S1 Mapping
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S1 Mapping is a laboratory method used for locating the start and end points of transcripts and for mapping introns. This technique is used for quantifying the amount of mRNA transcripts, it can therefore identify the level of transcription of the gene in the cell at a given time.
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S1 Nuclease Mapping
S1 nuclease mapping is a laboratory technique used to locate the 5' end of an RNA transcript within a mixture by using the S1 nuclease. The S1 nuclease is an endonuclease that degrades single-stranded DNA and RNA but does not degrade double-stranded DNA or RNA-DNA hybrids. In S1 mapping, a transcript is hybridized to a DNA template and treated with S1 nuclease, which degrades any unhybridized RNA. This allows mapping the 5' end of the transcript to the DNA template. S1 nuclease mapping can determine the exact locations of start and end points of transcription and any splice points within transcripts.
Phagemid vector
This document discusses the phagemid vector pBluescript, which can be used in either the positive or negative orientation. pBluescript is a phagemid vector that can be used to clone DNA fragments for sequencing, mutagenesis, protein expression or other molecular biology experiments. References for further information about pBluescript are provided.
Lectut btn-202-ppt-l23. labeling techniques for nucleic acids
Nucleic acid probes can be labeled with radioisotopes or nonisotopic labels for use in hybridization techniques. Common labeling methods include radioactive labeling with 32P or 3H, or nonisotopic labeling with biotin, digoxigenin, or fluorescein. Labeled probes are used to detect complementary DNA or RNA sequences and can be DNA, RNA, or oligonucleotide probes. Probes are prepared through various techniques such as PCR, random priming, or in vitro transcription and must be purified before use and stored appropriately.
Chromosome walking
Chromosome walking is a technique used to analyze large regions of DNA by identifying overlapping clones. It involves selecting a starting clone, subcloning fragments from the ends, and using those fragments to screen a genomic library and identify neighboring clones with overlapping sequences. This process is repeated to "walk" along the chromosome. Chromosome walking was developed in the 1980s and is useful for finding mutations associated with genetic diseases and detecting single nucleotide polymorphisms, though it has limitations with repetitive sequences.
Express sequence tags
ESTs are short sequences of DNA derived from cDNA clones that represent gene expression in particular cells or tissues. They provide a simple and inexpensive way to discover new genes and map their positions in genomes. To create an EST, mRNA is converted to cDNA and then sequenced, yielding short expressed DNA sequences. ESTs are deposited in public databases like NCBI's dbEST and can help identify genes, construct genome maps, and characterize expressed genes through clustering, assembly, and mapping to genomic sequences. However, isolating mRNA from some tissues can be difficult and ESTs alone do not indicate the genes they were derived from.
Site directed mutagenesis
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.
Protein dna interactions
Protein-DNA interactions can be either specific or non-specific. Specific interactions involve transcription factors that regulate gene expression by binding to DNA motifs through domains like helix-loop-helix, leucine zipper, or zinc finger motifs. Non-specific interactions involve histones that help structure DNA into nucleosomes within chromatin and can be chemically modified through methylation, demethylation, acetylation, and phosphorylation.
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.
Recommended
S1 Nuclease Mapping
S1 nuclease mapping is a laboratory technique used to locate the 5' end of an RNA transcript within a mixture by using the S1 nuclease. The S1 nuclease is an endonuclease that degrades single-stranded DNA and RNA but does not degrade double-stranded DNA or RNA-DNA hybrids. In S1 mapping, a transcript is hybridized to a DNA template and treated with S1 nuclease, which degrades any unhybridized RNA. This allows mapping the 5' end of the transcript to the DNA template. S1 nuclease mapping can determine the exact locations of start and end points of transcription and any splice points within transcripts.
Phagemid vector
This document discusses the phagemid vector pBluescript, which can be used in either the positive or negative orientation. pBluescript is a phagemid vector that can be used to clone DNA fragments for sequencing, mutagenesis, protein expression or other molecular biology experiments. References for further information about pBluescript are provided.
Lectut btn-202-ppt-l23. labeling techniques for nucleic acids
Nucleic acid probes can be labeled with radioisotopes or nonisotopic labels for use in hybridization techniques. Common labeling methods include radioactive labeling with 32P or 3H, or nonisotopic labeling with biotin, digoxigenin, or fluorescein. Labeled probes are used to detect complementary DNA or RNA sequences and can be DNA, RNA, or oligonucleotide probes. Probes are prepared through various techniques such as PCR, random priming, or in vitro transcription and must be purified before use and stored appropriately.
Chromosome walking
Chromosome walking is a technique used to analyze large regions of DNA by identifying overlapping clones. It involves selecting a starting clone, subcloning fragments from the ends, and using those fragments to screen a genomic library and identify neighboring clones with overlapping sequences. This process is repeated to "walk" along the chromosome. Chromosome walking was developed in the 1980s and is useful for finding mutations associated with genetic diseases and detecting single nucleotide polymorphisms, though it has limitations with repetitive sequences.
Express sequence tags
ESTs are short sequences of DNA derived from cDNA clones that represent gene expression in particular cells or tissues. They provide a simple and inexpensive way to discover new genes and map their positions in genomes. To create an EST, mRNA is converted to cDNA and then sequenced, yielding short expressed DNA sequences. ESTs are deposited in public databases like NCBI's dbEST and can help identify genes, construct genome maps, and characterize expressed genes through clustering, assembly, and mapping to genomic sequences. However, isolating mRNA from some tissues can be difficult and ESTs alone do not indicate the genes they were derived from.
Site directed mutagenesis
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.
Protein dna interactions
Protein-DNA interactions can be either specific or non-specific. Specific interactions involve transcription factors that regulate gene expression by binding to DNA motifs through domains like helix-loop-helix, leucine zipper, or zinc finger motifs. Non-specific interactions involve histones that help structure DNA into nucleosomes within chromatin and can be chemically modified through methylation, demethylation, acetylation, and phosphorylation.
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.
Shuttle vector
1. Yeast plasmids like the 2 micron circle have been extensively studied and developed into yeast cloning vectors.
2. Shuttle vectors like YEp vectors contain selectable marker genes like LEU2 and bacterial plasmid origins of replication like pBR322, allowing them to replicate in both E. coli and yeast.
3. The 2 micron circle is a 6kb endogenous yeast plasmid that replicates autonomously through an ARS sequence and is maintained at 50-100 copies per cell.
Methylases
This document discusses methylases, which are enzymes that add methyl groups to DNA. Specifically:
- Methylases transfer methyl groups from S-adenosylmethionine to adenine or cytosine bases within their recognition sequence on DNA. This methylation protects the DNA from restriction endonucleases.
- The methylase and restriction enzyme of a bacterial species together form the restriction-modification system, with the methylase protecting the host DNA.
- Methylases are of interest because methylation of some restriction enzyme recognition sites protects the DNA from being cleaved by that enzyme. This allows study of DNA isolated from strains expressing common methylases like Dam or Dcm.
Synthesis of c dna
The document discusses the process of synthesizing cDNA from mRNA. It involves isolating mRNA, using reverse transcriptase to copy the mRNA into single-stranded cDNA, then converting it to double-stranded cDNA using DNA polymerase. The double-stranded cDNA can then be inserted into a vector and used to create a cDNA library through cloning in bacteria or phage. The library can be screened by hybridization or assays to identify clones containing genes of interest.
SAGE (Serial analysis of Gene Expression)
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.
Protein protein interaction
Protein-protein interactions are important for many biological processes. There are various types of interactions depending on their composition and duration. Methods to study interactions include yeast two-hybrid, co-immunoprecipitation, affinity chromatography, and chromatin immunoprecipitation. Databases such as IntAct and MINT provide repositories for protein interaction data.
Yeast Artificial Chromosomes (YACs)
Yeast artificial chromosomes (YACs) are engineered DNA molecules that can clone and replicate large DNA sequences in yeast cells. YACs contain essential yeast elements like a centromere and telomeres that allow them to behave like natural yeast chromosomes. YACs can clone very large inserts of up to 10 megabases of foreign DNA, making them useful for generating whole genome libraries.
Genomic and c dna library
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.
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Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
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Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
Whole genome shotgun sequencing
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.
Molecular probes
This document discusses molecular probes, including their definition, types, preparation, and labeling. It describes the three main types of probes - oligonucleotide probes, DNA probes, and RNA probes. It explains how to prepare probes from genomic DNA, cDNA, synthetic oligonucleotides, and RNA. Methods of radioactive labeling including nick translation and oligonucleotide labeling are covered. Non-radioactive labeling using biotin and digoxigenin is also discussed. Finally, applications of molecular probes in identification of recombinant clones, fingerprinting, in situ hybridization, and medical research are summarized.
bacterial artificial chromosome & yeast artificial chromosome
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2D-PAGE is a technique used to separate complex protein mixtures based on isoelectric point and molecular weight. It involves two sequential steps - isoelectric focusing and SDS-PAGE. In isoelectric focusing, proteins are separated based on their isoelectric point in an immobilized pH gradient. They are then separated by SDS-PAGE based on their molecular weight. The separated proteins can then be visualized through staining and identified through mass spectrometry. While useful for proteomic analysis, 2D-PAGE has limitations such as low reproducibility and dynamic range.
DNA footprinting
DNA footprinting is a technique used to identify protein binding regions on DNA. It involves treating DNA with nucleases like DNase I, which will degrade the DNA except for regions bound by proteins. These protected regions, called footprints, can identify transcription factor binding sites that regulate gene expression. The technique was originally developed in 1978 to study the binding specificity of the lac repressor protein, and it provides information on DNA-protein interactions and transcriptional regulation.
Genomic library construction
A DNA library is a collection of DNA fragments that have been cloned into vectors. DNA libraries allow researchers to isolate and study specific DNA fragments of interest. To create a genomic library, DNA is extracted from an organism, cut into fragments, inserted into vectors, and introduced into host bacteria to generate clones containing all the organism's DNA sequences. This library can then be screened to identify and study particular genes. DNA libraries provide an efficient way to store, isolate, and analyze DNA sequences.
YEAST TWO HYBRID SYSTEM
Simply describe the yeast tow hybrid,where you can find definition ,principle,procedure,advantages and disadvantages .
Synthesis and cloning of c dna
This document provides information about synthesizing and cloning cDNA from mRNA. It describes how cDNA libraries are constructed by isolating mRNA, synthesizing cDNA via reverse transcription, treating cDNA ends, ligating the cDNA to vectors, and transforming the vectors into host cells. The key steps include mRNA purification, first and second strand cDNA synthesis, linker ligation, vector ligation, and transformation. cDNA libraries are useful for eukaryotic gene analysis as they contain only expressed genes without introns.
mRNA Isolation
1. mRNA isolation is the process of extracting messenger RNA from biological samples. It is important for research and industry applications as mRNA provides insight into which genes are being expressed and translated into proteins.
2. Total RNA is first extracted using Trizol, which separates RNA, DNA and proteins. mRNA is then isolated from total RNA using biotin-labeled oligo dT probes that selectively bind to the poly-A tail of mRNA molecules.
3. The mRNA-probe complexes are immobilized on magnetic beads coated with streptavidin. This allows the mRNA to be separated and purified from other RNAs through magnetic separation washes. The purified mRNA can then be used in applications like RT-PCR and protein
T dna & transposone tagging 1 (2)
Gene tagging uses recognizable DNA fragments like T-DNA or transposons to disrupt gene function and identify genes responsible for mutant phenotypes. T-DNA tagging in plants involves random integration of Agrobacterium T-DNA that can disrupt genes and create mutants. Transposon tagging relies on the ability of transposons to move within genomes and disrupt gene function. Both techniques have been used successfully to isolate numerous plant genes involved in traits like color and development.
Labelling of dna
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.
Studying gene expression and function
The document discusses various methods for studying gene expression and function, including analyzing RNA transcripts. It describes techniques like northern hybridization, DNA-mRNA hybridization, S1 nuclease mapping, primer extension, and PCR that can be used to study transcripts and locate start/stop points. The document also covers methods for studying gene regulation, such as identifying protein binding sites through gel retardation assays and footprinting, and using deletion analysis to identify control sequences.
Transcription.ppt
Transcription is the process by which RNA polymerase converts DNA into RNA. It involves 3 stages: initiation, elongation, and termination. Initiation begins at promoter sequences on DNA and requires a sigma factor. During elongation, RNA polymerase proofreads as it synthesizes RNA to ensure accuracy. Termination occurs when the polymerase reaches termination sequences and releases the RNA transcript.
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Shuttle vector
1. Yeast plasmids like the 2 micron circle have been extensively studied and developed into yeast cloning vectors.
2. Shuttle vectors like YEp vectors contain selectable marker genes like LEU2 and bacterial plasmid origins of replication like pBR322, allowing them to replicate in both E. coli and yeast.
3. The 2 micron circle is a 6kb endogenous yeast plasmid that replicates autonomously through an ARS sequence and is maintained at 50-100 copies per cell.
Methylases
This document discusses methylases, which are enzymes that add methyl groups to DNA. Specifically:
- Methylases transfer methyl groups from S-adenosylmethionine to adenine or cytosine bases within their recognition sequence on DNA. This methylation protects the DNA from restriction endonucleases.
- The methylase and restriction enzyme of a bacterial species together form the restriction-modification system, with the methylase protecting the host DNA.
- Methylases are of interest because methylation of some restriction enzyme recognition sites protects the DNA from being cleaved by that enzyme. This allows study of DNA isolated from strains expressing common methylases like Dam or Dcm.
Synthesis of c dna
The document discusses the process of synthesizing cDNA from mRNA. It involves isolating mRNA, using reverse transcriptase to copy the mRNA into single-stranded cDNA, then converting it to double-stranded cDNA using DNA polymerase. The double-stranded cDNA can then be inserted into a vector and used to create a cDNA library through cloning in bacteria or phage. The library can be screened by hybridization or assays to identify clones containing genes of interest.
SAGE (Serial analysis of Gene Expression)
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.
Protein protein interaction
Protein-protein interactions are important for many biological processes. There are various types of interactions depending on their composition and duration. Methods to study interactions include yeast two-hybrid, co-immunoprecipitation, affinity chromatography, and chromatin immunoprecipitation. Databases such as IntAct and MINT provide repositories for protein interaction data.
Yeast Artificial Chromosomes (YACs)
Yeast artificial chromosomes (YACs) are engineered DNA molecules that can clone and replicate large DNA sequences in yeast cells. YACs contain essential yeast elements like a centromere and telomeres that allow them to behave like natural yeast chromosomes. YACs can clone very large inserts of up to 10 megabases of foreign DNA, making them useful for generating whole genome libraries.
Genomic and c dna library
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.
Vector engineering and codon optimization
Eukaryotic expression vectors, Prokaryotic expression vectors, synthetic promoters, vector engineering, and host-specific codon optimization.
Dna modifying enzymes
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
Whole genome shotgun sequencing
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.
Molecular probes
This document discusses molecular probes, including their definition, types, preparation, and labeling. It describes the three main types of probes - oligonucleotide probes, DNA probes, and RNA probes. It explains how to prepare probes from genomic DNA, cDNA, synthetic oligonucleotides, and RNA. Methods of radioactive labeling including nick translation and oligonucleotide labeling are covered. Non-radioactive labeling using biotin and digoxigenin is also discussed. Finally, applications of molecular probes in identification of recombinant clones, fingerprinting, in situ hybridization, and medical research are summarized.
bacterial artificial chromosome & yeast artificial chromosome
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
2d Page
2D-PAGE is a technique used to separate complex protein mixtures based on isoelectric point and molecular weight. It involves two sequential steps - isoelectric focusing and SDS-PAGE. In isoelectric focusing, proteins are separated based on their isoelectric point in an immobilized pH gradient. They are then separated by SDS-PAGE based on their molecular weight. The separated proteins can then be visualized through staining and identified through mass spectrometry. While useful for proteomic analysis, 2D-PAGE has limitations such as low reproducibility and dynamic range.
DNA footprinting
DNA footprinting is a technique used to identify protein binding regions on DNA. It involves treating DNA with nucleases like DNase I, which will degrade the DNA except for regions bound by proteins. These protected regions, called footprints, can identify transcription factor binding sites that regulate gene expression. The technique was originally developed in 1978 to study the binding specificity of the lac repressor protein, and it provides information on DNA-protein interactions and transcriptional regulation.
Genomic library construction
A DNA library is a collection of DNA fragments that have been cloned into vectors. DNA libraries allow researchers to isolate and study specific DNA fragments of interest. To create a genomic library, DNA is extracted from an organism, cut into fragments, inserted into vectors, and introduced into host bacteria to generate clones containing all the organism's DNA sequences. This library can then be screened to identify and study particular genes. DNA libraries provide an efficient way to store, isolate, and analyze DNA sequences.
YEAST TWO HYBRID SYSTEM
Simply describe the yeast tow hybrid,where you can find definition ,principle,procedure,advantages and disadvantages .
Synthesis and cloning of c dna
This document provides information about synthesizing and cloning cDNA from mRNA. It describes how cDNA libraries are constructed by isolating mRNA, synthesizing cDNA via reverse transcription, treating cDNA ends, ligating the cDNA to vectors, and transforming the vectors into host cells. The key steps include mRNA purification, first and second strand cDNA synthesis, linker ligation, vector ligation, and transformation. cDNA libraries are useful for eukaryotic gene analysis as they contain only expressed genes without introns.
mRNA Isolation
1. mRNA isolation is the process of extracting messenger RNA from biological samples. It is important for research and industry applications as mRNA provides insight into which genes are being expressed and translated into proteins.
2. Total RNA is first extracted using Trizol, which separates RNA, DNA and proteins. mRNA is then isolated from total RNA using biotin-labeled oligo dT probes that selectively bind to the poly-A tail of mRNA molecules.
3. The mRNA-probe complexes are immobilized on magnetic beads coated with streptavidin. This allows the mRNA to be separated and purified from other RNAs through magnetic separation washes. The purified mRNA can then be used in applications like RT-PCR and protein
T dna & transposone tagging 1 (2)
Gene tagging uses recognizable DNA fragments like T-DNA or transposons to disrupt gene function and identify genes responsible for mutant phenotypes. T-DNA tagging in plants involves random integration of Agrobacterium T-DNA that can disrupt genes and create mutants. Transposon tagging relies on the ability of transposons to move within genomes and disrupt gene function. Both techniques have been used successfully to isolate numerous plant genes involved in traits like color and development.
Labelling of dna
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.
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bacterial artificial chromosome & yeast artificial chromosome
bacterial artificial chromosome & yeast artificial chromosome
Similar to S1 Mapping
Studying gene expression and function
The document discusses various methods for studying gene expression and function, including analyzing RNA transcripts. It describes techniques like northern hybridization, DNA-mRNA hybridization, S1 nuclease mapping, primer extension, and PCR that can be used to study transcripts and locate start/stop points. The document also covers methods for studying gene regulation, such as identifying protein binding sites through gel retardation assays and footprinting, and using deletion analysis to identify control sequences.
Transcription.ppt
Transcription is the process by which RNA polymerase converts DNA into RNA. It involves 3 stages: initiation, elongation, and termination. Initiation begins at promoter sequences on DNA and requires a sigma factor. During elongation, RNA polymerase proofreads as it synthesizes RNA to ensure accuracy. Termination occurs when the polymerase reaches termination sequences and releases the RNA transcript.
Transcription.ppt
Photosystem II captures and transfers energy.
– chlorophyll absorbs
energy from sunlight
– energized electrons
enter electron
transport chain
– water molecules are
split
– oxygen is released as
waste
– hydrogen ions are
transported across
thylakoid membrane
4.1 Chemical Energy and ATP
• Photosystem I captures energy and produces energycarrying molecules.
– chlorophyll absorbs
energy from sunlight
– energized electrons
are used to make
NADPH
– NADPH is transferred
to light-independent
reactions
4.1 Chemical Energy and ATP
• The light-dependent reactions produce ATP.
– hydrogen ions flow through a channel in the thylakoid
membrane
– ATP synthase attached to the channel makes ATP
4.1 Chemical Energy and ATP
• Light-independent
reactions occur in the
stroma and use CO2
molecules.
The second stage of photosynthesis uses energy from
the first stage to make sugars.
4.1 Chemical Energy and ATP
• A molecule of glucose is formed as it stores some of the
energy captured from sunlight.
– carbon dioxide molecules enter the Calvin Photosystem II captures and transfers energy.
– chlorophyll absorbs
energy from sunlight
– energized electrons
enter electron
transport chain
– water molecules are
split
– oxygen is released as
waste
– hydrogen ions are
transported across
thylakoid membrane
4.1 Chemical Energy and ATP
• Photosystem I captures energy and produces energycarrying molecules.
– chlorophyll absorbs
energy from sunlight
– energized electrons
are used to make
NADPH
– NADPH is transferred
to light-independent
reactions
4.1 Chemical Energy and ATP
• The light-dependent reactions produce ATP.
– hydrogen ions flow through a channel in the thylakoid
membrane
– ATP synthase attached to the channel makes ATP
4.1 Chemical Energy and ATP
• Light-independent
reactions occur in the
stroma and use CO2
molecules.
The second stage of photosynthesis uses energy from
the first stage to make sugars.
4.1 Chemical Energy and ATP
• A molecule of glucose is formed as it stores some of the
energy captured from sunlight.
– carbon dioxide molecules enter the Calvin Photosystem II captures and transfers energy.
– chlorophyll absorbs
energy from sunlight
– energized electrons
enter electron
transport chain
– water molecules are
split
– oxygen is released as
waste
– hydrogen ions are
transported across
thylakoid membrane
4.1 Chemical Energy and ATP
• Photosystem I captures energy and produces energycarrying molecules.
– chlorophyll absorbs
energy from sunlight
– energized electrons
are used to make
NADPH
– NADPH is transferred
to light-independent
reactions
4.1 Chemical Energy and ATP
• The light-dependent reactions produce ATP.
– hydrogen ions flow through a channel in the thylakoid
membrane
– ATP synthase attached to the channel makes ATP
4.1 Chemical Energy and ATP
• Light-independent
reactions occur in the
stroma and use CO2
molecules.
The second stage of photosynthesis uses energy frvf
Lectut btn-202-ppt-l30. applications of pcr-ii
The document discusses various applications of polymerase chain reaction (PCR) including cDNA synthesis and rapid amplification of cDNA ends (RACE) to sequence mRNA ends, error-prone PCR for random mutagenesis, PCR-based molecular markers for genome mapping and analyzing genetic variation, analysis of fossil DNA and environments using PCR, medical diagnosis using PCR, and applications in forensic science.
Basic information of s1 nuclease
The S1 nuclease was extracted from Aspergill suoryzae. The S1 nuclease is a specific
single-stranded endonuclease. It can degrade single-stranded DNA and
single-stranded RNA to produce 5'-single-stranded nucleotides or oligonucleotides.
transcription.pptx
transcription is the process of synthesis of mRNA from DNA.
Click on the page and get the solution of your query.
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The document summarizes the process of transcription in cells. It defines transcription as the process by which RNA is synthesized using DNA as a template. It describes the basic structure of genes and the roles of different types of RNA including mRNA, tRNA, rRNA and snRNA. The transcription process involves RNA polymerase binding to the promoter region and synthesizing RNA along the DNA template, adding complementary bases. Elongation continues until a terminator sequence is reached, at which point transcription ends and the RNA transcript is released.
Prokaryotic transcription
Prokaryotes are organisms that consist of a single prokaryotic cell. Eukaryotic cells are found in plants, animals, fungi, and protists. They range from 10–100 μm in diameter, and their DNA is contained within a membrane-bound nucleus.Prokaryotes do not have membrane-enclosed nuclei. Therefore, the processes of transcription, translation, and mRNA degradation can all occur simultaneously.
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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.
enzyme used in RTD.pptx
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Protein synthesis mechanism with reference of Translation and Transcription d...
This fie is about Protein synthesis mechanism with reference of Translation and Transcription, their sequential steps in details.
Gene expression
Gene expression is the process by which genetic information encoded in DNA is converted into structures and functions within cells. It involves three main steps: replication, transcription, and translation. Replication copies DNA to produce identical daughter molecules. Transcription converts the DNA sequence into messenger RNA (mRNA). Translation then uses the mRNA to produce proteins through the joining of amino acids specified by the mRNA's codon sequence. These three steps together allow genetic information to direct the production of the RNA and protein molecules that drive cellular functions and inheritance of traits.
Long non coding RNA lncRNAs
Almost 98 of the human genome does not encode proteins
o The non coding transcripts less than 200 bases are called small non
coding RNA and comprise of tRNA, rRNA, miRNA, snoRNA, piwi
interacting RNA (pi RNA)
o RNA molecules that are of more than 200 bases in length are known
as long non coding RNA (
o lncRNAs are more than 200 nucleotides in length and also can be
more than 2 Kb
o Such long noncoding RNAs usually have limited coding potential due
to the absence of open reading frames, 3 UTR and termination
region while their coding potential is less than 100 amino acids
Transcription in eukariotes by kk sahu
INTRODUCTION
A STRUCTURAL GENE
EUKARYOTIC RNAPs
MACHANISM OF TRANSCRIPTION IN EUKARYOTES:
- INITIATION
-ELONGATION
-TERMINATION
RNA SPLISING
DIFFERENT BETWEEN PROKARYOTIC & EUKARYOTIC TRANSCRIPTION
BIBLIOGRAPHY
Transcription - DNA dependent RNA synthesis.pptx
The process by which an RNA copy of a gene is made or it’s a DNA dependent RNA synthesis.
Transcription resembles replication
In its fundamental chemical mechanism
Its polarity (direction of synthesis)
Its use of a template
Transcription differs from replication
It does not requires a primer
It involves only limited segments of a DNA molecule
Within transcribed segments only one DNA strand serves as a template for synthesis of RNA.
NORTHERN BLOTTING.pptx
This document describes the process of northern blotting. Northern blotting is used to detect specific RNA molecules and analyze gene expression. It involves separating RNA samples by size using gel electrophoresis, transferring them to a membrane, then using a labeled probe to detect the RNA of interest. The key steps are isolating RNA from tissue or cells, separating the RNA by size on a gel, blotting the RNA onto a membrane, hybridizing the membrane with a labeled probe, washing off excess probe, and visualizing where the probe bound to detect the RNA of interest. Northern blotting allows researchers to study gene expression patterns in different tissues, developmental stages, or disease states.
Transcription b.pharm
This document discusses the process of transcription from DNA to RNA. It begins with an overview of transcription in prokaryotes and eukaryotes. In prokaryotes, RNA polymerase binds to promoter sequences and transcribes DNA into RNA. Eukaryotes have three RNA polymerases that recognize different promoters. The document then covers the stages of transcription including initiation, elongation, and termination. It describes the requirements for transcription including the DNA template, RNA polymerase, and ribonucleotide substrates. Differences between DNA and RNA as well as between prokaryotic and eukaryotic transcription are also summarized.
RNA Synthesis (Transcription).pptx
RNA is synthesized through a process called transcription. During transcription, RNA polymerase binds to a gene's promoter and copies the gene sequence into RNA. The RNA transcript is then processed through splicing, capping, polyadenylation, and other modifications before being used to produce proteins. Key steps in transcription include initiation at the promoter, elongation as RNA polymerase moves along the DNA, and termination once the full sequence has been copied.
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S1 Mapping
- 1. S1 MAPPING Presented by Wardah Shah Roll no. 07 Submitted to Dr. Abrar Qureshi Dept. Of Biotechnology
- 2. INTRODUCTION • Gene expression is the process by which information encoded in a gene is used in the synthesis of a functional gene product, that is, protein in case of mRNA transcript translation. • To understand how a gene is expressed, the RNA transcript must be studied, in particularly, 1. The removal of introns, 2. The exact locations of start and end points of transcription, 3. The signals that determine the start and end of transcription.
- 3. • S1 nuclease is a restriction enzyme that cleaves only single stranded nucleic acids, ssDNA or ssRNA, leading to formation of 5' phosphomononucleotide product or 5' phosphooligonucleotide product. • It has been isolated from Aspargillus oryzae. S1 ENDONUCLEASE
- 4. Properties of S1 Nuclease: • At high ionic strength, low pH (4-4.5) and in the presence of Zn ions (cofactor), S1 nuclease digests ssDNA very efficiently. • It is relatively stable against denaturing agents like urea, SDS and formaldehyde. • It removes single stranded regions from dsDNA. (conversion of cohesive ends to blunt ends) • Cleaves hairpin loops generated during synthesis of DNA. • Used I nuclease mapping techniques and nuclease protection assay
- 5. S1 Mapping: • Technique developed by Berk and Sharp by the study of adenovirus mRNAs • S1 Mapping is a laboratory method used for locating the start and end points of transcripts and for mapping introns. • This technique is used for quantifying the amount of mRNA transcripts, it can therefore identify the level of transcription of the gene in the cell at a given time.
- 6. DNA-mRNA Hybridization: • When hybrid is formed between complementary DNA strand and its mRNA transcript, then the boundaries between double and single stranded regions marks the start and end points of the mRNA. • Introns, which are present in DNA, but not in the mRNA, will loop out as additional single stranded regions. • On treatment with S1 Nuclease, all single stranded regions are cleaved out, leaving behind the hybrid regions. • Upon treatment with alkali, the RNA strands are degraded and the DNA fragments are recovered and their sizes measured on gel electrophoresis. • LIMITATION: Order of DNA sequence cannot be determined.
- 7. LOCATING START SITE: • Modifications to hybridization technique allow the precise start and end points of the transcript and of any introns it contains to be mapped onto the DNA sequence • The figure shows an example of S1 mapping for locating the start site of a transcript. • Here, a 400 bp fragment is created by restriction digestion by Sau3A. This fragment contains the start site of the gene. • The fragment is inserted into M13 cloning vector. The mRNA transcript is made to anneal with the singe stranded DNA in the vector. • The transcript anneals only to the complementary sequence. Rest of the single stranded vector and single stranded mRNA is cleaves by action of S1 endonuclease. • The double stranded hybrid is treated with alkali to degrade the RNA and recover the DNA which is run on gel electrophoresis. • The size of this fragment corresponds to the distance between the transcription start point and right hand Sau3A site. • The same strategy is used to locate the end site.
- 8. SUMMARY • In S1 mapping, a labeled DNA probe is used to detect 5’- or 3’-end of a transcript. • Hybridization of the probe to the transcript protects a portion of the probe from digestion by S1 nuclease, specific for single-stranded polynucleotides. • Length of the section of probe protected by the transcript locates the end of the transcript relative to the known location of an end of the probe. • Amount of probe protected is proportional to concentration of transcript, so S1 mapping can be quantitative.
- 9. THANK YOU