. It summarizes that biodegradation of PLA mainly occurs through hydrolysis of ester bonds and is induced by microorganisms like certain actinomycetes, bacteria, and fungi. Key factors like temperature, pH, humidity, and oxygen levels also affect the degradation rate. While PLA is biodegradable, the process is often slow under natural conditions.
Alzhemier's disease and koraskoff syndromeSabahat Ali
Alzheimer's disease, Korsakoff's syndrome, and dreaming are compared and contrasted. Alzheimer's disease results from neuronal death and synapse loss, causing memory loss and dementia. Korsakoff's syndrome is caused by thiamine deficiency and can be reversed if treated early. Dreams occur during REM sleep and may help with memory consolidation. Both diseases involve memory loss and neuronal/synaptic changes, while dreaming is a normal process that occurs during sleep and differs in its effects on memory and brain activity.
Nerve cells, Nervous communication & its link to the celllular signallingSabahat Ali
The document discusses the structure and function of neurons. It notes that neurons are specialized cells that communicate via electrical and chemical signals. They contain dendrites that receive signals, a cell body, and an axon that transmits signals. At synapses, chemical neurotransmitters transmit signals between neurons or to other cell types. Neurons form circuits that allow for complex coordinated responses. The action potential involves changes in ion channel permeability that propagate electrical signals rapidly along axons. Calcium acts as an important intracellular messenger in neurons and other cell types, often working through the calcium sensor protein calmodulin.
Peptide hormones and catecholamines allow for rapid responses to environmental changes. They are stored in secretory vesicles and released via exocytosis within seconds or minutes in response to stimulation. This causes short-term effects that are terminated once the hormones are degraded. In contrast, steroid hormones and thyroid hormones are synthesized from cholesterol or thyroglobulin precursors within cells. They diffuse out of cells and circulate in the blood bound to carrier proteins. This allows their effects to last longer, from hours to days, but production and release takes longer than for peptide hormones and catecholamines. The different hormone types thus allow for both rapid short-term responses and longer-term regulatory effects.
Cells in multicellular organisms communicate through elaborate signaling networks involving hundreds of signaling molecules. These molecules allow cells to regulate development, growth, and coordinated function. Signaling occurs through paracrine, synaptic, and endocrine mechanisms using molecules like hormones, neurotransmitters, and growth factors. Target cells contain receptors that recognize signaling molecules with high specificity and affinity. While some responses are rapid, others involve long-term changes through regulated synthesis, release, and degradation of signaling compounds.
Folding depends upon sequence of Amino Acids not the Composition. Folding starts with the secondary structure and ends at quaternary structure.
Denaturation occur at secondary, tertiary & quaternary level but not at primary level.
Tertiary Structure basically of Hydrophobic interactions, (interactions in side chains), hydrogen bonding, salt bridges, Vander Waals interactions.
e.g. Globular proteins & Fibrous Proteins
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 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.
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.
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.
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.
Evidence of Jet Activity from the Secondary Black Hole in the OJ 287 Binary S...Sérgio Sacani
Wereport the study of a huge optical intraday flare on 2021 November 12 at 2 a.m. UT in the blazar OJ287. In the binary black hole model, it is associated with an impact of the secondary black hole on the accretion disk of the primary. Our multifrequency observing campaign was set up to search for such a signature of the impact based on a prediction made 8 yr earlier. The first I-band results of the flare have already been reported by Kishore et al. (2024). Here we combine these data with our monitoring in the R-band. There is a big change in the R–I spectral index by 1.0 ±0.1 between the normal background and the flare, suggesting a new component of radiation. The polarization variation during the rise of the flare suggests the same. The limits on the source size place it most reasonably in the jet of the secondary BH. We then ask why we have not seen this phenomenon before. We show that OJ287 was never before observed with sufficient sensitivity on the night when the flare should have happened according to the binary model. We also study the probability that this flare is just an oversized example of intraday variability using the Krakow data set of intense monitoring between 2015 and 2023. We find that the occurrence of a flare of this size and rapidity is unlikely. In machine-readable Tables 1 and 2, we give the full orbit-linked historical light curve of OJ287 as well as the dense monitoring sample of Krakow.
Candidate young stellar objects in the S-cluster: Kinematic analysis of a sub...Sérgio Sacani
Context. The observation of several L-band emission sources in the S cluster has led to a rich discussion of their nature. However, a definitive answer to the classification of the dusty objects requires an explanation for the detection of compact Doppler-shifted Brγ emission. The ionized hydrogen in combination with the observation of mid-infrared L-band continuum emission suggests that most of these sources are embedded in a dusty envelope. These embedded sources are part of the S-cluster, and their relationship to the S-stars is still under debate. To date, the question of the origin of these two populations has been vague, although all explanations favor migration processes for the individual cluster members. Aims. This work revisits the S-cluster and its dusty members orbiting the supermassive black hole SgrA* on bound Keplerian orbits from a kinematic perspective. The aim is to explore the Keplerian parameters for patterns that might imply a nonrandom distribution of the sample. Additionally, various analytical aspects are considered to address the nature of the dusty sources. Methods. Based on the photometric analysis, we estimated the individual H−K and K−L colors for the source sample and compared the results to known cluster members. The classification revealed a noticeable contrast between the S-stars and the dusty sources. To fit the flux-density distribution, we utilized the radiative transfer code HYPERION and implemented a young stellar object Class I model. We obtained the position angle from the Keplerian fit results; additionally, we analyzed the distribution of the inclinations and the longitudes of the ascending node. Results. The colors of the dusty sources suggest a stellar nature consistent with the spectral energy distribution in the near and midinfrared domains. Furthermore, the evaporation timescales of dusty and gaseous clumps in the vicinity of SgrA* are much shorter ( 2yr) than the epochs covered by the observations (≈15yr). In addition to the strong evidence for the stellar classification of the D-sources, we also find a clear disk-like pattern following the arrangements of S-stars proposed in the literature. Furthermore, we find a global intrinsic inclination for all dusty sources of 60 ± 20◦, implying a common formation process. Conclusions. The pattern of the dusty sources manifested in the distribution of the position angles, inclinations, and longitudes of the ascending node strongly suggests two different scenarios: the main-sequence stars and the dusty stellar S-cluster sources share a common formation history or migrated with a similar formation channel in the vicinity of SgrA*. Alternatively, the gravitational influence of SgrA* in combination with a massive perturber, such as a putative intermediate mass black hole in the IRS 13 cluster, forces the dusty objects and S-stars to follow a particular orbital arrangement. Key words. stars: black holes– stars: formation– Galaxy: center– galaxies: star formation
Sexuality - Issues, Attitude and Behaviour - Applied Social Psychology - Psyc...PsychoTech 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!
2. Introduction:
• NEBcutter accepts an input sequence, which can be pasted in, picked
up from a local file or retrieved from NCBI as a GenBank file via its
accession number. Various options are available to select the size of
ORFs to be displayed and the set of restriction enzymes to be used.
The program calculates the positions of all restriction enzyme sites
noting those that might potentially be blocked by overlapping
methylation and finds the ORFs in the sequence. It then displays a
schematic diagram of the sequence, the long ORFs, based on the
rules described in the Methods and all restriction enzymes that cut it
just once.
2
13. • How to calculate the size of fragments produced by restriction
digestion?
Subtracting the initial base number from ending base number and
then Adding one.
For example: BamHI Finds its restriction sites at 5506 bp and 22346bp
so the size of its fragment will be 16840bp as explained below:
BamHI-BamHI
5506-22346
22346-5506=16840
16840+1=16841bp
13
Editor's Notes
This digestion involves digesting the sequence with all the available enzymes on NEB.
The required enzymes are selected by viewing their restriction sites and other characteristics from the list of available enzymes,more than one or two enzymes can be selected from the list.