Southern blotting
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Southern blotting is a technique used to detect specific DNA sequences within DNA samples. It involves digesting DNA samples with restriction enzymes, separating the fragments via gel electrophoresis, transferring the fragments to a membrane, and using radioactive probes to hybridize to complementary DNA sequences, which are then visualized. The key steps are digesting DNA, separating fragments by size via gel electrophoresis, transferring fragments to a membrane, hybridizing with radioactive probes, and detecting hybridized probes.
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Northen blotting
Northern blotting is a technique used to detect specific RNA sequences in a sample. It involves isolating RNA, separating it via gel electrophoresis, transferring it to a membrane, then hybridizing probes with complementary sequences to the target RNA. This allows visualization of gene expression patterns between tissues, developmental stages, and disease states from the detected RNA sequences.
Southern blotting
Southern blotting is a technique used to detect specific DNA sequences. It involves separating DNA fragments by size via gel electrophoresis, transferring them to a membrane, and using a labeled probe to hybridize to the target sequence. The probe binds only to complementary DNA fragments, allowing their detection and location on the membrane via autoradiography. Key steps include restriction enzyme digestion of DNA, gel electrophoresis, membrane transfer, probe hybridization, and x-ray film development to visualize hybridized fragments. Southern blotting is used for applications like mutation detection, gene rearrangement studies, and forensic analysis.
Northern blotting
Northern blotting is a technique used to detect specific mRNAs in a sample. It involves separating RNA fragments by size through gel electrophoresis, transferring them to a membrane, then using a complementary DNA or RNA probe to identify the mRNA of interest. It allows researchers to examine gene expression by measuring mRNA levels and determine tissue specificity and regulatory factors of gene expression. While it provides useful information, it also has some limitations such as risk of mRNA degradation and relatively low sensitivity compared to PCR-based methods.
Lecture on DNA sequencing
The document discusses different methods of DNA sequencing including the Maxam-Gilbert and Sanger chain termination methods as well as newer next generation sequencing techniques. It describes the principles, steps, and significance of the Maxam-Gilbert and Sanger methods and how next generation sequencing improved DNA sequencing by allowing millions of DNA molecules to be sequenced simultaneously in an automated process.
RFLP
This document discusses Restriction Fragment Length Polymorphism (RFLP) analysis, which is a technique used to differentiate organisms by analyzing patterns in their DNA after digestion with restriction enzymes. RFLP analysis can be used for various applications like determining paternity, detecting mutations, and genetic mapping. The process involves digesting DNA with restriction enzymes, running the fragments on a gel, transferring the DNA to a membrane, and using probes to detect polymorphisms and produce an autoradiogram showing differences in fragment patterns. As an example, the document describes using PCR-RFLP to rapidly screen for a BRCA2 mutation by taking advantage of a restriction site change caused by the mutation.
Sanger sequencing
Sanger sequencing is a method of DNA sequencing developed by Frederick Sanger in 1977 that was widely used for 25 years. It involves making copies of a DNA region using DNA polymerase and chain-terminating dideoxynucleotides that are labeled with different colored dyes. This produces fragmented DNA of different lengths that can be separated by size to determine the DNA sequence. Sanger sequencing is useful for sequencing single genes or short sequences but is limited to read lengths of 300-1000 base pairs. It has been replaced by next generation sequencing methods for most applications.
Blotting techniques
This document discusses various blotting techniques used to detect DNA, RNA, and proteins, including Southern blotting, Northern blotting, and Western blotting. It provides detailed descriptions of the procedures for each technique, including separating biomolecules by electrophoresis, transferring them to a membrane, hybridizing probes, and detecting bound probes. The techniques allow visualization and analysis of specific biomolecules within complex samples.
Northern blot
Northern blotting is a technique used to detect specific RNA molecules in a sample. It involves separating RNA by electrophoresis, transferring it to a membrane, then using a probe with a complementary nucleotide sequence to detect the target RNA. Key steps include isolating high quality RNA, separating it by denaturing gel electrophoresis, blotting it onto a membrane, fixing the RNA to the membrane by UV crosslinking or baking, then detecting it using a labeled probe via hybridization. Northern blotting allows analysis of RNA expression of particular genes and is useful for studying gene expression.
Recommended
Northen blotting
Northern blotting is a technique used to detect specific RNA sequences in a sample. It involves isolating RNA, separating it via gel electrophoresis, transferring it to a membrane, then hybridizing probes with complementary sequences to the target RNA. This allows visualization of gene expression patterns between tissues, developmental stages, and disease states from the detected RNA sequences.
Southern blotting
Southern blotting is a technique used to detect specific DNA sequences. It involves separating DNA fragments by size via gel electrophoresis, transferring them to a membrane, and using a labeled probe to hybridize to the target sequence. The probe binds only to complementary DNA fragments, allowing their detection and location on the membrane via autoradiography. Key steps include restriction enzyme digestion of DNA, gel electrophoresis, membrane transfer, probe hybridization, and x-ray film development to visualize hybridized fragments. Southern blotting is used for applications like mutation detection, gene rearrangement studies, and forensic analysis.
Northern blotting
Northern blotting is a technique used to detect specific mRNAs in a sample. It involves separating RNA fragments by size through gel electrophoresis, transferring them to a membrane, then using a complementary DNA or RNA probe to identify the mRNA of interest. It allows researchers to examine gene expression by measuring mRNA levels and determine tissue specificity and regulatory factors of gene expression. While it provides useful information, it also has some limitations such as risk of mRNA degradation and relatively low sensitivity compared to PCR-based methods.
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The document discusses different methods of DNA sequencing including the Maxam-Gilbert and Sanger chain termination methods as well as newer next generation sequencing techniques. It describes the principles, steps, and significance of the Maxam-Gilbert and Sanger methods and how next generation sequencing improved DNA sequencing by allowing millions of DNA molecules to be sequenced simultaneously in an automated process.
RFLP
This document discusses Restriction Fragment Length Polymorphism (RFLP) analysis, which is a technique used to differentiate organisms by analyzing patterns in their DNA after digestion with restriction enzymes. RFLP analysis can be used for various applications like determining paternity, detecting mutations, and genetic mapping. The process involves digesting DNA with restriction enzymes, running the fragments on a gel, transferring the DNA to a membrane, and using probes to detect polymorphisms and produce an autoradiogram showing differences in fragment patterns. As an example, the document describes using PCR-RFLP to rapidly screen for a BRCA2 mutation by taking advantage of a restriction site change caused by the mutation.
Sanger sequencing
Sanger sequencing is a method of DNA sequencing developed by Frederick Sanger in 1977 that was widely used for 25 years. It involves making copies of a DNA region using DNA polymerase and chain-terminating dideoxynucleotides that are labeled with different colored dyes. This produces fragmented DNA of different lengths that can be separated by size to determine the DNA sequence. Sanger sequencing is useful for sequencing single genes or short sequences but is limited to read lengths of 300-1000 base pairs. It has been replaced by next generation sequencing methods for most applications.
Blotting techniques
This document discusses various blotting techniques used to detect DNA, RNA, and proteins, including Southern blotting, Northern blotting, and Western blotting. It provides detailed descriptions of the procedures for each technique, including separating biomolecules by electrophoresis, transferring them to a membrane, hybridizing probes, and detecting bound probes. The techniques allow visualization and analysis of specific biomolecules within complex samples.
Northern blot
Northern blotting is a technique used to detect specific RNA molecules in a sample. It involves separating RNA by electrophoresis, transferring it to a membrane, then using a probe with a complementary nucleotide sequence to detect the target RNA. Key steps include isolating high quality RNA, separating it by denaturing gel electrophoresis, blotting it onto a membrane, fixing the RNA to the membrane by UV crosslinking or baking, then detecting it using a labeled probe via hybridization. Northern blotting allows analysis of RNA expression of particular genes and is useful for studying gene expression.
Northern blotting
The northern blot is a technique used to detect specific RNA sequences and analyze gene expression. It involves separating RNA fragments by size through electrophoresis, transferring them to a membrane, then using a probe with a radioactive or fluorescent tag to detect the target RNA sequences through hybridization. This allows researchers to determine which tissues or cell types express certain genes and how expression levels vary. While it only examines one or a few genes at a time, northern blotting provides advantages like direct measurement of RNA size and ability to store membranes for future analysis.
Shahbaz Str
This document discusses short tandem repeats (STRs) which are repeating sequences of 1-10 DNA bases that can be used as genetic markers. STRs are efficiently amplified by PCR and analyzed using fluorescent detection systems or silver-stained gels to identify alleles based on the number of repeats. STR testing results in peak patterns that are converted to genotypes for comparison between laboratories. STRs provide marker loci that can be used for linkage analysis to map genes, determine ancestry from Y-chromosome or surname tests, and for quality assurance such as confirming tissue samples.
Blotting techniques1
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
Northern blotting
Northern blotting is a technique developed in 1977 to detect specific RNA sequences through electrophoresis, transfer to a membrane, and hybridization with a probe. RNA is extracted from tissue samples, separated by size via electrophoresis in an agarose gel, and transferred by capillary action to a nylon or nitrocellulose membrane where it binds. Probes are then hybridized to the RNA and detected through radiolabeling or fluorescence to study gene expression under different conditions.
Presentation blotting
The document discusses various molecular techniques including blotting, probes, and polymerase chain reaction (PCR). It describes Southern blotting for detecting DNA, Northern blotting for RNA, and Western blotting for proteins. It explains how probes are used to identify specific DNA or RNA sequences in Southern and Northern blotting. The key steps of PCR are outlined, including denaturation, annealing of primers, and extension of DNA copies. Applications of these techniques include gene discovery, mutation detection, forensics, diagnosis of genetic disorders, and more. PCR has revolutionized research and diagnostics due to its speed, sensitivity, and specificity.
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Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
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Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
Northern blotting
Northern blotting is a technique used to detect RNA in a sample. It involves isolating RNA from cells, separating RNA fragments by size using gel electrophoresis, transferring the RNA fragments to a membrane, then using a labeled probe to detect specific RNA sequences through hybridization and visualization. The northern blot allows researchers to study gene expression at the mRNA level and determine which genes are expressed under different conditions or in different tissues.
polymorphism
Genetic polymorphisms are normal variations that occur in more than 1% of the population. Single nucleotide polymorphisms are the most common type, involving a change in a single DNA base pair. Polymorphisms can occur in coding and regulatory regions, and may affect protein function or expression levels. The interleukin-1 gene cluster contains polymorphisms that have been associated with increased severity of periodontitis, where individuals with certain IL-1 genotypes experience more rapid bone and tissue loss around teeth. Understanding genetic predispositions can help with early detection and prevention of more serious disease.
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DNA SEQUENCING METHOD
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Pcr primer design
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Microarray (DNA and SNP microarray)
DNA microarrays, also known as DNA chips, allow researchers to analyze large numbers of DNA sequences simultaneously. They contain thousands of unique DNA probes immobilized on a solid surface in an organized grid pattern. Unknown DNA samples are labeled with fluorescent dyes and hybridized to the probes on the chip. The bound DNA is then detected using lasers and analyzed by a computer to determine which genes are present or expressed in the sample. There are two main types of DNA chips - cDNA-based chips containing amplified cDNA probes, and oligonucleotide-based chips containing short, synthesized DNA sequences. DNA microarrays have many applications including gene expression profiling, comparative genomics, disease diagnosis, and drug discovery.
Genome organisation
The document provides information about eukaryotic genome organization. It discusses that eukaryotic DNA is organized into chromosomes that are linear molecules located within the nucleus. The genome contains both coding and non-coding DNA sequences. It also describes various repetitive elements like transposons that make up a significant portion of the genome. Mobile elements can move within genomes and have contributed to genetic variation.
southern blotting.pptx
Blotting
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier.
The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.
Types of blotting techniques
Southern Blotting
Northern Blotting
Western Blotting
A Southern blot is a method used
in molecular biology for detection of a specific DNA sequence in DNA samples.
Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
The method is named after its inventor, the British biologist Edwin Mellor Southern.
- Methods in Southern blotting
- Advantages and disadvantages
Southern blotting
Southern Blotting is a technique developed by E.M. SOUTHERN BLOTTING in 1975. It includes Principle, steps and applications of southern blotting.
Pcr
The polymerase chain reaction (PCR) is a technique used to amplify specific DNA fragments. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. During each cycle, the DNA strand is separated from its complement at a high temperature and two new strands are synthesized from the original copies at a lower temperature, thereby exponentially increasing the number of target DNA copies. Real-time PCR allows for quantification of the target DNA by detecting fluorescence at each cycle, while reverse transcription PCR is used to transcribe RNA into DNA.
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The methods used for DNA finger printing are the same Molecular markers...so for detailed note on the steps which is explained in DNA typing can be used to study the performance pf markers too...
dna sequencing methods
1. DNA sequencing involves determining the order of nucleotide bases in DNA. The original chain termination or dideoxy method developed by Sanger is still widely used for small DNA segments.
2. Whole genome shotgun sequencing breaks large genomes into fragments that are sequenced and then reassembled, allowing sequencing of entire genomes.
3. Pyrosequencing is a sequencing by synthesis method that uses a bioluminescent reaction to determine nucleotides added, enabling accurate and fast sequencing.
Southern blot power point
Southern blotting is a DNA analysis technique developed by Edwin Southern in 1975 that involves separating DNA fragments by size using gel electrophoresis, transferring them to a membrane, and using a radioactive probe to detect complementary DNA sequences by their position on the membrane after developing an X-ray film. Southern blots are commonly used to verify that a desired DNA sequence has been successfully inserted into the genome of a genetically modified organism.
Southern Blotting Technique
This document describes the process of Southern blotting, which is used to detect specific DNA sequences in a sample. It involves purifying DNA from cells, cutting the DNA into fragments using restriction enzymes, separating the fragments by size via gel electrophoresis, transferring them to a membrane, then using a radioactive probe to detect fragments that hybridize based on complementary base pairing. The technique allows identification of mutations, deletions and other genetic variations, and has applications in medical diagnosis, forensics and other areas requiring DNA analysis.
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Northern blotting
The northern blot is a technique used to detect specific RNA sequences and analyze gene expression. It involves separating RNA fragments by size through electrophoresis, transferring them to a membrane, then using a probe with a radioactive or fluorescent tag to detect the target RNA sequences through hybridization. This allows researchers to determine which tissues or cell types express certain genes and how expression levels vary. While it only examines one or a few genes at a time, northern blotting provides advantages like direct measurement of RNA size and ability to store membranes for future analysis.
Shahbaz Str
This document discusses short tandem repeats (STRs) which are repeating sequences of 1-10 DNA bases that can be used as genetic markers. STRs are efficiently amplified by PCR and analyzed using fluorescent detection systems or silver-stained gels to identify alleles based on the number of repeats. STR testing results in peak patterns that are converted to genotypes for comparison between laboratories. STRs provide marker loci that can be used for linkage analysis to map genes, determine ancestry from Y-chromosome or surname tests, and for quality assurance such as confirming tissue samples.
Blotting techniques1
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
Northern blotting
Northern blotting is a technique developed in 1977 to detect specific RNA sequences through electrophoresis, transfer to a membrane, and hybridization with a probe. RNA is extracted from tissue samples, separated by size via electrophoresis in an agarose gel, and transferred by capillary action to a nylon or nitrocellulose membrane where it binds. Probes are then hybridized to the RNA and detected through radiolabeling or fluorescence to study gene expression under different conditions.
Presentation blotting
The document discusses various molecular techniques including blotting, probes, and polymerase chain reaction (PCR). It describes Southern blotting for detecting DNA, Northern blotting for RNA, and Western blotting for proteins. It explains how probes are used to identify specific DNA or RNA sequences in Southern and Northern blotting. The key steps of PCR are outlined, including denaturation, annealing of primers, and extension of DNA copies. Applications of these techniques include gene discovery, mutation detection, forensics, diagnosis of genetic disorders, and more. PCR has revolutionized research and diagnostics due to its speed, sensitivity, and specificity.
Techniques of DNA Extraction, Purification and Quantification
Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
Components of DNA extraction solutions
Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
Northern blotting
Northern blotting is a technique used to detect RNA in a sample. It involves isolating RNA from cells, separating RNA fragments by size using gel electrophoresis, transferring the RNA fragments to a membrane, then using a labeled probe to detect specific RNA sequences through hybridization and visualization. The northern blot allows researchers to study gene expression at the mRNA level and determine which genes are expressed under different conditions or in different tissues.
polymorphism
Genetic polymorphisms are normal variations that occur in more than 1% of the population. Single nucleotide polymorphisms are the most common type, involving a change in a single DNA base pair. Polymorphisms can occur in coding and regulatory regions, and may affect protein function or expression levels. The interleukin-1 gene cluster contains polymorphisms that have been associated with increased severity of periodontitis, where individuals with certain IL-1 genotypes experience more rapid bone and tissue loss around teeth. Understanding genetic predispositions can help with early detection and prevention of more serious disease.
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Dna libraries
DNA libraries allow for the storage and organization of genetic information, similar to how physical libraries store books. There are two main types of DNA libraries: genomic libraries, which are created from genomic DNA and contain entire genes with exons and introns, and cDNA libraries, which are created from mRNA and contain only exons. To create a genomic library, genomic DNA is isolated, fragmented, and inserted into cloning vectors within host bacteria. For cDNA libraries, mRNA is isolated, reverse transcribed into cDNA, which is then amplified and inserted into vectors. Both library types are screened to find clones containing desired DNA sequences.
DNA SEQUENCING METHOD
DNA sequencing determines the order of nucleotide bases in a DNA molecule. The first methods were developed in the 1970s by Maxam and Gilbert (chemical method) and Sanger (chain termination/dideoxy method). Sanger's method is now most commonly used and involves DNA synthesis with chain termination by dideoxynucleotides to generate fragments of different lengths that can be separated and read to determine the DNA sequence. DNA sequencing has revolutionized biological sciences by enabling diagnosis of genetic diseases, identification of disease-causing mutations, and mapping of genomes. It provides benefits for medicine, forensics, and agriculture.
Pcr primer design
The Polymerase Chain Reaction (PCR) revolutionized life sciences by providing an efficient means of amplifying DNA. Invented in 1983 by Kary Mullis, PCR uses the DNA polymerase from Thermus aquaticus bacteria and cycles of heating and cooling to amplify a targeted region of DNA. The primary materials needed for PCR are DNA nucleotides, template DNA, primers that are complementary to the template DNA, and a heat-stable DNA polymerase. PCR changed molecular biology techniques and applications.
Microarray (DNA and SNP microarray)
DNA microarrays, also known as DNA chips, allow researchers to analyze large numbers of DNA sequences simultaneously. They contain thousands of unique DNA probes immobilized on a solid surface in an organized grid pattern. Unknown DNA samples are labeled with fluorescent dyes and hybridized to the probes on the chip. The bound DNA is then detected using lasers and analyzed by a computer to determine which genes are present or expressed in the sample. There are two main types of DNA chips - cDNA-based chips containing amplified cDNA probes, and oligonucleotide-based chips containing short, synthesized DNA sequences. DNA microarrays have many applications including gene expression profiling, comparative genomics, disease diagnosis, and drug discovery.
Genome organisation
The document provides information about eukaryotic genome organization. It discusses that eukaryotic DNA is organized into chromosomes that are linear molecules located within the nucleus. The genome contains both coding and non-coding DNA sequences. It also describes various repetitive elements like transposons that make up a significant portion of the genome. Mobile elements can move within genomes and have contributed to genetic variation.
southern blotting.pptx
Blotting
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier.
The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.
Types of blotting techniques
Southern Blotting
Northern Blotting
Western Blotting
A Southern blot is a method used
in molecular biology for detection of a specific DNA sequence in DNA samples.
Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
The method is named after its inventor, the British biologist Edwin Mellor Southern.
- Methods in Southern blotting
- Advantages and disadvantages
Southern blotting
Southern Blotting is a technique developed by E.M. SOUTHERN BLOTTING in 1975. It includes Principle, steps and applications of southern blotting.
Pcr
The polymerase chain reaction (PCR) is a technique used to amplify specific DNA fragments. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. During each cycle, the DNA strand is separated from its complement at a high temperature and two new strands are synthesized from the original copies at a lower temperature, thereby exponentially increasing the number of target DNA copies. Real-time PCR allows for quantification of the target DNA by detecting fluorescence at each cycle, while reverse transcription PCR is used to transcribe RNA into DNA.
Dna finger printing
The methods used for DNA finger printing are the same Molecular markers...so for detailed note on the steps which is explained in DNA typing can be used to study the performance pf markers too...
dna sequencing methods
1. DNA sequencing involves determining the order of nucleotide bases in DNA. The original chain termination or dideoxy method developed by Sanger is still widely used for small DNA segments.
2. Whole genome shotgun sequencing breaks large genomes into fragments that are sequenced and then reassembled, allowing sequencing of entire genomes.
3. Pyrosequencing is a sequencing by synthesis method that uses a bioluminescent reaction to determine nucleotides added, enabling accurate and fast sequencing.
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Techniques of DNA Extraction, Purification and Quantification
Techniques of DNA Extraction, Purification and Quantification
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Southern blot power point
Southern blotting is a DNA analysis technique developed by Edwin Southern in 1975 that involves separating DNA fragments by size using gel electrophoresis, transferring them to a membrane, and using a radioactive probe to detect complementary DNA sequences by their position on the membrane after developing an X-ray film. Southern blots are commonly used to verify that a desired DNA sequence has been successfully inserted into the genome of a genetically modified organism.
Southern Blotting Technique
This document describes the process of Southern blotting, which is used to detect specific DNA sequences in a sample. It involves purifying DNA from cells, cutting the DNA into fragments using restriction enzymes, separating the fragments by size via gel electrophoresis, transferring them to a membrane, then using a radioactive probe to detect fragments that hybridize based on complementary base pairing. The technique allows identification of mutations, deletions and other genetic variations, and has applications in medical diagnosis, forensics and other areas requiring DNA analysis.
Southern blot method
The document describes the Southern blot technique, which was discovered in 1973 by Edwin Southern at Edinburgh University. The Southern blot allows for the detection of specific DNA sequences and involves separating DNA fragments using gel electrophoresis, transferring them to a membrane, incubating the membrane with a probe that binds to complementary DNA sequences, and revealing the location of the probe using a colorless substrate and X-ray film.
Blotting techniques
This document describes three types of blotting techniques - Southern blotting, Northern blotting, and Western blotting. Southern blotting is used to detect DNA fragments separated by agarose gel electrophoresis. Northern blotting detects specific RNA sequences separated by gel electrophoresis. Western blotting identifies proteins separated by SDS-PAGE gel using an antibody probe. The document provides detailed procedures and applications for each type of blotting.
Blotting techniques includes southren,northern,western and dot blotting
This document describes various blotting techniques used to detect specific DNA or RNA sequences, including Southern blotting, Northern blotting, Western blotting, and dot blotting. Southern blotting involves transferring DNA fragments separated by gel electrophoresis to a membrane and probing for specific sequences. Northern blotting is similar but uses RNA. Western blotting detects specific proteins. Dot blotting detects sequences in non-fractionated samples by directly applying samples to a membrane. These techniques allow for detection and analysis of genetic material.
Southern blotting
Southern blotting is a technique used to detect specific DNA sequences. It involves extracting DNA from cells, cutting the DNA with restriction enzymes, separating fragments via electrophoresis, transferring DNA to a membrane, then using a labeled probe to hybridize and identify the target sequence via autoradiography. Southern blotting allows identification of mutations, deletions and rearrangements, and is used for cancer prognosis, genetic disease diagnosis, DNA fingerprinting and more. It is an effective detection method but is also complex, labor-intensive, and time-consuming.
Southern northern and western blotting
Southern, Northern, and Western blotting are techniques to detect specific biomolecules separated by gel electrophoresis. Southern blotting detects DNA using DNA probes, Northern blotting detects RNA using RNA or DNA probes, and Western blotting detects proteins using antibodies. All three techniques involve transferring the separated biomolecules from a gel to a membrane, probing with a labeled detection molecule, washing unbound probes, and detecting signals.
Southern blot
The document summarizes the Southern blot technique. It involves digesting DNA with restriction enzymes, separating fragments by size via gel electrophoresis, transferring fragments to a membrane, and detecting a specific DNA sequence using a radiolabeled probe. The Southern blot allows determining the size of a restriction fragment, measuring amounts between samples, and locating a specific sequence within a complex DNA mixture. It has applications in gene discovery, mapping, identification of transgenic genes, and DNA fingerprinting.
Western Blot
The document provides a detailed overview of the Western blot technique. It describes the key steps as:
1) Tissue preparation and protein extraction from samples.
2) Separation of proteins by gel electrophoresis based on molecular weight.
3) Transfer of proteins from the gel to a membrane for detection.
4) Blocking of the membrane to prevent non-specific antibody binding.
5) Detection of target proteins using primary and secondary antibodies, and various detection methods.
Northern blotting
Northern blotting is a technique used to detect specific RNA sequences in a sample. It involves separating RNA samples by size through gel electrophoresis, transferring them to a nylon membrane, and using a labeled probe to detect the target RNA sequence through hybridization. The target RNA will be visible as a band on the membrane where the probe has bound. This allows researchers to observe gene expression levels under different conditions like development or disease.
Northern blotting
Northern blotting (RNA Dectection by botting process)
Southern and northern blotting comparison (similarities and differences)
Southern blot
Southern Blot is a technique invented by Edwin Southern in the 1970s to locate specific DNA sequences within a larger sample of DNA. It works by cutting DNA with restriction enzymes, separating the fragments via gel electrophoresis, transferring them to a membrane, and using a radioactive or enzyme-labeled probe to bind to complementary DNA sequences, revealing their locations. Southern Blot can be used to map genes, for forensic analysis, and to discover new genes or detect diseases.
Southern blot
Southern blotting is a technique developed in the 1970s by Edward Southern to detect specific DNA sequences within a complex DNA sample by separating DNA fragments by size after restriction enzyme digestion, transferring them to a membrane, and using a labeled probe to bind to the target sequence for visualization. It is commonly used for gene discovery, mapping, evolution and development studies, diagnostics, and ensuring successful incorporation of DNA into a host genome.
Blotting techniques in Biotechnology
This document provides an overview of blotting techniques, including transfer methods, membranes used, and applications of Southern blotting, Northern blotting, and Western blotting. It discusses how blotting involves transferring proteins, DNA, or RNA from a gel onto a membrane. It describes the capillary, electro-blotting, and vacuum blot transfer processes and membranes like nitrocellulose, nylon, and PVDF. It then explains the specific methods and applications of Southern blotting for detecting DNA, Northern blotting for detecting RNA, and Western blotting for detecting proteins.
Fundamentos de técnicas blotting: Southern, Northern, Western.
El documento describe las técnicas de Southern blot, Northern blot y Western blot. Estas técnicas comienzan con la separación de moléculas (ADN, ARN o proteínas) mediante electroforesis en gel. Luego, las moléculas se transfieren a una membrana para identificar fragmentos específicos mediante sondas marcadas o anticuerpos. El Southern blot se usa para detectar secuencias de ADN, el Northern blot para detectar genes transcriptos, y el Western blot para detectar proteínas mediante anticuerpos.
Western blot.
El Western blot es una técnica que permite detectar proteínas específicas en una muestra mediante la separación de las proteínas por electroforesis en un gel y su posterior transferencia a una membrana. La membrana se incuba con anticuerpos primarios y secundarios para identificar las proteínas de interés, y las bandas se detectan comúnmente por quimioluminiscencia o fluorescencia.
Fluorescent in-situ Hybridization (FISH)
Fluorescent in-situ hybridization (FISH) is a technique that uses fluorescent probes that bind to only parts of the genome to detect and localize specific DNA sequences on chromosomes. It involves denaturing and hybridizing a fluorescent probe to a complementary DNA or RNA target sequence on preserved tissue. The bound probe can then be visualized with a fluorescent microscope to identify specific gene mutations, abnormalities, or other chromosome changes to aid in cancer diagnosis, prenatal testing, and other clinical applications.
Dna fingerprinting
DNA fingerprinting is a method of identifying individuals based on unique characteristics in their DNA. Alec Jeffreys developed this method in the 1980s to solve criminal cases using DNA profiling techniques. Modern DNA profiling uses short tandem repeats that are variable between individuals and can be used to determine biological relationships in forensic investigations and parental testing. DNA profiling analysis involves extracting DNA from samples, amplifying targeted regions, separating the fragments by size, and comparing the profiles to determine matches or exclusions.
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Blotting techniques includes southren,northern,western and dot blotting
Blotting techniques includes southren,northern,western and dot blotting
Fundamentos de técnicas blotting: Southern, Northern, Western.
Fundamentos de técnicas blotting: Southern, Northern, Western.
Similar to Southern blotting
Prabhakar singh ii sem-paper v-blotting techniques
Prabhakar singh ii sem-paper v-blotting techniquesDepartment of Biochemistry, Veer Bahadur Singh Purvanchal Univarsity, Jaunpur
1. Blotting techniques like Southern, Northern, and Western blotting are used to transfer and detect DNA, RNA, and proteins separated by gel electrophoresis.
2. Southern blotting involves transferring DNA fragments to a membrane, then using labeled probes to detect specific DNA sequences through hybridization and autoradiography. It is useful for mapping genes.
3. Northern and Western blotting follow similar procedures to detect RNA and proteins, respectively.Blotting techniques.pptx
Blotting techniques involve the separation (via electrophoresis) and transfer of DNA, RNA, or proteins onto a blotting membrane. This separation is generally followed by complexing of the target with a labeled molecule for detection.
Used extensively in analysis of:
DNA.
RNA.
Proteins.
Blotting techniques
The document discusses various blotting techniques used to transfer and detect DNA, RNA, and proteins, including Southern blotting, Northern blotting, and Western blotting. Southern blotting was developed by Edwin Southern in 1975 to detect specific DNA sequences and involves transferring DNA from a gel to a membrane. Northern blotting, developed in 1979, detects specific RNA sequences and involves transferring RNA. Western blotting, developed in 1981, detects specific proteins and involves separating proteins by gel electrophoresis, transferring them to a membrane, and using antibodies to detect proteins of interest. These techniques are widely used analytical tools.
Lecture 5 dna finger, foot printing rflp
This document discusses DNA fingerprinting techniques including restriction fragment length polymorphism (RFLP) and DNA footprinting. RFLP involves using restriction enzymes to cut DNA at specific sites, resulting in fragments of varying lengths that can be used to differentiate individuals. DNA footprinting identifies the specific binding sites of DNA-binding proteins by detecting regions of DNA that are protected from cleavage by bound proteins. The document provides detailed explanations of the principles and procedures of these techniques.
Blotting techniques (manish)
The document discusses various blotting techniques used to transfer and detect DNA, RNA, and proteins, including Southern blotting for DNA, Northern blotting for RNA, and Western blotting for proteins. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, and using a probe to detect specific sequences. It allows researchers to identify genes, map genomes, and study evolution and disease.
Southern-blotting-and-Western-blotting.pptx
1. DNA or proteins are separated by gel electrophoresis.
2. The molecules are then transferred to a membrane through blotting.
3. For Southern blotting, DNA is detected using labeled probes that hybridize to complementary DNA sequences. For Western blotting, proteins are detected using primary and secondary antibodies that bind to the protein of interest.
4. These techniques are used for applications like identifying genes, detecting infectious diseases and genetic disorders, and forensic analysis.
Nucleic acid hybridization
Nucleic acid hybridization is a technique used to identify specific DNA sequences. It involves denaturing DNA or RNA samples and probes, followed by annealing of the probes to complementary sequences. There are two main types: Southern blotting separates DNA fragments by gel electrophoresis before hybridization with probes, while Northern blotting separates RNA this way. Both techniques allow detection of specific sequences through the use of labeled probes.
Souther and northern blotting systems
Southern blotting and Northern blotting are techniques used to detect specific DNA and RNA sequences. Southern blotting involves transferring DNA fragments separated by gel electrophoresis to a membrane, then using a labeled probe to identify fragments by hybridization. Northern blotting follows similar steps but detects RNA, requiring formaldehyde treatment to denature RNA. Both techniques allow identification of specific molecules in complex mixtures.
Blotting techniques
This document describes various blotting techniques used to detect specific DNA or RNA sequences, including Southern blotting, Northern blotting, Western blotting, and dot blotting. Southern blotting involves transferring DNA fragments separated by gel electrophoresis to a membrane and probing for specific sequences. Northern blotting is similar but uses RNA. Western blotting detects specific proteins. Dot blotting detects sequences in non-fractionated samples by directly applying samples to a membrane. These techniques allow for detection and analysis of genes and their expression.
VBC-321_BLOTTING_TECHNIQUES.pptx
1. Blotting techniques such as Southern, Northern, and Western blotting allow for the transfer of DNA, RNA, and proteins from a gel to a membrane for detection.
2. The Southern blot detects DNA using hybridization with a labeled probe. The Northern blot detects RNA and the Western blot detects proteins using antibodies.
3. These techniques separate biomolecules by size then transfer and detect them on a membrane using probes or antibodies, allowing analysis of complex samples.
blooting image.pptx
1. DNA or proteins are separated by gel electrophoresis.
2. The molecules are then transferred to a membrane through either southern or western blotting.
3. For southern blotting, DNA is detected through hybridization with a labeled probe. For western blotting, specific proteins are detected through interaction with labeled antibodies.
4. These techniques are used for diagnostic purposes and research applications such as identifying DNA sequences or proteins of interest.
SOUTHERN BLOTTING SMG
Southern blotting is a technique used to detect specific DNA sequences. It involves separating DNA fragments by electrophoresis in an agarose gel, transferring the fragments to a nitrocellulose membrane, and using a radioactive DNA probe to hybridize to complementary DNA sequences on the membrane. The probe-hybridized DNA can then be visualized using autoradiography. Key steps include cleaving DNA with restriction enzymes, gel electrophoresis, transferring DNA from the gel to nitrocellulose via capillary action, fixing the DNA to the membrane by baking, hybridizing with a radioactive probe, and detecting hybridized fragments using autoradiography.
Northern, southern and western blotting
The document discusses various techniques used for nucleic acid hybridization, including Southern blotting, Northern blotting, dot blot hybridization, and in situ hybridization. Southern blotting involves separating DNA fragments by size, transferring them to a membrane, and using a labeled probe to detect complementary DNA sequences. It can be used to detect mutations. Northern blotting is similar but detects RNA. Dot blot hybridization spots DNA/RNA samples directly onto a membrane. In situ hybridization detects nucleic acids within intact cells using labeled probes. Microarrays allow simultaneous screening of thousands of genes using hybridization on an array.
Lecture 3 southern blotting
This document discusses blotting techniques, specifically Southern blotting. It defines Southern blotting as a technique developed by Professor Sir Edwin Southern in 1975 to detect specific DNA sequences. The technique involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, then using a labeled probe to detect complementary DNA sequences via hybridization. Southern blotting is used for applications like gene discovery, mapping, diagnostics, and DNA fingerprinting.
Blotting & its types
Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow for the detection of specific DNA, RNA, and protein sequences by transferring them from a separation gel onto a membrane and using probes to detect the targets. Southern blotting detects DNA using DNA probes, Northern blotting detects RNA using RNA or DNA probes, and Western blotting detects proteins using antibodies. These techniques are used for applications like detecting gene expression, RNA splicing, and confirming diseases.
Blotting techniques1
This document discusses different blotting techniques used to detect DNA, RNA, and proteins. Southern blotting is used to detect DNA and was developed by Edwin Southern in 1975. It involves separating DNA fragments by size, transferring them to a membrane, and using a probe to detect specific sequences. Northern blotting detects RNA and was developed in 1979. It similarly separates RNA and uses a probe to detect sequences of interest. Western blotting detects proteins and was developed in 1981. It separates proteins by SDS-PAGE gel electrophoresis, transfers them to a membrane, and uses an antibody probe to detect the target protein. These techniques are widely used in research and diagnostics.
Blotting techniques
Southern, Northern, and Western blotting techniques allow for the detection of specific DNA, RNA, and protein fragments, respectively. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, then using a labeled probe for detection via hybridization. Northern blotting is similar but detects RNA. Western blotting separates proteins via gel electrophoresis, transfers them to a membrane, then uses primary and secondary antibodies for detection. These techniques are used for applications like gene mapping and the study of gene expression.
Presentation1
This presentation provides an overview of northern blotting. Northern blotting is a technique used to detect RNA in a sample. It involves separating RNA fragments via 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 cells, separating fragments by size via gel electrophoresis, transferring RNA to a membrane, hybridizing the membrane with a labeled probe, and detecting the RNA of interest on the membrane. Northern blotting allows researchers to study gene expression patterns and RNA splicing.
Rflp technology
RFLP is a technique that differentiates organisms by analyzing patterns in DNA fragments produced after digestion with restriction enzymes. If two organisms differ in the distance between restriction sites, the lengths of fragments produced will differ. These patterns can differentiate species and strains. RFLP detection relies on comparing band profiles after digestion and gel electrophoresis to see length polymorphisms, which can then be examined further through hybridization and visualization. RFLPs have applications in forensics, disease detection, and human population genetics.
Blotting techniques
This document summarizes several blotting techniques used in molecular biology to detect biomolecules like DNA, RNA, and proteins. Southern blotting detects specific DNA sequences, Northern blotting detects RNA, and Western blotting detects proteins. Eastern blotting detects post-translational modifications to proteins. Dot blotting directly applies biomolecule samples to a membrane as dots to detect presence or absence without separation by size. These techniques involve transferring molecules to a membrane, probing with a labeled probe, washing, and detecting hybridized probes.
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Southern blotting
- 3. Southern blotting is a process that transfer the DNA fragments that separated by electrophoresis into a nitrocellulose membrane/ nylon.
- 4. To determine the DNA sequence between 2 DNA samples . Whether the DNA sequence is present or not. There is also lot of blotting methods like nothern,southern,western blotting.
- 5. Southern blotting involved in five main processes 1.digest DNA 2.RUN digest DNA into agarose gel 3.Denature the DNA 4.Transfer the denatured DNA to the membrane 5. hybridization. 6.visualization
- 6. 1. DNA DIGEST Digest the SAMPLE BY USING specific Restriction enzymes . 2.TRANSFER DNA digest into agarose gel 3.denature of DNA separate double-stranded DNA into single-stranded DNA. Only ssDNA can transfer.(0.5M NAOH,0.2M HCL)
- 8. Nitrocellulose membrane or nylon can be used for this step. because they have small pores that allows the capillary motion. Transfer is usually done by capillary force CAN use a vacuum blot apparatus instead of capillary action
- 10. AFTER THAT treat the membrane with UV light or bake the sample in 800c. This causes cross links the DNA to the membrane. (via non-covalent semi permanent bonds) Now the DNA became immobilized in membrane.
- 12. We need a radio active probe to determine presence of a specific sequence in the DNA FRAGMENT. The blotted DNA fragments then dipped into a buffer solution and mix with radio active probes and sealed and rotated..
- 13. We want to make sure there is enough probes to bind with DNA fragments. In autoradiograph THEY ENCOUNTERED A PROBLEM !!!! the probes that didn’t bound with DNA fragments also there. this effect our final result.(probes bounded with membrane) So they introduced a technique call prehybridization.
- 14. This step involved in closing the binding site of menbrane other than DNA fragments. For this a special prehybridization solution is used. Such as-1.polyvinylpyrrolidone 2.bovine serum albumin 3.dried milk 4.other organism DNA (salmon sperm DNA) After this, membrane is washed and ready for hybridization.
- 15. Visualize your radioactively labeled target sequence BY autoradiograph
- 16. Using nylon instead of nitrocellulose membrane is speed up the movements of DNA.(membrane=18 h,nylon=2h) Electro blotting –using electrophorosis between gel to nylon speed up the DNA movements. Vacum blotting
Editor's Notes
- PRO HYBRIDIZATION ALSO THERE.