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Restriction fragment length polymorphism (RFLP) analysis detects variations in DNA fragment length to distinguish individuals. It involves incubating DNA with restriction endonucleases that cut DNA at specific recognition sites. Differences in the resulting fragment patterns between individuals arise from variations in restriction enzyme recognition sites. RFLP analysis can be used for genetic fingerprinting, paternity testing, and studying diversity. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) are PCR-based methods that detect polymorphisms without prior DNA sequence knowledge and are faster than RFLP analysis.
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PCR can be used for a variety of applications including isolating and cloning genes, cDNA synthesis, mutagenesis, molecular markers, analyzing ancient DNA, medical diagnostics, and forensics. Molecular biologists use PCR to identify specific genes and make multiple copies. Forensic technicians use PCR to help identify suspects and victims based on DNA amplification patterns. PCR has impacted many areas of life and promises further exciting discoveries.
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Handout available at
https://drive.google.com/open?id=0B52D4j3rC4WDN2hRV25CaW9kb0E
Define DNA sequencing.
Describe the history of DNA sequencing.
Describe the steps of Sanger sequencing.
Define next generation sequencing.
Describe the steps of next generation DNA sequencing.
Describe use of DNA sequencing.
Sanger Technique
The Sanger technique is a method for DNA sequencing developed in 1977 that uses chain-terminating dideoxynucleotides. It relies on the fact that DNA synthesis will stop if a dideoxynucleotide is incorporated instead of a normal deoxynucleotide. The technique involves incubating a single-stranded DNA with the four dideoxynucleotides separately, which results in DNA fragments of different lengths that can be separated via gel electrophoresis to determine the sequence.
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DNA sequencing techniques allow determining the sequence of nucleotides in a DNA molecule. In 1977, Sanger and Gilbert independently developed methods for DNA sequencing. Sanger's dideoxy method, also known as chain termination method, uses DNA polymerase and a mixture of dNTPs and ddNTPs, which lack a 3'-OH group and terminate DNA strand extension. Electrophoresis then separates DNA fragments by size to reveal the sequence. Sanger's method became the most widely used technique and helped enable major genome sequencing projects like the Human Genome Project.
Molecular Markers and Applications-Lecture.pdf
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DNA sequencing involves determining the order of nucleotides in DNA. It is used to diagnose diseases, synthesize DNA for analysis, understand how the body works, and more. Several methods of DNA sequencing exist, including Sanger sequencing, Illumina sequencing using fluorescently labeled nucleotides, Roche 454 sequencing detecting light from nucleotide reactions, and Ion Torrent sequencing measuring pH changes from nucleotide binding. DNA sequencing is becoming faster and cheaper due to parallelization and new technologies.
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Genome sequencing is the process of determining the order of nucleotide bases - A, C, G, and T - that make up an organism's DNA. Shotgun sequencing involves randomly breaking the genome into small fragments, sequencing those pieces, and reassembling the sequence by identifying overlapping regions. It was originally used by Sanger to sequence small genomes like viruses and bacteria. There are two main methods - hierarchical shotgun sequencing for larger genomes containing repeats, and whole genome shotgun sequencing for smaller genomes.
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Pyrosequencing slide presentation rev3.
Pyrosequencing was evaluated as an alternative to Sanger sequencing for HIV drug resistance genotyping. Three assays were designed to detect mutations in the protease gene using a 356 bp amplicon and three sequencing primers. The assays demonstrated good linearity and sensitivity below 5% in quantifying mixed bases. However, read lengths were limited due to signal degradation, making it difficult to sequence large amplicons. Overall, pyrosequencing showed promise as a method for HIV genotyping but further optimization was needed to improve read lengths and mixed variant detection.
Pyrosequencing
Pyrosequencing is a sequencing method that detects DNA polymerase activity by measuring the release of pyrophosphate using a cascade of enzymatic reactions that generate visible light. It utilizes emulsion PCR to amplify DNA fragments on beads in microreactors. The beads are then loaded into wells and sequenced by sequentially adding nucleotides and detecting light produced upon incorporation using a CCD camera. Key advantages are its accuracy, high throughput of up to 48,000 probes per day, and ease of automation. However, it requires specialized equipment and software.
Nanoball squencing
DNA nanoball sequencing is a high-throughput sequencing method that determines the entire genomic sequence of an organism. It uses rolling circle replication to amplify small DNA fragments into DNA nanoballs. Fluorescent probes then bind to complementary DNA sequences on the nanoballs, and the probes' colors are recorded to identify the sequence. The method allows for high DNA concentration and adding new probes without disrupting the reaction. It has been used to identify mutations in cancer samples and estimate inter-generational mutation rates.
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Next generation sequencing techniques allow DNA to be sequenced much more quickly and cheaply than previous Sanger sequencing. The document describes three main next generation sequencing methods: Illumina sequencing uses reversible dye-terminator nucleotides and DNA polymerase to sequence DNA clusters on a flowcell; Roche 454 sequencing is based on pyrosequencing and detects pyrophosphate release during nucleotide incorporation to sequence DNA; SOLiD sequencing uses DNA ligase and emulsion PCR to immobilize DNA on beads for sequencing. These new techniques have revolutionized genomics research by increasing sequencing speed and reducing costs.
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This document provides an overview of DNA sequencing:
- It discusses the history of DNA sequencing, from the early 1970s methods to modern techniques. The Sanger and Maxam-Gilbert methods were among the first developed.
- DNA sequencing involves determining the order of nucleotides (A, T, C, G) in a DNA molecule. This provides important information for research and applications in diagnostics, biotechnology, forensics, and more.
- The document outlines some of the major DNA sequencing techniques and methods, including Sanger sequencing and Maxam-Gilbert sequencing. It also discusses next-generation sequencing approaches.
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This document discusses the history and various methods of DNA sequencing. It begins with a brief overview of DNA sequencing and its uses. It then outlines some of the major developments in DNA sequencing techniques, including the earliest RNA sequencing in 1972, Sanger sequencing in 1977, and the first complete genome of Haemophilus influenzae in 1995. The document proceeds to provide more detailed explanations of several DNA sequencing methods, such as Sanger sequencing, pyrosequencing, shotgun sequencing, Illumina sequencing, and SOLiD sequencing.
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DNA sequencing is a technique to find out the exact arrangement of Nucleotides to make one strand of DNA. DNA sequencing helps in numerous ways from sequence information to paternity testing, mutation detection etc. Traditionally two approaches were used to solve the problem. First is based of enzymes and Second is based on ddNTPs to sequence the DNA using gel electrophoresis technique.
Pyrosequencing
Pyrosequencing is a sequencing by synthesis method launched in 2005 that detects the release of pyrophosphate during the synthesis of multiple DNA strands of varying lengths, allowing for low-cost and fast sequencing with short read lengths.
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Introduction DNA Sequencing
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Introduction to Genome Sequencing
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DNA sequencing determines the order of nucleotides in a DNA molecule. The Sanger dideoxy chain termination method is commonly used and involves DNA polymerase, dNTPs, and chain-terminating ddNTPs. This generates DNA fragments of different lengths that can be separated by gel electrophoresis and used to determine the DNA sequence. Modern sequencing uses fluorescent dye-labeled ddNTPs and capillary electrophoresis for higher throughput automated sequencing. DNA sequencing is important for understanding genetic disorders and developing treatments.
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PCR can be used for a variety of applications including isolating and cloning genes, cDNA synthesis, mutagenesis, molecular markers, analyzing ancient DNA, medical diagnostics, and forensics. Molecular biologists use PCR to identify specific genes and make multiple copies. Forensic technicians use PCR to help identify suspects and victims based on DNA amplification patterns. PCR has impacted many areas of life and promises further exciting discoveries.
Next generation sequencing
Handout available at
https://drive.google.com/open?id=0B52D4j3rC4WDN2hRV25CaW9kb0E
Define DNA sequencing.
Describe the history of DNA sequencing.
Describe the steps of Sanger sequencing.
Define next generation sequencing.
Describe the steps of next generation DNA sequencing.
Describe use of DNA sequencing.
Sanger Technique
The Sanger technique is a method for DNA sequencing developed in 1977 that uses chain-terminating dideoxynucleotides. It relies on the fact that DNA synthesis will stop if a dideoxynucleotide is incorporated instead of a normal deoxynucleotide. The technique involves incubating a single-stranded DNA with the four dideoxynucleotides separately, which results in DNA fragments of different lengths that can be separated via gel electrophoresis to determine the sequence.
PCR
PCR is a technique used to exponentially amplify a specific DNA sequence using DNA polymerase. It was invented in 1983 by Kary Mullis and has revolutionized molecular biology. PCR uses DNA polymerase, primers, dNTPs, and thermal cycling to amplify the target DNA sequence. It has numerous applications including disease detection, genetic fingerprinting, sequencing, and classification of organisms.
2 whole genome sequencing and analysis
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Lectut btn-202-ppt-l31. dna sequencing-i
DNA sequencing techniques allow determining the sequence of nucleotides in a DNA molecule. In 1977, Sanger and Gilbert independently developed methods for DNA sequencing. Sanger's dideoxy method, also known as chain termination method, uses DNA polymerase and a mixture of dNTPs and ddNTPs, which lack a 3'-OH group and terminate DNA strand extension. Electrophoresis then separates DNA fragments by size to reveal the sequence. Sanger's method became the most widely used technique and helped enable major genome sequencing projects like the Human Genome Project.
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DNA Sequencing : Maxam Gilbert and Sanger Sequencing
DNA Sequencing : Maxam Gilbert and Sanger Sequencing
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A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter. It can be used to reveal certain characteristics about the respective source. DNA, for example, is a molecular marker containing information about genetic disorders, genealogy and the evolutionary history of life. Specific regions of the DNA (genetic markers) are used to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding). Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological molecular markers are also used, for example, in environmental studies.
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Molecular markers are identifiable DNA sequences used to locate genes associated with specific traits or genetic conditions.
A molecular marker is a specific gene fragment present at a specific position called ‘locus’ (pleural loci) in the genome of a cell.
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Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
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this presentation is about the molecular markers as we all know the molecular markers are the DNA sequences it can be easily detected and its inheritance is easily monitored.so the main basics of the molecular markers is the polymorphic nature so it can used as molecular markers.and this will gives you the idea about AFLP, RFLP, RAPD, SNPS,ETC.
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PCR is a technique used to amplify specific DNA sequences. It involves using DNA polymerase to replicate target DNA sequences across multiple temperature cycles. Key points:
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2. It was invented by Kary Mullis in 1983 and revolutionized molecular biology. Mullis shared the 1993 Nobel Prize in Chemistry for his invention.
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Molecular markers are DNA sequences that can be used to detect genetic variation between individuals. There are several types of molecular markers including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs). Molecular markers have various applications including measuring genetic diversity, fingerprinting, marker-assisted selection, and identification of genotypes. They provide powerful tools for genetic analysis in plants and animals.
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DNA profiling process, RFLP analysis, STR analysis by PCR, basic principle of dna fingerprinting, advantages and disadvantages of RFLP and STR analysis
Molecular marker by anil bl gather
1. Molecular markers are DNA sequences that can be used to identify specific locations or genes on chromosomes. Different types of molecular markers include isozymes, restriction fragment length polymorphisms (RFLPs), variable number of tandem repeats (VNTRs), and single nucleotide polymorphisms (SNPs).
2. Molecular markers have a variety of uses in genetics and forensic analysis. RFLPs and VNTRs can be used for DNA fingerprinting and identifying individuals. SNP markers allow for analysis of genetic variations between individuals and populations. Molecular markers are also used for genome mapping and marker-assisted breeding in plants and animals.
3. There are several techniques used to detect molecular markers, including gel electrophoresis, Southern blot
DNA MARKERS 2023 DNA FINGERPRINTING TYPE OF METHODS OF DNA FINGERPRINTING
Molecular techniques allow for analysis of protein and DNA interactions through techniques like biochips, polymerase chain reaction (PCR), and quantitative real-time PCR. DNA fingerprinting and markers like RAPD, RFLP, AFLP, and SSR can be used to study genetics. Other techniques mentioned include site directed mutagenesis, reverse genetics, gene knockouts using RNAi and gene silencing, and gene therapy. Omics techniques like metagenomics, transcriptomics, and proteomics are also introduced.
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.
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This document provides information on three molecular biology techniques: polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and random amplified polymorphic DNA (RAPD). It describes the history, principles, requirements, procedures, applications, and variations of PCR. It also briefly discusses the principles, procedures, advantages, and limitations of RFLP and RAPD techniques.
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Rflp
- 1. REASTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS
- 2. • Definition : used to detect variation in length of DNA (polymorphisms) in order to distinguish individuals, populations, or species or to pinpoint the locations of genes within a sequence. • Restriction endonuclease enzyme recognisation site : in DND • Variations in pattern of restriction endonuclease enzyme recognisation in two different DNA is due to variable number of tandem repeats(VNTRs)
- 3. • Hybridization based method • Cannot run pcr as require more amount of DNA
- 4. • If DNA is incubated with specific restriction endonuclease enzyme produce cut after recognising specific sequence on DNA sequence. • EcoRV restriction endonuclease site TWO DIFFERENT DNA SEGMENT INCUBATE WITH SAME RESTRICTION ENDONUCLEASE ENZYME DIFFERENT DNA FRAGMENTS agarose gel electrophoresis , Southern blotting respectively
- 5. Applications 1) genetic fingerprinting : criminal identification 2) Paternity testing 3) Diversity or phylogeny study
- 6. Random amplification of polymorphic DNA (RAPD) • PCR based method • Random primer is used to compare polymorphism in different DNA • 1990 BY WILLIUM et.al • Lab technique by which amplify unknown sequence
- 7. STEPS RAPD • DNA ISOLATION DENATURATION ADD RANDOM PRIMER PUT IN PCR MACHINE GELE ELECTROPHORESIS
- 8. Amplified fragment length polymorphism (AFLP) • AFLP is a technique used to detect polymorphisms in DNA when no information about the genome is known • Method based on pcr • Involve use of rflp and pcr techniques • Faster and less labor require • Use for geneting mapping
- 9. • Developed in 1990s by Keygene • Uses two different restriction enzyme (6 base cutter and 4 base cutter)
- 10. • 6 base cutter (EcoRI) and 4 base cutter (Mse1) cut only complementary DNA sequences.
- 11. Restriction digestion by two different restriction enzyme (6 base cutter (EcoR1) and 4 base cutter (Mse1) DNA isolation 5’ GAATTC 3’ 3’CTTAAG5’ 5’ TTAA 3’ 3’AATT 5’ DNA with complementary sites GAATTC…..//……TTAA…..// CTTAAG…//….AATT…// RESTRICTIO N PRODUCT AATTC…//…T G….//…AAT LIGATIONS WITH ADOPTORS TO COMPLEMENTORY DNA Pcr amplification 2 times to get more numbers of fragments EcoR1 and Mse1 Adaptor specific primers (consisting of complementary to restriction ,adaptors . Add adenine at 3 prime end and cytocin at 5 prime end on both primers respectively Selective amplifications EcoR1: consisting of complementary to restriction ,adaptors and AGC Mse1: : consisting of complementary to restriction ,adaptors and CAG GEL ELECTROPHORESIS
- 12. • THANK YOU