Restriction fragment length polymorphism
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Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the English scientist Alec Jeffreys during research into hereditary diseases. It is used for the analysis of unique patterns in DNA fragments in order to genetically differentiate between organisms – these patterns are called Variable Number of Tandem Repeats (VNTRs). Genetic polymorphism is defined as the inherited genetic differences among individuals in over 1% of normal population. The RFLP technique exploits these differences in DNA sequences to recognize and study both intraspecies and interspecies variation.
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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.
RFLP
RFLP is a technique that uses restriction enzymes to cut DNA into fragments at specific nucleotide sequences. These fragments are then separated by gel electrophoresis and analyzed by hybridization with labeled probes. Differences in the fragment patterns between individuals or strains can be used for applications like genome mapping, disease analysis, and criminal investigations. RFLP provides semi-dominant markers but requires high quality DNA and is a long, labor-intensive process.
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1) DNA sequencing refers to determining the order of nucleotide bases (A, G, C, T) in a DNA molecule. This provides essential genetic information for growth and development.
2) Two major early methods for DNA sequencing were the chemical cleavage method developed by Maxam and Gilbert in 1977 and the chain termination method developed by Sanger. Sanger's method became more popular due to fewer toxic chemicals.
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Rflp
Restriction fragment length polymorphism (RFLP) is a technique used to detect differences in DNA sequences among individuals. It involves cutting DNA with restriction enzymes, separating the fragments by size through gel electrophoresis, and comparing fragment lengths between individuals or populations. RFLP analysis was an early DNA profiling method and is still used for genome mapping, disease risk assessment, and paternity testing by detecting differences in restriction fragment sizes.
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This document discusses Restriction Fragment Length Polymorphism (RFLP) analysis. RFLP is a technique used to detect genetic mutations and variations between individuals. It works by digesting DNA with restriction enzymes, which cut the DNA into fragments of varying lengths. These fragments are then separated via gel electrophoresis and analyzed to detect any length polymorphisms between individuals, indicating genetic differences. RFLP has applications in forensics, mutation detection, and requires isolating DNA, restriction digestion, gel electrophoresis, Southern blotting, and DNA hybridization.
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This document discusses various molecular marker techniques used in genetics, including their discovery and applications. It covers:
- RFLP (restriction fragment length polymorphism), the first widely used molecular marker technique, which detects variations in DNA fragments after restriction enzyme digestion.
- RAPD (random amplified polymorphic DNA) which uses random primers to amplify variable DNA regions by PCR for genetic mapping.
- AFLP (amplified fragment length polymorphism) which combines restriction enzyme digestion and PCR amplification to generate multiple polymorphic DNA fragments.
- SSR (simple sequence length polymorphism) markers which detect variations in short tandem repeats useful for genetic linkage maps.
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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.
RFLP
RFLP is a technique that uses restriction enzymes to cut DNA into fragments at specific nucleotide sequences. These fragments are then separated by gel electrophoresis and analyzed by hybridization with labeled probes. Differences in the fragment patterns between individuals or strains can be used for applications like genome mapping, disease analysis, and criminal investigations. RFLP provides semi-dominant markers but requires high quality DNA and is a long, labor-intensive process.
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Polymerase chain reaction (PCR) amplifies a specific DNA sequence in vitro. It involves repeated cycles of heating and cooling DNA in the presence of primers, DNA polymerase, and nucleotides. Each cycle doubles the number of DNA copies. After many cycles, a single copy of DNA can be amplified exponentially to generate millions of copies. PCR has numerous applications including disease diagnosis, genetic fingerprinting, gene cloning, forensics, and molecular biology research.
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1) DNA sequencing refers to determining the order of nucleotide bases (A, G, C, T) in a DNA molecule. This provides essential genetic information for growth and development.
2) Two major early methods for DNA sequencing were the chemical cleavage method developed by Maxam and Gilbert in 1977 and the chain termination method developed by Sanger. Sanger's method became more popular due to fewer toxic chemicals.
3) Modern DNA sequencing often uses fluorescent dye-labeled chain terminators and capillary electrophoresis. Each dye fluoresces at a different wavelength, allowing all four reactions to occur in one tube. This high-throughput automated approach has accelerated genomic research.
Rflp
Restriction fragment length polymorphism (RFLP) is a technique used to detect differences in DNA sequences among individuals. It involves cutting DNA with restriction enzymes, separating the fragments by size through gel electrophoresis, and comparing fragment lengths between individuals or populations. RFLP analysis was an early DNA profiling method and is still used for genome mapping, disease risk assessment, and paternity testing by detecting differences in restriction fragment sizes.
VNTR- Minisatellite
The document discusses repetitive DNA elements in human chromosomes, focusing on tandem repeats classified as satellites, minisatellites, and microsatellites. It describes the characteristics of each type of repeat, including length, copy number, location, and uses. Variable number tandem repeats (VNTRs) are highlighted as being highly polymorphic due to variation in repeat number between individuals, making them useful for genetic analysis and forensic identification.
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This document discusses Restriction Fragment Length Polymorphism (RFLP) analysis. RFLP is a technique used to detect genetic mutations and variations between individuals. It works by digesting DNA with restriction enzymes, which cut the DNA into fragments of varying lengths. These fragments are then separated via gel electrophoresis and analyzed to detect any length polymorphisms between individuals, indicating genetic differences. RFLP has applications in forensics, mutation detection, and requires isolating DNA, restriction digestion, gel electrophoresis, Southern blotting, and DNA hybridization.
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This document discusses various molecular marker techniques used in genetics, including their discovery and applications. It covers:
- RFLP (restriction fragment length polymorphism), the first widely used molecular marker technique, which detects variations in DNA fragments after restriction enzyme digestion.
- RAPD (random amplified polymorphic DNA) which uses random primers to amplify variable DNA regions by PCR for genetic mapping.
- AFLP (amplified fragment length polymorphism) which combines restriction enzyme digestion and PCR amplification to generate multiple polymorphic DNA fragments.
- SSR (simple sequence length polymorphism) markers which detect variations in short tandem repeats useful for genetic linkage maps.
- SNPs (single nucleotide polymorphisms)
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RFLP
Molecular marker
Method of RFLP
Various applications of RFLP
Advantages and Disadvantages of RFLP
molecular marker RFLP, and application
INTRODUCTION
RFLP
Analysis technique
APPLICATION OF RFLP
FORENSIC
DISEASE PROGNOSIS
GENETIC COUNSELING
PEDIGREE
VARIETAL
Advantages of RFLP
Disadvantages of RFLP
CONCLUSIONS
REFERENCES
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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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Fred Sanger developed the chain termination method of DNA sequencing that is still widely used today. It involves making multiple copies of a DNA fragment, conducting separate sequencing reactions with each of the four dideoxynucleotides, and using gel electrophoresis or capillary electrophoresis to determine the sequence based on fragment sizes. While it can sequence fragments up to 900 base pairs, Sanger sequencing is time-consuming and expensive for large genomes. However, it provides high quality sequence data and remains useful for targeted sequencing applications.
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1. The document discusses various blotting techniques including Southern blotting, Northern blotting, and Western blotting. It provides a brief history and overview of each technique.
2. Southern blotting is used to detect DNA, Northern blotting detects RNA, and Western blotting detects proteins. The techniques involve separating biomolecules by electrophoresis, transferring them to a membrane, then using a probe for detection.
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BCHM 415- RFLP.pptx
Restriction Fragment Length Polymorphism (RFLP) is a technique used to differentiate organisms by analyzing patterns in their DNA generated by restriction enzymes. RFLP involves digesting DNA with restriction enzymes, separating the fragments via gel electrophoresis, transferring the fragments to a membrane, and detecting specific fragments using labelled probes. RFLP can be used for applications like determining paternity in criminal cases by comparing DNA fragment patterns between individuals. While useful, RFLP has limitations like being slow, requiring large DNA samples, and not allowing for automation.
Rflp
RFLP is a technique that uses restriction enzymes to cut DNA into fragments of varying lengths that can then be used to differentiate between organisms. It involves digesting DNA with restriction enzymes, separating the fragments via gel electrophoresis, transferring the fragments to a membrane, and detecting specific fragments with labelled probes. RFLP has applications in paternity testing, disease detection, genetic mapping, and studying genetic diversity in populations. However, it is also a slow and cumbersome technique that requires a large DNA sample.
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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.
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Molecular marker
Method of RFLP
Various applications of RFLP
Advantages and Disadvantages of RFLP
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INTRODUCTION
RFLP
Analysis technique
APPLICATION OF RFLP
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DISEASE PROGNOSIS
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Advantages of RFLP
Disadvantages of RFLP
CONCLUSIONS
REFERENCES
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Restriction enzymes are endonucleases found in bacteria and archaea that provide defense against viruses by selectively cutting invading viral DNA. Over 3,000 restriction enzymes have been identified, with some being commercially available. They recognize specific DNA sequences and cut the phosphodiester bonds within or near the recognition site. Restriction Fragment Length Polymorphism (RFLP) analyzes patterns from cleavage of DNA with restriction enzymes to differentiate organisms. RFLPs have forensic and medical applications such as paternity testing and disease detection.
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Restriction Digestion: DNA from a biological sample (such as blood or tissue) is broken into smaller fragments using restriction enzymes, which cut the DNA at specific sequences.
Electrophoresis: The DNA fragments are separated based on their molecular weights using gel electrophoresis. This process allows smaller fragments to move faster than larger fragments.
Transfer to Membrane: The DNA fragments are transferred from the gel onto a solid membrane, typically a positively charged nylon membrane, using capillary action.
Hybridization: The membrane is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The probe is designed to be complementary to the target DNA sequence, allowing it to bind to the specific DNA fragment on the membrane.
Detection: The bound probe is detected using methods such as X-ray film, phosphorimaging, or chemiluminescent substrates, depending on the type of label used.
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3. These techniques separate biomolecules by size then transfer and detect them on a membrane using probes or antibodies, allowing analysis of complex samples.
Blotting techniques
This document provides information on immunoblotting techniques such as Western blotting. It discusses the basic workflow of Western blotting which includes sample preparation, gel electrophoresis, protein transfer, blocking and antibody probing, detection, and analysis. It also summarizes other blotting techniques like Southern blot which is used to detect specific DNA sequences. The applications of Western and Southern blotting in research and clinical diagnosis are also briefly mentioned.
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
The document describes various blotting techniques used to detect DNA, RNA, and proteins. It discusses Southern blotting, which is used to detect specific DNA sequences through separation, transfer, and hybridization. The overall steps involve DNA fragmentation, gel electrophoresis, transfer to a membrane, hybridization with a probe, washing, and detection. Northern blotting is similar but detects RNA and uses formaldehyde during gel electrophoresis. Western blotting detects specific proteins by separating proteins by size on a gel, transferring them to a membrane, labeling with antibodies, washing, and detecting the bound antibodies. These techniques allow for detection of specific biomolecules.
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.
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Southern blotting Technique (Nucleic acid hybridization).pptx
Southern blotting Technique (Nucleic acid hybridization).pptx
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Warfare is defined as methods of fighting war. Biological warfare has been used throughout history, such as during the 14th century Black Plague where infected corpses were catapulted over city walls. In World War I, Germany aimed to use anthrax, cholera, and plague as biological weapons against several countries. Biological, chemical, nuclear, and cyber warfare are all methods that have been used or proposed for use in warfare.
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Algae has significant potential as a biofuel feedstock due to its high oil yield per acre, ability to grow in diverse environments unsuitable for other crops, and ability to solve land use issues. Algae can produce 30 times more oil per acre than soybeans or palm, and yields of up to 94,000 liters per acre are possible. It does not require prime farmland and can use wastewater, making it less likely to drive food prices up. The oil produced from algae has benefits like being non-polluting and high in oxygen with zero sulfur content. While challenges remain in harvesting, oil extraction and generating fuel at scale, algae biofuel holds promise as one of the leading renewable fuels due
Endoplasmic Reticulum and Golgi Apparatus.pptx
The document summarizes key aspects of the endoplasmic reticulum (ER), Golgi apparatus, and their discovery. It discusses how the ER was first observed in 1945 and forms an interconnected network involved in protein transport and synthesis. The Golgi apparatus was discovered in 1898 and helps process and package proteins and lipids. It has a stacked structure and modifies proteins as they pass through. Both organelles are essential in the transport and processing of molecules within eukaryotic cells.
Mechanism of Generation and Propagation of Nerve Impulse.pptx
1. Neurons are specialized cells that carry signals in the nervous system. A neuron consists of a cell body, dendrites which receive signals, and an axon which conducts signals.
2. When a neuron is at rest, it maintains a negative resting membrane potential through ion gradients. When stimulated, voltage-gated sodium channels open, sodium rushes in, and the membrane depolarizes in an action potential.
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Mechanism of Generation and Propagation of Nerve Impulse.docx
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Endomembrane system
The Endomembrane system is composed of the different membranes that are suspended in the cytoplasm within a eukaryotic cell. These membranes divide the cell into functional and structural compartments, or organelles.
In simple words Endomembrane System is network of single membrane bound cell organelles performing coordinated functions.
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Mechanism of Generation and Propagation of Nerve Impulse.pptx
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Nobel Prize winners in the field of Human Physiology.docx
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Equivariant neural networks and representation theory
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Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
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Restriction fragment length polymorphism
- 1. Faculty of Biosciences, Institute of Biosciences and Technology, Shri Ramswaroop Memorial University By Abhinav Baranwal Roll No. 202110902120035 BSc. (H) Biotechnology 1st Year Submitted To Dr. Nishu Mittal
- 2. • Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the English scientist Alec Jeffreys during research into hereditary diseases. It is used for the analysis of unique patterns in DNA fragments in order to genetically differentiate between organisms – these patterns are called Variable Number of Tandem Repeats (VNTRs). • Genetic polymorphism is defined as the inherited genetic differences among individuals in over 1% of normal population. The RFLP technique exploits these differences in DNA sequences to recognize and study both intraspecies and interspecies variation. Restriction Fragment Length Polymorphism (RFLP)
- 4. Principles of RFLP • RFLP is an enzymatic procedure for separation and identification of desired fragments of DNA. • Using Restriction Endonuclease enzymes fragments of DNA is obtained and the desired fragment is detected by using restriction probes. • May be used to differentiated two organism by analysis of patterns derived from cleavage of their DNA.
- 5. Procedures or Steps of RFLP Test Step I: Restriction digest •Extraction of desired fragments of DNA using restriction endonuclease (RE). •The enzyme RE has specific restriction site on the DNA, so it cut DNA into fragments. Different size of fragments are generated along with the specific desired fragments. Step II: Gel electrophoresis •The digested fragment are run in polyacrylamide gel electrophoresis or Agarose gel electrophoresis to separate the fragments on the basis of length or size or molecular weight. •Different size of fragments form different bands.
- 6. Step IV: Blotting •The single stranded DNA obtained are transferred into charge membrane i.e. Nitrocellulose paper by the process called capillary blotting or electro-blotting. Step III: Denaturation •The gel is placed in sodium hydroxide (NaOH) solution for denaturation so that single stranded DNA are formed. Step V: Baking and blocking •The nitrocellulose paper transferred with DNA is fixed by autoclaving. •Then the membrane is blocked by using bovine serum albumin or casein to prevent binding of labelled probe nonspecifically to the charged membrane.
- 7. Step VI: Hybridization and visualization •The labelled RFLP probe is hybridized with DNA on the nitrocellulose paper. •The RFLP probes are complimentary as well as labelled with radioactive isotopes so they form color band under visualization by autoradiography.
- 8. Applications of RFLP •To determine the status of genetic diseases such as Cystic Fibrosis in an individual. •To determine or confirm the source of a DNA sample such as in paternity tests or criminal investigations. •In genetic mapping to determine recombination rates that show the genetic distance between the loci. •To identify a carrier of a disease-causing mutation in a family.