How to design a DNA primer on NCBI.pptx
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Designing a nucleotide primer using bioinformatic tools on NCBI could be a good way to decide which primer to design for gene amplification. This presentation was designed for computational biology students at UPES to give a step by step method for designing a Primer on NCBI.
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DNA manipulating enzymes include nucleases, ligases, polymerases and modifying enzymes. Restriction endonucleases are enzymes that cut DNA molecules at specific recognition sequences. They produce either blunt or sticky ends. Restriction endonucleases are classified as type I, II or III, with type II having widespread use in recombinant DNA technology by allowing precise cutting of DNA molecules at specific sites.
Gene cloning strategies
Gene cloning strategies depend on whether genomic or cDNA libraries are being constructed. Shotgun cloning is used to construct genomic libraries by fragmenting genomic DNA and inserting all fragments into vectors at once. cDNA libraries are constructed by reverse transcribing mRNA to cDNA, which is then cloned into vectors. Both library types are screened to identify overlapping clones that are assembled into contigs representing the entire genome.
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Pyrosequencing is a sequencing method that detects DNA polymerase activity by measuring the release of pyrophosphate using a cascade of enzymatic reactions that generate visible light. It utilizes emulsion PCR to amplify DNA fragments on beads in microreactors. The beads are then loaded into wells and sequenced by sequentially adding nucleotides and detecting light produced upon incorporation using a CCD camera. Key advantages are its accuracy, high throughput of up to 48,000 probes per day, and ease of automation. However, it requires specialized equipment and software.
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Genome sequencing
The document discusses genome sequencing and related topics. It begins by defining what a genome is - the complete set of DNA in an organism. It then discusses the different types of genomes, such as prokaryotic and eukaryotic, including nuclear, mitochondrial, and chloroplast genomes. The document also defines genomics as the comprehensive study of whole genomes and all gene interactions, distinguishing it from traditional genetics which focuses on single genes. It outlines some key milestones in genomic sequencing and the technical foundations that enabled sequencing whole genomes. Finally, it describes the main approaches used for genome sequencing projects, including hierarchical shotgun sequencing and whole genome shotgun sequencing.
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cDNA Library Construction
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DNA microarray
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.
Recommended
DNA manipulation
DNA manipulating enzymes include nucleases, ligases, polymerases and modifying enzymes. Restriction endonucleases are enzymes that cut DNA molecules at specific recognition sequences. They produce either blunt or sticky ends. Restriction endonucleases are classified as type I, II or III, with type II having widespread use in recombinant DNA technology by allowing precise cutting of DNA molecules at specific sites.
Gene cloning strategies
Gene cloning strategies depend on whether genomic or cDNA libraries are being constructed. Shotgun cloning is used to construct genomic libraries by fragmenting genomic DNA and inserting all fragments into vectors at once. cDNA libraries are constructed by reverse transcribing mRNA to cDNA, which is then cloned into vectors. Both library types are screened to identify overlapping clones that are assembled into contigs representing the entire genome.
Pyrosequencing
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Asymetric -PCR
What is PCR
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Types of PCR
Asymmetric PCR
Applications of PCR
Advantages of PCR
Limitations of PCR
DNA Template
Primers
Taq polymerase
Deoxynucleoside
triphosphates(dNTPs)
Buffer solution
Divalent cations(eg.Mg2+ )
Genome sequencing
The document discusses genome sequencing and related topics. It begins by defining what a genome is - the complete set of DNA in an organism. It then discusses the different types of genomes, such as prokaryotic and eukaryotic, including nuclear, mitochondrial, and chloroplast genomes. The document also defines genomics as the comprehensive study of whole genomes and all gene interactions, distinguishing it from traditional genetics which focuses on single genes. It outlines some key milestones in genomic sequencing and the technical foundations that enabled sequencing whole genomes. Finally, it describes the main approaches used for genome sequencing projects, including hierarchical shotgun sequencing and whole genome shotgun sequencing.
Pyrosequencing
Pyrosequencing is a sequencing by synthesis technique that uses a luciferase enzyme system to monitor DNA synthesis. It works by adding DNA polymerase and a single nucleotide to the DNA fragments, generating pyrophosphate that is converted to light. The light is detected and identifies the nucleotide incorporated. Pyrosequencing has applications in cDNA analysis, mutation detection, re-sequencing of disease genes, and identifying single nucleotide polymorphisms and typing bacteria and viruses.
cDNA Library Construction
Creation of a cDNA library starts with mRNA instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA (i.e., cDNA). A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.
DNA microarray
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.
Primer design
Primers are short DNA sequences used to initiate DNA replication via polymerase chain reaction (PCR). Good primer design is important for PCR to work properly. Primers should be 18-24 base pairs in length and have a GC content and melting temperature that allows for specific annealing. Software tools can help design primers that meet characteristics like avoiding primer-dimer formations and complementarity at the 3' end. Common steps in primer design include specifying the target DNA, selecting primer length and melting temperature parameters, and filtering results.
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Genome sequencing is the process of determining the order of nucleotide bases - A, C, G, and T - that make up an organism's DNA. Shotgun sequencing involves randomly breaking the genome into small fragments, sequencing those pieces, and reassembling the sequence by identifying overlapping regions. It was originally used by Sanger to sequence small genomes like viruses and bacteria. There are two main methods - hierarchical shotgun sequencing for larger genomes containing repeats, and whole genome shotgun sequencing for smaller genomes.
Shotgun and clone contig method
In shotgun sequencing the genome is broken randomly into short fragments (1 to 2 kbp long) suitable for sequencing. The fragments are ligated into a suitable vector and then partially sequenced. Around 400–500 bp of sequence can be generated from each fragment in a single sequencing run. In some cases, both ends of a fragment are sequenced. Computerized searching for overlaps between individual sequences then assembles the complete sequence.
Pcr primer design
Primer design is a key step in PCR that requires considering various factors to optimize the reaction. These include primer length, melting temperature, GC content, specificity, and potential for secondary structures. Well-designed primers are unique in targeting a single region, have compatible melting temperatures, and do not form hairpins or primer dimers that could inhibit the reaction. Choosing appropriate primers is essential for successful PCR amplification.
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Genetic and physical mapping are two types of genome mapping. Genetic mapping uses pedigree analysis and breeding experiments to determine sequence features, while physical mapping uses molecular techniques. Restriction mapping, radiation hybrid mapping, and STS mapping are techniques used to construct physical maps in the absence of complete DNA sequencing. Restriction mapping identifies restriction sites, radiation hybrid mapping analyzes fragments from irradiated cells hybridized with hamster cells, and STS mapping tags genomic sites using PCR primers. These physical mapping strategies provide distance and order estimates between DNA sequences to construct frameworks for sequencing.
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Ligase enzyme
DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between DNA fragments, joining two DNA strands together. It plays an important role in DNA replication by joining Okazaki fragments and filling in gaps, as well as in DNA repair and genetic engineering techniques like cloning. The most commonly used DNA ligase is from bacteriophage T4, which utilizes ATP as a cofactor and works efficiently at lower temperatures to ligate DNA strands with either sticky or blunt ends.
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Fred Sanger developed the chain termination method of DNA sequencing that is still widely used today. It involves making multiple copies of a DNA fragment, conducting separate sequencing reactions with each of the four dideoxynucleotides, and using gel electrophoresis or capillary electrophoresis to determine the sequence based on fragment sizes. While it can sequence fragments up to 900 base pairs, Sanger sequencing is time-consuming and expensive for large genomes. However, it provides high quality sequence data and remains useful for targeted sequencing applications.
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This document provides instructions for using Primer-BLAST to check the specificity of designed primers. It describes two main functions of Primer-BLAST: designing new primers and checking their specificity using sequence databases; and checking the specificity of already designed primers. The steps outlined are to input primer sequences, select the nr database and desired organism, and click "Get Primers" to obtain results, including matched organisms/genes and predicted primer-template matches. The goal is to use Primer-BLAST to verify primers only amplify the intended target sequences.
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Primers are short DNA sequences used to initiate DNA replication via polymerase chain reaction (PCR). Good primer design is important for PCR to work properly. Primers should be 18-24 base pairs in length and have a GC content and melting temperature that allows for specific annealing. Software tools can help design primers that meet characteristics like avoiding primer-dimer formations and complementarity at the 3' end. Common steps in primer design include specifying the target DNA, selecting primer length and melting temperature parameters, and filtering results.
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Genomic and cDNA libraries are collections of clones containing DNA fragments from an organism. A genomic DNA library contains all fragments of the genomic DNA, while a cDNA library contains only coding sequences synthesized from expressed mRNA. Genomic libraries are suitable for prokaryotes due to their small genomes but eukaryotic genomes require too many clones, so cDNA libraries are preferred for eukaryotic gene cloning. cDNA libraries represent the expressed genes and contain only coding regions without introns.
shotgun sequncing
Genome sequencing is the process of determining the order of nucleotide bases - A, C, G, and T - that make up an organism's DNA. Shotgun sequencing involves randomly breaking the genome into small fragments, sequencing those pieces, and reassembling the sequence by identifying overlapping regions. It was originally used by Sanger to sequence small genomes like viruses and bacteria. There are two main methods - hierarchical shotgun sequencing for larger genomes containing repeats, and whole genome shotgun sequencing for smaller genomes.
Shotgun and clone contig method
In shotgun sequencing the genome is broken randomly into short fragments (1 to 2 kbp long) suitable for sequencing. The fragments are ligated into a suitable vector and then partially sequenced. Around 400–500 bp of sequence can be generated from each fragment in a single sequencing run. In some cases, both ends of a fragment are sequenced. Computerized searching for overlaps between individual sequences then assembles the complete sequence.
Pcr primer design
Primer design is a key step in PCR that requires considering various factors to optimize the reaction. These include primer length, melting temperature, GC content, specificity, and potential for secondary structures. Well-designed primers are unique in targeting a single region, have compatible melting temperatures, and do not form hairpins or primer dimers that could inhibit the reaction. Choosing appropriate primers is essential for successful PCR amplification.
High-Throughput Sequencing
Talk on High-Throughput Sequencing: Overview and Selected Applications for Masters students. Nov 9th 2011
Serial analysis of gene expression
Serial Analysis of Gene Expression (SAGE) is a method that allows for the quantitative analysis of gene expression patterns in cells or tissues without prior knowledge of gene sequences. It works by extracting short sequence tags (10-14 base pairs) from the 3' end of transcripts and linking them together in long strings that can be cloned and sequenced simultaneously. This allows for many transcripts to be analyzed from a single sequencing reaction and provides quantitative data on gene expression levels based on tag frequencies. SAGE provides an accurate way to discover genes and analyze overall expression patterns without needing to know full mRNA sequences in advance.
PHYSICAL MAPPING STRATEGIES IN GENOMICS
Genetic and physical mapping are two types of genome mapping. Genetic mapping uses pedigree analysis and breeding experiments to determine sequence features, while physical mapping uses molecular techniques. Restriction mapping, radiation hybrid mapping, and STS mapping are techniques used to construct physical maps in the absence of complete DNA sequencing. Restriction mapping identifies restriction sites, radiation hybrid mapping analyzes fragments from irradiated cells hybridized with hamster cells, and STS mapping tags genomic sites using PCR primers. These physical mapping strategies provide distance and order estimates between DNA sequences to construct frameworks for sequencing.
PCR Primer desining
Primer desing tutorial,primer cane be desinged using web sources,details properties of PCR primers etc
Ri Plasmid
The Ri plasmid is a plasmid found in Agrobacterium rhizogenes bacteria that causes hairy root disease in plants. The plasmid contains genes that allow it to integrate portions of its DNA (T-DNA regions) into the plant genome. These integrated genes cause uncontrolled root growth and the formation of hairy roots. The Ri plasmid shares similarities with the Ti plasmid found in Agrobacterium tumefaciens, including virulence genes that mediate the transfer of T-DNA into plant cells and opine synthesis genes. Integration of the T-DNA from the Ri plasmid alters plant hormone production and induces hairy root formation.
Bacteriophage vectors
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
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Gene prediction methods vijay
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
Est database
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.
Structural genomics
Structural genomics aims to determine the 3D structure of all proteins in a genome. It uses high-throughput methods like X-ray crystallography and NMR on a genomic scale. This allows determination of protein structures for entire proteomes. It provides insights into protein function and can aid drug discovery by identifying potential drug targets like in Mycobacterium tuberculosis. Structural genomics leverages completed genome sequences to clone and express all encoded proteins for structural characterization.
Gene identification using bioinformatic tools.pptx
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.
Ligase enzyme
DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between DNA fragments, joining two DNA strands together. It plays an important role in DNA replication by joining Okazaki fragments and filling in gaps, as well as in DNA repair and genetic engineering techniques like cloning. The most commonly used DNA ligase is from bacteriophage T4, which utilizes ATP as a cofactor and works efficiently at lower temperatures to ligate DNA strands with either sticky or blunt ends.
DNA Sequencing- Sanger's Method
Fred Sanger developed the chain termination method of DNA sequencing that is still widely used today. It involves making multiple copies of a DNA fragment, conducting separate sequencing reactions with each of the four dideoxynucleotides, and using gel electrophoresis or capillary electrophoresis to determine the sequence based on fragment sizes. While it can sequence fragments up to 900 base pairs, Sanger sequencing is time-consuming and expensive for large genomes. However, it provides high quality sequence data and remains useful for targeted sequencing applications.
Comparative genomics
Comparative genomics involves comparing genomes to discover similarities and differences. It can provide insights into evolutionary relationships, help predict gene function, and aid in drug discovery. The first step is often aligning genome sequences using tools like BLAST or MUMmer. Genomes can then be compared at various levels, such as overall nucleotide statistics, genome structure, and coding/non-coding regions. Comparing gene and protein content across genomes helps predict functions. Conserved genomic features across species also aid prediction. Insights into genome evolution come from studying molecular events like inversions and duplications. Comparative genomics has impacted phylogenetics and drug target identification.
cDNA synthesis
The document discusses various methods for synthesizing complementary DNA (cDNA) from messenger RNA (mRNA). It describes the basic three step process of first-strand cDNA synthesis using reverse transcriptase, removal of the RNA template, and second-strand cDNA synthesis using DNA polymerase. Early methods used hairpin priming of the second strand but were later improved using oligo-dT tailing and oligo-dG priming to avoid 5' end losses. Other methods discussed include oligo-capping to select for full-length mRNAs and RACE (rapid amplification of cDNA ends) to amplify cDNA fragments from both ends of transcripts.
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DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCING
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Gene identification using bioinformatic tools.pptx
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一.毕业证【q微741003700】成绩单、使馆认证、教育部认证、雅思托福成绩单、学生卡等！
二.真实使馆公证(即留学回国人员证明,不成功不收费)
三.真实教育部学历学位认证（教育部存档！教育部留服网站永久可查）
四.办理各国各大学文凭(一对一专业服务,可全程监控跟踪进度)
如果您处于以下几种情况：
◇在校期间，因各种原因未能顺利毕业……拿不到官方毕业证【q/微741003700】
◇面对父母的压力，希望尽快拿到；
◇不清楚认证流程以及材料该如何准备；
◇回国时间很长，忘记办理；
◇回国马上就要找工作，办给用人单位看；
◇企事业单位必须要求办理的
◇需要报考公务员、购买免税车、落转户口
◇申请留学生创业基金
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
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How to design a DNA primer on NCBI.pptx
- 1. How to design nucleotide Primer on NCBI 1 Okechukwu Francis Programme: PhD Biotechnology SCHOOL OF HEALTH SCIENCE AND TECHNOLOGY (SoHST)
- 2. NCBI provides a variety of tools for DNA primer design, including Primer-BLAST, which integrates the primer design and sequence search steps into a single tool. Here are the general steps for designing a DNA primer on NCBI using Primer-BLAST: Access Primer-BLAST: Go to the NCBI Primer-BLAST website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and enter the target DNA sequence in FASTA format. Choose the Target Region: Select the specific region of the DNA sequence that you want to amplify by specifying the start and end positions. Specify Primer Parameters: Enter the desired primer parameters, such as primer length, melting temperature, and GC content. Select the Organism: Choose the organism for which you want to design the primers. This step is optional but can improve the specificity of the primers.
- 4. Run Primer-BLAST: Click the “Get Primer" button to run Primer-BLAST and wait for NCBI to generate a list of candidate primers.
- 5. Analyze Results: The results page displays the primer sequences, melting temperatures, and other relevant information. The tool also provides a summary of the specificity of each primer based on its alignment with the target sequence and other sequences in the NCBI database.
- 7. Refine Primers: Use the "Edit Sequence" feature to refine the target region or the primer parameters and run Primer-BLAST again to generate new candidate primers. Test the Primers: Once the primers are designed, it is important to experimentally validate their efficiency and specificity using PCR. NCBI also provides other primer design tools, such as the Primer3 program, which can be accessed through the NCBI website or downloaded as standalone software. Additionally, there are other commercial primer design tools available that can provide additional features and options for primer design.