This document provides an overview of basic molecular genetic methodologies and their applications in studying atherosclerosis. It describes several key techniques used in molecular genetics research, such as polymerase chain reaction (PCR), gel electrophoresis, Southern blotting, and DNA sequencing. It also discusses methods for detecting genetic variations like single nucleotide polymorphisms. The document then covers various applications of these techniques in genomic analysis and molecular studies of cardiovascular diseases like atherosclerosis.
DNA replication in eukaryotes proceeds in a coordinated process of initiation, elongation, and termination. Initiation begins at specific origin of replication sequences and involves the assembly of pre-replication complexes. During elongation, helicases unwind the DNA strands exposing templates for DNA polymerase enzymes to synthesize new daughter strands. Termination occurs when the polymerases reach the end regions called telomeres, which are then extended by the enzyme telomerase to allow replication to continue.
RNA is a polymer made of ribonucleotides linked together. There are three main classes of RNA - transfer RNA, ribosomal RNA, and messenger RNA. In eukaryotes, primary transcripts undergo processing including capping, polyadenylation, and splicing before being transported to the cytoplasm for translation. MicroRNAs and small interfering RNAs are types of small regulatory RNAs that cause inhibition of gene expression through post-transcriptional gene silencing. Both miRNAs and siRNAs have potential applications as therapeutic targets in humans.
This document describes the process of DNA replication in eukaryotes. It occurs in S phase of the cell cycle and involves three main stages: initiation, formation of the initiation complex, and elongation. Initiation requires the assembly of pre-replication complexes containing ORC, Cdc6, Cdt1 and MCM proteins. In S phase, Cdc45 and GINS are recruited to form the initiation complex. Elongation proceeds bidirectionally from replication forks, with leading strand synthesis continuous and lagging strand discontinuous via Okazaki fragments. Replication terminates at telomeres.
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.
The document discusses techniques for DNA sequencing, including early methods developed in the 1970s by Maxam and Gilbert as well as Sanger. It provides details on how both methods work, such as using specific chemical or enzymatic reactions to generate labeled DNA fragments of different lengths corresponding to nucleotide positions in the sequence. The document also describes how these methods were later automated, using fluorescent tags on dideoxynucleotides and capillary electrophoresis to simultaneously sequence multiple samples in a single gel. This allowed rapid determination of thousands of nucleotides and enabled large genome sequencing projects such as the Human Genome Project.
DNA repair mechanisms identify and correct damage to DNA that occurs due to normal cellular processes and environmental factors. There are two main types of DNA damage: endogenous damage caused by normal cellular processes and exogenous damage caused by external agents like UV radiation and chemicals. The main repair mechanisms are base excision repair, nucleotide excision repair, direct repair via photolyases, and error-prone repair systems like SOS repair. Together, these pathways maintain genome integrity by repairing different types of DNA lesions.
DNA replication in eukaryotes proceeds in a coordinated process of initiation, elongation, and termination. Initiation begins at specific origin of replication sequences and involves the assembly of pre-replication complexes. During elongation, helicases unwind the DNA strands exposing templates for DNA polymerase enzymes to synthesize new daughter strands. Termination occurs when the polymerases reach the end regions called telomeres, which are then extended by the enzyme telomerase to allow replication to continue.
RNA is a polymer made of ribonucleotides linked together. There are three main classes of RNA - transfer RNA, ribosomal RNA, and messenger RNA. In eukaryotes, primary transcripts undergo processing including capping, polyadenylation, and splicing before being transported to the cytoplasm for translation. MicroRNAs and small interfering RNAs are types of small regulatory RNAs that cause inhibition of gene expression through post-transcriptional gene silencing. Both miRNAs and siRNAs have potential applications as therapeutic targets in humans.
This document describes the process of DNA replication in eukaryotes. It occurs in S phase of the cell cycle and involves three main stages: initiation, formation of the initiation complex, and elongation. Initiation requires the assembly of pre-replication complexes containing ORC, Cdc6, Cdt1 and MCM proteins. In S phase, Cdc45 and GINS are recruited to form the initiation complex. Elongation proceeds bidirectionally from replication forks, with leading strand synthesis continuous and lagging strand discontinuous via Okazaki fragments. Replication terminates at telomeres.
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.
The document discusses techniques for DNA sequencing, including early methods developed in the 1970s by Maxam and Gilbert as well as Sanger. It provides details on how both methods work, such as using specific chemical or enzymatic reactions to generate labeled DNA fragments of different lengths corresponding to nucleotide positions in the sequence. The document also describes how these methods were later automated, using fluorescent tags on dideoxynucleotides and capillary electrophoresis to simultaneously sequence multiple samples in a single gel. This allowed rapid determination of thousands of nucleotides and enabled large genome sequencing projects such as the Human Genome Project.
DNA repair mechanisms identify and correct damage to DNA that occurs due to normal cellular processes and environmental factors. There are two main types of DNA damage: endogenous damage caused by normal cellular processes and exogenous damage caused by external agents like UV radiation and chemicals. The main repair mechanisms are base excision repair, nucleotide excision repair, direct repair via photolyases, and error-prone repair systems like SOS repair. Together, these pathways maintain genome integrity by repairing different types of DNA lesions.
• Plasmids are extra-chromosomal genetic elements that replicate independently of the host chromosome.
• They are small, circular (some are linear), double-stranded DNA molecules that exist in bacterial cells and in some eukaryotes.
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.
Rolling circle replication is a form of DNA replication that occurs in circular DNA like plasmids, bacteriophages, and viroids. It involves five key steps: 1) a circular double-stranded DNA template is nicked at a single origin site, 2) the exposed 3' end is used to initiate replication of the leading strand while displacing the 5' end, 3) the displaced single-stranded DNA acts as the lagging strand and is replicated through Okazaki fragments, 4) both the displaced and unnicked strands are fully replicated, and 5) the displaced strand is rejoined to form a circular DNA product.
The trp operon contains a cluster of genes involved in tryptophan biosynthesis that are under the control of a single promoter. It was the first repressible operon discovered in E. coli in 1953. The trp operon contains structural genes that encode enzymes for tryptophan synthesis, as well as a promoter, operator, and regulatory genes. Tryptophan acts as an effector molecule that binds to the repressor protein, increasing its affinity for the operator sequence and repressing transcription when tryptophan is present. The trp operon is also regulated by transcriptional attenuation, where tryptophan levels affect the formation of termination or anti-termination hairpin loops in the mRNA.
DNA Sequencing : Maxam Gilbert and Sanger SequencingVeerendra Nagoria
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.
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.
description of mechanism of transcription in prokaryotes and eukaryotes with clear explanation and clear pictures and also mentiong of different promotors and enhancers and silencers
Meselson and Stahl conducted an experiment using E. coli bacteria to test the hypothesis that DNA replicates semi-conservatively. They grew the bacteria in medium containing a heavy isotope of nitrogen, then switched the bacteria to medium with a light isotope. Analysis of DNA densities over multiple generations provided evidence that DNA replication results in one old strand and one new strand in each daughter molecule, supporting the semi-conservative model.
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.
This document summarizes DNA replication in eukaryotic cells. It describes that replication occurs through replicons to overcome the slower polymerases. Replication is initiated at specific sites called autonomous replicating sequences (ARS) where the origin recognition complex (ORC) binds. Elongation uses DNA polymerases α, δ, and ε and occurs semi-discontinuously with Okazaki fragments on the lagging strand. Termination involves removing RNA primers with RNase H and sealing fragments with DNA ligase. Multiple enzymes are involved in each phase including MCM helicase, primase, DNA ligase, and DNA polymerases.
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.
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine.
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.
The document discusses DNA sequencing methods. It describes two early conventional methods: Sanger chain termination sequencing and Maxam-Gilbert chemical degradation. Sanger's method uses dideoxynucleotides as chain terminators and is more efficient with fewer toxic chemicals. The document also explains pyrosequencing, an early next-generation sequencing technique that detects pyrophosphate release upon nucleotide incorporation.
DNA sequencing techniques have evolved over time. The dideoxy method developed by Sanger is useful for sequencing short DNA fragments of 500-750bp by terminating the chain with ddNTPs. Whole genome sequencing became possible using shotgun sequencing, which breaks the genome into random fragments that are sequenced and then reassembled. More recently, pyrosequencing was developed for sequencing by synthesis and allows accurate, parallel, and automated sequencing without the need for gel electrophoresis.
DNA sequencing is the process of determining the order of nucleotides in a DNA molecule. The Sanger method, developed in 1977, was the most widely used sequencing technique for 25 years. It utilizes chain termination with dideoxynucleotides which lack a 3' OH group, preventing formation of a phosphodiester bond and terminating strand elongation. Four reactions are run in parallel with each dideoxynucleotide labeled with a different color. Gel electrophoresis separates the terminated fragments by size, allowing the DNA sequence to be read by matching fragment sizes to nucleotide colors.
This document provides an overview of DNA sequencing. It begins by defining DNA and its structure. It then explains that DNA sequencing is the process of determining the order of nucleotide bases in a DNA strand. Two common methods of DNA sequencing are described in detail: the Maxam-Gilbert chemical cleavage method and the Sanger dideoxy chain termination method. The document concludes by discussing some applications of DNA sequencing in fields like forensics, medical research, and agriculture.
Culture independent methods for detection & enumeration of gut microfloraAmna Jalil
This document discusses various culture-independent methods for detecting and enumerating gut microflora, including: 1) Design of PCR primers and hybridization probes to target specific species or groups; 2) PCR-ELISA to combine PCR amplification with ELISA detection; 3) Sequence analysis of randomly amplified 16S rRNA genes; 4) Temperature gradient gel electrophoresis (TGGE) and denaturing high performance liquid chromatography (DHPLC) to separate amplified DNA fragments by size; 5) Terminal restriction fragment length polymorphism (T-RFLP) to generate terminal restriction fragments for profiling; 6) Oligonucleotide arrays and quantitative real-time PCR for rapid, accurate detection of thousands of bacteria; 7) Fluorescence in
Metagenomics is a set of techniques used to study microbial communities through direct collection and analysis of environmental DNA samples. It allows researchers to study millions of microbial organisms and genetic fragments simultaneously without needing to culture individual microbes in the lab. The main procedures involve sampling an environment, filtering out particles by size, extracting and sequencing DNA fragments. Two common sequencing methods are shotgun sequencing and high-throughput sequencing using platforms like Illumina or SOLiD. Projects like MetaHIT use metagenomics to study the human gut microbiome and its role in health and disease. Potential applications include contributions to earth sciences, life sciences, biomedicine, bioenergy, biotechnology, and microbial forensics.
• Plasmids are extra-chromosomal genetic elements that replicate independently of the host chromosome.
• They are small, circular (some are linear), double-stranded DNA molecules that exist in bacterial cells and in some eukaryotes.
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.
Rolling circle replication is a form of DNA replication that occurs in circular DNA like plasmids, bacteriophages, and viroids. It involves five key steps: 1) a circular double-stranded DNA template is nicked at a single origin site, 2) the exposed 3' end is used to initiate replication of the leading strand while displacing the 5' end, 3) the displaced single-stranded DNA acts as the lagging strand and is replicated through Okazaki fragments, 4) both the displaced and unnicked strands are fully replicated, and 5) the displaced strand is rejoined to form a circular DNA product.
The trp operon contains a cluster of genes involved in tryptophan biosynthesis that are under the control of a single promoter. It was the first repressible operon discovered in E. coli in 1953. The trp operon contains structural genes that encode enzymes for tryptophan synthesis, as well as a promoter, operator, and regulatory genes. Tryptophan acts as an effector molecule that binds to the repressor protein, increasing its affinity for the operator sequence and repressing transcription when tryptophan is present. The trp operon is also regulated by transcriptional attenuation, where tryptophan levels affect the formation of termination or anti-termination hairpin loops in the mRNA.
DNA Sequencing : Maxam Gilbert and Sanger SequencingVeerendra Nagoria
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.
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.
description of mechanism of transcription in prokaryotes and eukaryotes with clear explanation and clear pictures and also mentiong of different promotors and enhancers and silencers
Meselson and Stahl conducted an experiment using E. coli bacteria to test the hypothesis that DNA replicates semi-conservatively. They grew the bacteria in medium containing a heavy isotope of nitrogen, then switched the bacteria to medium with a light isotope. Analysis of DNA densities over multiple generations provided evidence that DNA replication results in one old strand and one new strand in each daughter molecule, supporting the semi-conservative model.
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.
This document summarizes DNA replication in eukaryotic cells. It describes that replication occurs through replicons to overcome the slower polymerases. Replication is initiated at specific sites called autonomous replicating sequences (ARS) where the origin recognition complex (ORC) binds. Elongation uses DNA polymerases α, δ, and ε and occurs semi-discontinuously with Okazaki fragments on the lagging strand. Termination involves removing RNA primers with RNase H and sealing fragments with DNA ligase. Multiple enzymes are involved in each phase including MCM helicase, primase, DNA ligase, and DNA polymerases.
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.
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine.
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.
The document discusses DNA sequencing methods. It describes two early conventional methods: Sanger chain termination sequencing and Maxam-Gilbert chemical degradation. Sanger's method uses dideoxynucleotides as chain terminators and is more efficient with fewer toxic chemicals. The document also explains pyrosequencing, an early next-generation sequencing technique that detects pyrophosphate release upon nucleotide incorporation.
DNA sequencing techniques have evolved over time. The dideoxy method developed by Sanger is useful for sequencing short DNA fragments of 500-750bp by terminating the chain with ddNTPs. Whole genome sequencing became possible using shotgun sequencing, which breaks the genome into random fragments that are sequenced and then reassembled. More recently, pyrosequencing was developed for sequencing by synthesis and allows accurate, parallel, and automated sequencing without the need for gel electrophoresis.
DNA sequencing is the process of determining the order of nucleotides in a DNA molecule. The Sanger method, developed in 1977, was the most widely used sequencing technique for 25 years. It utilizes chain termination with dideoxynucleotides which lack a 3' OH group, preventing formation of a phosphodiester bond and terminating strand elongation. Four reactions are run in parallel with each dideoxynucleotide labeled with a different color. Gel electrophoresis separates the terminated fragments by size, allowing the DNA sequence to be read by matching fragment sizes to nucleotide colors.
This document provides an overview of DNA sequencing. It begins by defining DNA and its structure. It then explains that DNA sequencing is the process of determining the order of nucleotide bases in a DNA strand. Two common methods of DNA sequencing are described in detail: the Maxam-Gilbert chemical cleavage method and the Sanger dideoxy chain termination method. The document concludes by discussing some applications of DNA sequencing in fields like forensics, medical research, and agriculture.
Culture independent methods for detection & enumeration of gut microfloraAmna Jalil
This document discusses various culture-independent methods for detecting and enumerating gut microflora, including: 1) Design of PCR primers and hybridization probes to target specific species or groups; 2) PCR-ELISA to combine PCR amplification with ELISA detection; 3) Sequence analysis of randomly amplified 16S rRNA genes; 4) Temperature gradient gel electrophoresis (TGGE) and denaturing high performance liquid chromatography (DHPLC) to separate amplified DNA fragments by size; 5) Terminal restriction fragment length polymorphism (T-RFLP) to generate terminal restriction fragments for profiling; 6) Oligonucleotide arrays and quantitative real-time PCR for rapid, accurate detection of thousands of bacteria; 7) Fluorescence in
Metagenomics is a set of techniques used to study microbial communities through direct collection and analysis of environmental DNA samples. It allows researchers to study millions of microbial organisms and genetic fragments simultaneously without needing to culture individual microbes in the lab. The main procedures involve sampling an environment, filtering out particles by size, extracting and sequencing DNA fragments. Two common sequencing methods are shotgun sequencing and high-throughput sequencing using platforms like Illumina or SOLiD. Projects like MetaHIT use metagenomics to study the human gut microbiome and its role in health and disease. Potential applications include contributions to earth sciences, life sciences, biomedicine, bioenergy, biotechnology, and microbial forensics.
The document discusses probiotics, which are live microorganisms that provide health benefits when consumed. It defines probiotics and lists common types including various Lactobacillus and Bifidobacterium bacteria. The document then outlines uses of probiotics for humans, cattle/poultry, and aquaculture. It provides a table of commercial probiotic strains and discusses specific probiotic supplements including Bio-K+, Bio-K+ Fruity, Bio-K+ Dairy Free, and Bio-Kaps Regular Strength.
Metagenomics is the study of genetic material recovered directly from environmental samples without culturing. This field enables research on uncultured organisms and microbial communities. There are three main metagenomic approaches: biochemical, whole genome shotgun sequencing, and 16s rRNA sequencing. Metagenomics is being applied to study human microbiomes, discover new genes and enzymes, monitor environmental impacts, and characterize uncultured microbes. Future directions include identifying more novel products from uncultured bacteria and improving culture methods and bioinformatics tools.
The document discusses how to choose a probiotic supplement. Key factors include choosing strains that are beneficial for your specific needs, selecting a probiotic that is effective for the desired health benefits, and choosing ones that are safe, acid/bile resistant, and reliably deliver live cultures. The document recommends considering strain origin, ability to colonize the gut, and individual dietary needs or preferences when selecting a probiotic supplement.
This document provides an overview of probiotics, focusing on the bacteria Lactobacillus and Bifidobacterium. It discusses the history of probiotics, why they are important for human health, examples of foods containing probiotics, and their mechanisms of action. The document also covers commercial probiotic strains, genetically engineered probiotics, prebiotics, and Indian probiotic manufacturers.
DNA Fingerprinting & its techniques by Shiv Kalia (M.Pharma in Analytical Che...Shiv Kalia
DNA fingerprinting and below mention content widely cover in this presentation
History & Introduction of DNA fingerprinting
How was the first DNA fingerprint produced?
Types of DNA Based Markers
Polymerase Chain Reaction (PCR)
PCR based Methodology of DNA fingerprinting
Electrophoresis
Utility of DNA Based Markers
Various DNA Fingerprinting Techniques Advantages & Disadvantages
Authentication of Various Ayurvedic Herbs by DNA Fingerprinting
Advantages of DNA fingerprinting in Plants
Disadvantages of DNA fingerprinting in Plants
CONCLUSION
PCR is a technique used to amplify a specific DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample to denature and separate the DNA strands, followed by primer annealing and polymerase extension. This results in exponential amplification of the target DNA sequence. PCR requires a DNA template, DNA polymerase, primers, nucleotides, and repeated cycling between high and low temperatures. It has applications in research, forensics, medicine and molecular biology.
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:
1. PCR amplifies specific DNA segments through repeated cycles of heating and cooling. This allows millions of copies of the target DNA to be generated.
2. It was invented by Kary Mullis in 1983 and revolutionized molecular biology. Mullis shared the 1993 Nobel Prize in Chemistry for his invention.
3. PCR has many applications including pathogen detection, genetic testing, forensics, and molecular cloning. It is a core technique in molecular biology and genetics research.
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.
The document discusses polymerase chain reaction (PCR), a laboratory technique used to amplify a specific segment of DNA. It was invented in 1983 by Kary Mullis, who was awarded the Nobel Prize in 1993. PCR works by repeating cycles of heating and cooling of the DNA sample to separate the double helix, followed by use of DNA polymerase to make copies of the targeted region. This process can generate millions of copies of the targeted DNA sequence. The document outlines the components and steps of PCR, including primers, DNA polymerase, and thermal cycling. It discusses some applications of PCR such as detecting low-abundance DNA sequences, forensic analysis, and prenatal diagnosis.
Microarray and dna chips for transcriptome studyBia Khan
Microarrays and DNA chips can be used to study transcriptomes by comparing gene expression profiles. They work by immobilizing reference cDNA or oligonucleotides on a glass slide, then hybridizing labeled cDNA from the cells of interest. This allows determining which genes are expressed and their relative expression levels based on fluorescence intensities. While powerful, the method has complications like cross-hybridization of similar mRNAs and experimental errors. Normalization procedures help account for these. Yeast is commonly used as a model organism in transcriptome studies due to its stable yet responsive gene expression. Applications include stem cell research, cancer studies, and embryonic development.
This document discusses various molecular methods used in molecular genetics and molecular biology. It begins by categorizing common techniques into diagnostic methods, analysis and sequencing, microarray methods, and proteomics. Several key diagnostic techniques are then described in more detail, including gel electrophoresis, hybridization, PCR, immunoprecipitation, and karyotyping. Recombinant DNA technology basics like cloning genes, cDNA, and purposes of cloning are explained. Other analysis methods such as sequencing, microarrays, RNA-Seq, and techniques like RNAi, knockout methods, and liposomes are also summarized.
DNA fingerprinting is a technique developed in 1984 that analyzes variable regions in genetic material to distinguish one person from another. It involves isolating DNA from a sample, cutting it with restriction enzymes, sorting by size, and probing specific locations to generate a unique pattern. This technique uses variations in the number of short tandem repeats between individuals and has been used successfully in criminal cases and establishing paternity. The most common DNA fingerprinting methods are electrophoresis, polymerase chain reaction, restriction fragment length polymorphism, random amplified polymorphic DNA, and amplified fragment length polymorphism. DNA fingerprinting has applications in diagnosing inherited disorders, developing cures, and identifying criminals using biological evidence.
DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCINGPuneet Kulyana
This presentation will give you a brief idea about the various DNA sequencing methods and various strategies used for genome sequencing and much more vital information related to gene expression and analysis
The analysis of global gene expression and transcription factor regulation, global approaches to alternative splicing and its regulation, long noncoding RNAs, gene expression models of signalling pathways, from gene expression to disease phenotypes, introduction to isoform sequencing, systematic and integrative analysis of gene expression to identify feature genes underlying human diseases.
Transcriptomics is the study of RNA in cells and tissues. The transcriptome refers to the complete set of transcripts in a cell under a specific condition. Understanding the transcriptome reveals the functional elements of the genome and molecular constituents of cells. Techniques for studying the transcriptome include microarray analysis and RNA sequencing. Microarrays measure gene expression levels using fluorescently-labeled cDNA hybridized to probes on an array. RNA sequencing determines expression levels by sequencing individual cDNAs produced from target RNA. Transcriptomics provides insights into development, disease, and varying gene expression under different environmental conditions.
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.
This document discusses gene mapping and sequencing. It begins by defining genomics and genetic markers such as RFLP, SSLP, and SNP that are used to track inheritance. Gene mapping involves determining the locus and distance between genes on chromosomes, which is important for diagnosing genetic diseases. There are two main types of gene mapping: linkage mapping which measures recombination frequency to determine if genes are linked, and physical mapping which precisely locates DNA sequences on chromosomes using techniques like fluorescence in situ hybridization. The document also discusses methods for gene sequencing, including Sanger sequencing and Maxam-Gilbert sequencing, as well as newer techniques like shotgun sequencing and Illumina sequencing.
Polymerase chain reaction (PCR) is a method to rapidly amplify a specific DNA sample, allowing scientists to study very small amounts of DNA. PCR uses the enzyme DNA polymerase to assemble copies of DNA by denaturing and separating the DNA strands, annealing primers to the strands, and extending the primers to make new copies of DNA. This cycling process can produce millions of copies of DNA. PCR is used in applications like disease diagnosis, forensics, genetic testing, and evolutionary studies.
Microarrays allow researchers to study gene expression across thousands of genes at once. They work by immobilizing DNA probes on a solid surface, then exposing the surface to fluorescently labeled cDNA or cRNA from samples. The microarray is then scanned to see which probes fluoresce, indicating gene expression. Microarrays have many applications including disease diagnosis, drug discovery, and toxicology. While powerful, they also have limitations like expense and complexity of data analysis. Standards are being developed to allow use of microarray data in regulatory decision making.
This document discusses cancer genomics and tumor sequencing. It explains that tumor genotyping helps clinicians individualize cancer treatments by matching patients to the best treatment based on their tumor's DNA alterations. Next generation sequencing methods have made it possible to sequence entire cancer genomes and identify additional targets for new cancer therapies. Large-scale projects like The Cancer Genome Atlas and the International Cancer Genome Consortium are analyzing hundreds of cancer genomes to better understand the molecular changes driving different cancer types.
This document discusses DNA fingerprinting techniques for identifying herbal drugs, including those of natural origin. It describes several DNA-based marker techniques like RFLP, RAPD, AFLP, and ISSR that can generate unique DNA profiles to distinguish between plant species and identify adulteration. The document also provides a case study on using RAPD-PCR and six primer pairs to generate DNA fingerprints to identify the herbal plant Exacum lawii. The results found unique DNA banding profiles that can be used to authenticate Exacum lawii.
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.
This document discusses various methods for single nucleotide polymorphism (SNP) analysis, including their principles, advantages, and disadvantages. It describes SNP genotyping techniques like RFLP-PCR, TaqMan assays, microarrays, Sanger sequencing, SNaPshot, and next-generation sequencing. The key aspects are accuracy, throughput, cost, and ability to detect both known and unknown SNPs. The document emphasizes choosing methods based on required information extraction and cost effectiveness.
This document discusses developing an artificial intelligence system to predict short-term cardiovascular disease (CVD) events. The goal is to eradicate unexpected heart attacks by predicting risk similar to hurricane forecasts. Existing studies are cited that show over 50% of heart attacks are first symptoms of underlying disease. The document outlines previous work by SHAPE to define vulnerable patients and release guidelines. It proposes using machine learning on existing cohort data to develop algorithms predicting heart attacks within 12 months, and validate the system. The hope is this can trigger preventative actions and add over 10 years to life expectancy. Funding is needed to implement the proposed "Machine Learning Vulnerable Patient Project".
Triggers of cardiovascular events can include physical and emotional stress. Stress from events like earthquakes, blizzards, intense sporting games, and overexertion from activities like snow shoveling have been shown to increase the risk of acute cardiovascular outcomes like myocardial infarction. While modern therapies have improved cardiovascular health, research continues to show temporary increases in cardiovascular mortality associated with highly emotional sporting events even in recent years. Managing risk factors, reducing stress, and utilizing preventative therapies may help reduce the impact of triggers on cardiovascular health.
The document introduces the All of Us Research Program, which aims to collect health data from one million Americans to advance precision medicine research. It was announced by President Obama in 2015. The program receives funding from the federal government and private partners. It collects various types of health data from participants through surveys, health records, samples, and devices. The data is stored and shared securely while protecting privacy. The goal is to generate new medical discoveries and more personalized healthcare through collaboration between researchers and participants.
A machine learning model outperformed the ACC/AHA Pooled Cohort Equations Risk Calculator in detecting high-risk asymptomatic individuals and recommending statin treatment for cardiovascular disease prevention in the Multi-Ethnic Study of Atherosclerosis. The machine learning model used support vector machines and data augmentation to derive a CVD risk predictor from nine variables in the MESA study population. It demonstrated higher sensitivity, specificity, and AUC compared to the ACC/AHA risk calculator, recommending statin treatment for fewer individuals while missing fewer cardiovascular events.
This document discusses machine learning applications in cardiac imaging presented by Piotr Slomka. It describes how machine learning can improve image analysis, diagnosis, and risk prediction. Machine learning combines multiple data points like imaging and clinical data to predict outcomes. Deep learning can perform tasks like image segmentation. Machine learning provides quantitative scores that predict disease, need for intervention, or patient outcomes to help clinicians. The goal is to integrate machine learning into clinical decision making.
This document summarizes a post-mortem study examining the prevalence of inflammatory cells in non-ruptured atherosclerotic plaques. The study found that moderate or heavy staining for macrophages was present in 45% of femoral artery cross-sections and 84% of femoral arteries had at least one cross-section with moderate/heavy inflammation. There was no observed relationship between the degree of inflammation in the left and right coronary arteries within individuals, indicating the level of local inflammation is locally determined with little predictive value for other arteries.
The document provides guidelines for defining vulnerable plaque and vulnerable patients from the Association for Eradication of Heart Attack. It outlines major and minor histopathological and clinical criteria for vulnerable plaque including active inflammation, thin fibrous cap with large lipid core, endothelial denudation, and stenosis. Potential screening and diagnostic methods are discussed at the plaque, systemic, and blood levels ranging from non-invasive imaging to intravascular techniques. Different types of vulnerable plaque that can cause acute coronary events are also categorized.
Vulnerable plaque refers to dangerous forms of atherosclerotic plaques that can rupture or induce thrombosis, disrupting blood flow. The document discusses the history and research around vulnerable plaque, including pioneers in the field and emerging techniques to detect vulnerable plaque such as intravascular ultrasound, optical coherence tomography, and magnetic resonance imaging. It summarizes that vulnerable plaques are typically characterized by a thin fibrous cap, large lipid core, and presence of macrophages.
The document summarizes research on vulnerable plaques and markers of vulnerability. It finds that ruptured plaques are the most common type of culprit lesion, accounting for around 70% of cases. Major criteria for defining vulnerable plaque include outward remodeling, endothelial dysfunction, and a thin fibrous cap with a large lipid core. Both plaque morphology and activity need to be assessed to identify vulnerability.
This document contains a summary of a presentation on vulnerable patient syndrome. It includes PowerPoint slides and videos on defining and identifying vulnerable plaques and patients. It thanks sponsors for their support of the educational event. The slides define vulnerable plaques as those likely to rupture in the future, causing heart attacks, and provide criteria for identifying them based on morphology and activity. Biomarkers and conditions that increase plaque and myocardial vulnerability are also summarized. The presentation outlines a pyramid approach for screening, diagnosing, and treating vulnerable patients annually to help reduce heart attacks and their high costs.
This document discusses triggers for sudden cardiac arrest (SCA) and death (SCD). It notes that over 2/3 of SCD cases are unable to be predicted due to a lack of well-established risk factors. While population risk factors can identify at-risk groups, they cannot predict risk for individuals. The document explores various biological, anatomical, and environmental factors that can precipitate fatal arrhythmias and discusses how the timing of transient initiating events is critical for the development of ventricular tachyarrhythmias. It emphasizes that myocardial electrophysiological processes likely determine the onset or lack of VT/VF/SCD and that immediate access to automated external defibrillators is needed to save lives.
This document summarizes presentations from symposia on vulnerable plaque and discusses the relationship between plaque, blood, and patients in atherothrombosis. It notes that multiple factors like diabetes, smoking, and hyperlipidemia can make blood more thrombogenic and moderate the severity of acute events after plaque rupture. Statins, aspirin, and other drugs that target tissue factor or thrombin pathways may be promising antithrombotic agents by inhibiting thrombosis initiation and propagation.
The document discusses vulnerable plaque and challenges in detecting and treating it. It describes various imaging techniques for detecting vulnerable plaque such as thermography, MRI, CT angiography, and optical coherence tomography. However, it notes that while these can identify high-risk features, it remains unclear what exactly defines vulnerable plaque and whether imaging findings truly correlate with risk. The document also notes that while statins reduce events, the relationship between plaque burden and events is unclear, and better defining and detecting the disease is still needed before new therapies can be developed.
1) The study examined 92 hearts from patients with severe coronary artery disease who died suddenly. The hearts were sectioned and plaque types were classified.
2) The number of "vulnerable" plaques, particularly thin cap atheromas, was highest in hearts of patients who died from acute plaque rupture and lowest in those with incidental disease.
3) Thin cap atheromas and other unstable plaque types were concentrated in the proximal coronary segments, similar to the distribution of plaque ruptures. The study suggests vulnerable plaques contribute to acute coronary syndromes and are non-uniformly distributed within the coronary arteries.
1) Drug-coated stents, particularly those coated with sirolimus, have shown promise in reducing restenosis compared to bare metal stents. Sirolimus inhibits cell proliferation and has been shown in studies to reduce intimal hyperplasia and restenosis in animal models by 50% or more.
2) A study by Suzuki et al. found that a sirolimus-coated stent reduced restenosis by 50% through inhibiting cellular proliferation in a dose-dependent manner compared to a bare metal stent. Adding dexamethasone to the coating did not provide additional benefit.
3) If results of the RAVEL clinical trial showing "zero" restenosis out to 5 years
This document discusses drug-coated stents for preventing restenosis. It summarizes a study showing that stents coated with sirolimus via a polymer matrix reduced restenosis by 50% by inhibiting cell proliferation. Adding dexamethasone provided no additional benefit. Other studies also showed sirolimus inhibits smooth muscle cell proliferation. If results of the RAVEL trial showing "zero" restenosis at 210 days hold true long-term, sirolimus-coated stents may become the standard therapy for coronary revascularization. Questions are raised about whether coating vulnerable plaques could be a primary treatment and if multiple vulnerable plaques would all be stented.
1) Drug-coated stents, particularly those coated with sirolimus, have shown promise in reducing restenosis compared to bare metal stents. Sirolimus inhibits cell proliferation and has been shown in studies to reduce intimal hyperplasia and restenosis in animal models by 50% or more.
2) A study by Suzuki et al. found that a sirolimus-coated stent reduced restenosis by 50% through inhibiting cellular proliferation in a dose-dependent manner compared to a bare metal stent. Adding dexamethasone to the coating did not provide additional benefit.
3) If results of the RAVEL clinical trial showing "zero" restenosis out to 5 years
I. This document discusses various animal models that have been used to study atherosclerosis and plaque rupture, including quail, pigeons, chickens, dogs, monkeys, pigs, rats, rabbits, and mice. It provides details on the types of lesions developed and similarities to human disease for each model.
II. The double knockout LDL/apoE mice are highlighted as offering improvements in studying clinical complications of atherosclerosis like human heart disease. However, it is unclear how closely they model vulnerable plaques.
III. Questions are raised about how closely the coagulation systems of these animal models resemble humans and whether any model fully captures repeated plaque ruptures and the role of aging in natural history as seen in humans.
Trans-Blood Vision is a patented infrared technique that uses short-wave infrared wavelengths to see directly through blood. It has the potential to find vulnerable plaque lesions without first entering them, determine their size and surface characteristics in high resolution, and look at their material constituents both on and below the surface. While it cannot provide direct visual guidance for therapy or penetrate as deeply as ultrasound, combining it with augmentative technologies could allow for real-time multi-mode detection, analysis, and therapy guidance of vulnerable plaque lesions. The document concludes that Trans-Blood Vision warrants significant investigation, possibly in combination with other emerging technologies.
This study used intravascular ultrasound to examine arterial remodeling and plaque characteristics in 131 patients with either stable angina or recent unstable symptoms. Patients with unstable presentations had greater plaque burden at the culprit lesion despite similar luminal narrowing, and a greater extent of positive arterial remodeling compared to those with stable angina. The culprit lesions in unstable patients also showed a higher rate of echolucent plaque morphology. This suggests that bulky, remodeled plaques may be more vulnerable to rupture, leading to acute coronary syndromes. Further prospective study is needed to better understand the relationship between clinical presentation and plaque features.
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These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
- Video recording of this lecture in English language: https://youtu.be/kqbnxVAZs-0
- Video recording of this lecture in Arabic language: https://youtu.be/SINlygW1Mpc
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
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1. Basic Molecular GeneticBasic Molecular Genetic
StudiesStudies
in Atherosclerosisin Atherosclerosis
Methodology, Applications,Methodology, Applications,
and “Future Horizons”and “Future Horizons”
2. Risk factors: e.g.
hypertension, DM,
infection( HSV,CP
), smoking etc
Hyperlipidemia
• Diet
• Familial
lipid deposition
and removal
imbalance
Endothelia
l injury
Release of cytokines, adhesion
molecules vasoactive molecules and
growth factor
Lipid
accumulatio
n and
oxidation
Macrophage and T
cell accumulation
Chronic Active
Inflammation
SMC
proliferation
Intimal
thickening and
fibrous cap
formation
Core formation
Plaque formation
SMC and foam cell apoptosis, active macrophage
infiltration, and matrix degradation
Plaque rupture
A
T
H
E
R
O
S
C
L
E
R
O
S
I
S
A
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A
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L
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4. Polymerase chain reaction-PCR
• This is achieved by repeated rounds of three
different steps: heat denaturation of template
DNA,
• Annealing of two convergent oligonucleotide
primers to the opposite strands of the DNA
template
• 5’-3’ extension from each of the annealed primers
using a thermosable DNA polymerase
5. Chain Termination
• This is done to obtain DNA fragments of different
lengths
• For this reaction to occur four standard dNTPs
(dATP, dCTP, DGTP, dTTP) are included in the
reaction mixtures
6. Gel-Electrophoresis and
Autoradiography
• After the chain terminated strands are obtained,
they are run through an agarose gel and usually
stained with Ethidium bromide and visualize the
bands on a u.v transilluminator.
• Electrophoresis makes use of the fact that
molecules like DNA migrate in an electric field
inversely proportional to its molecular weight
towards the positive pole.
8. Southern Blot for DNA analysis
• The restriction fragments obtained by gel-
electrophoresis are subject to denaturation with
alkali and transferred to a nitrocellulose filter or
nylon membrane by blotting.
• The filter is them incubated under hybridization
conditions with a specific radiolabeled probe
usually generated from a cloned restriction
fragment.
9. • The DNA restriction fragment that is
complementary to the probe hybridizes, and its
location on the filter can be revealed by
autoradiography.
10. RT-PCR
a way to detect mRNA in cells
1. by using Reverse-
Transcriptase enzyme
a cDNA is made on the
template of RNA
2. PCR amplifies the
cDNA derived from
mRNA molecule
3. The amplified cDNA is
detected
mRNA
Standard PCR
primer
Bases
cDNA
Reverse
transcriptase
11. Methods to detect Sequence Variations
• Use of silver stainings to detect Nucleic acids
• Nonradioactive Method for the Detection of
Single-strand Conformational Polymorphisms.
• Temperature Gradient Gel Electrophoresis(TGGE)
for the detection of polymorphic DNA and RNA.
• TGGE in Quanttitative PCR of DNA and RNA
• Direct sequencing of PCR products
12. Analysis of Specific Nucleic
Acids in Complex Mixtures
• Southern Blotting Detects specific DNA
fragments
• Northern Blotting Detects Specific RNAs
• Nuclease Protection is used to Quantitate Specific
RNAs and Map the DNA Regions encoding them
13. Probes
• A probe is a relatively small piece of DNA that is
used to find another piece of DNA.
• In nucleic acid hybridization a DNA probe,
labeled radioactively or non-radioactively, seeks
out and finds complementary DNA In the target
DNA.
• The shortest useful probe is about 20 bases long
and is known as oligonucleotide probe.
• RNA molecules may also be used as probes and
are termed “riboprobes”.
14. Nucleic acid probes are segments of
DNA(/RNA) that :
1.1. have been labeledhave been labeled (with enzymes antigeneic
substrate , chemiluminescent moieties or
radiolabeled) and can bind with high specificityan bind with high specificity
to DNA/ RNA targets
2. probes can be 2 to 1000 bases
3. early DNA probes were labeled with 32 P, now
in most kits radioisotopes are replaced by
enzymes, affinity labeles and chemiluminescent
molecules. ( Enzymes like alkaline phosphatase ,
horseradish peroxidase , affinity labeles like
biotin, digoxigenin)
15. Hybridization
• The process of forming a double stranded DNA
molecule between a probe and a target.
• DNA is double stranded and can be made single
stranded.
• If a large excess of DNA, called a probe, that has
complementarity to a particular sequence of
interest is added to the target, they form a double
stranded structure and this is termed hybridization.
16. Nucleic Acid
Immunocytochemistry
This technique uses nucleic acid-antibody complexes
as probes and nucleic acids as targets. The
aspects of a nucleic acid immunocytochemistry
are as follows:
• Target is DNA or mRNA localized within a cell
• The recognition and detection of target nucleic
acid relies on base pairing(hybridization) of
complementary bases of the target nucleic acid
17. • The recognition and detection of target nucleic
acid relies on base pairing(hybridization) of
complementary bases of the target nucleic acid.
• The antibody moiety complexed to the nucleic
acid portion of the probe serves only as a signal-
generating system reporting that a target has
been found and hybridization has occurred.
18. Fluorescence In Situ
Hybridization (FISH)
• FISH is widely used to determine the
chromosomal map location and the relative
order of genes and DNA sequences within a
chromosomal band.
19. Recombinant DNA Technology
• Recombinant DNA is artificially created DNA for the
purpose of genetic engineering which can be introduced
into appropriate cells to form a clone of such cells.
• It makes use of two enzymes:
• Restriction enzymes: which cut the DNA from any
organism at specific sequences.
• Ligases: which can insert DNA restriction fragments into
replicating DNA molecules producing recombinant DNA.
20. DNA Cloning with Plasmid
Vectors
• Plasmids are extrachromosomal self-replicating DNA
molecules.
• E-coli Plasmids can be engineered for use as cloning
vectors.
• Replication origin(ORI) is a specific DNA sequence of 50-
100 base-pairs that must be present in a plasmid for it to
replicate.
• Cells to be transformed are selected and then under
favorable conditions, i.e;high concentration of certain
divalent cations,1 cell in about 10,000 or more cells
becomes competent to take up foreign DNA.
21.
22. Commercial Potential of
Recombinant DNA
• Genetic Engineering as a man-made entity has
been refined over the 25 years of its existence to
the point where medically and pharmaceutically
important reagents ( for example, insulin, and
interferon) as well as certain vaccines are now
routinely being made on a manufacturing scale.
• Genetic Engineering has been also used in
brewing, fermenting, wine making and other
fields.
23. Genomic analysis
1. DNA sequencing
2. Single molecule sequencing
3. SNP( single nucleotide polymorphism)
4. DNA array technology (Functional
Genomic analysis)
24. DNA sequencing
1. A chain terminators
stops DNA synthesis at
different sites.
2. A strand of DNA is
replicated
3. The chain terminator is
a nucleotide that is
similar but not the
same as original
nucleotide
4. The place of all A's,
T's, C's and G's are
detected by gel-
electrophoresis.
25. Single molecule sequencing
• Uses exonucleases to degrade DNA by cleaving
individual nucleotides from 5’-end of a DNA
molecule and detecting them
• The most common methods use DNA segments
made from nucleotides where four bases have
been differentially tagged with fluorescent labels
• When the tagged nucleotide is clipped, it flows
past a laser-based fluorescence detector
26. Single nucloetide polymorphism
• The most common type of stable genetic
variations, these point mutations can produce
different phenotypes and can be contributory
factors for human disease.
• SNP detections methods are diverse and beyond
the scope of this review.
• One notable approach uses matrix-assisted laser
desorption mass spectroscopy(MALDI-MS) on a
silicon chip for SNP detection; another system
employs electrical circury on siliccon microchips
to produce a fluorescent signal
27. Expressed Sequence Tags
• ESTs are identified using reverse transcriptase
(RT) to create cDNA sequnces from mRNA
present in a cell, allowing genes expressed in
different tissues or environmental conditions to be
easily amplified by PCR for further study
• Because ESTs might be only gene
fragments(typically 3’ or 5’0, they are more easily
generated than entire sequence information
28. Active and In-active mRNA
• Protein expression levels are not correlated with
mRNA expression levels
• Because protein rather than mRNA levels
determine phenotype, there are efforts underway
to investigate this difference by analysing the
translation state of mRNA
• Active and inactive ribosomes can be readily
separated using sucrose gradient centrifugation;
the fractions can then be identified using labeled
cDNA probes and used to interpret data based on
mRNA expression to estimate protein levels
29. DNA arrays
• DNA arrays consists of large number of DNA molecules
spotted in a systematic order on a solid substrate( such as
nylon membrane, glass slide, or silicon chip).
• Depending on the size of each DNA spot on the array,
DNA arrays can be categorized as microarrays(each DNA
spot has a diameter of <250 microns) and
macroarrays( spot diameter of>300 microns).
• When the solid substrate used is small in size, arrays are
also referred to as DNA chips
30. Microarray assays
• Traditional hybridization assays developed in the
1970s utilize flexible membranes such as
nitrocellulose and nylon, radioactivity, and
autoradiography.
• By contrast, microarray or biochip assays utilize
solid surfaces such as glass with fluorescent
labelling and detection, this miniaturized biochip
format has revolutionized biological analysis.
• Advantages of solid surface are it being non-
porous, allows using of small sample volumes,
rapid hybridization kinetics, uniform attachement
31. Microarray Fabrication
• There are three primary technologies used
presently in microarray manufacture include
photolithography, ink-jetting, mechanical
microspotting, and derivatives thereof.
32. • Microarray fabrication technologies
CRITERION PHOTOLITHOG
RAPHY
PIEZOELECTRIC MICROSPOTT
ING
Combinatorail
syntesis
Yes Yes No
Ink-jetting NO Yes No
Surface printing No No Yes
Masks needed Yes NO NO
Sample tracking NO NO Yes
Density(/cm) 244000 10000 6500
Array elements Oligos only Oligos and cDNAs Oligos and
cDNAs
Prototyping cost High Moderate Low
34. Applications of DNA array
• Gene expression profiling
• De novo gene sequencing
• Gene mutation analysis(SNP)
• Gene mapping and genotyping
35. Main steps in performing a DNA
array experiment
1. Printing target DNA or oligoneculotide onto a
substance,
2. Sample RNA isolation (either total RNA or
mRNA),
3. cDNA synthesis and labeling,
4. Hybridization of the labeled probe cDNA to
target cDNA on the substrate,
5. Imaging of hybridization results and image
analysis
36. Schematic of probe preparation , hybridization,
scanning and immage analysis
37. • cDNA microarray target printing apparatus. Computer-
controlled robotic cantilever arm, capable of moving in XYZ
directional planes, can be armed with up to 16 (two rows of eight)
`quill' print tips on the print head. In one automated print cycle,
the print head dips the quills into a set of target DNA wells
arrayed in 96 well microwell plates; then the print head traverses
the vacuum table and touches the quill tips to each glass slide in
succession ,depositing target DNA; the print head continues to the
wash/dry station where the tips are cleaned twice with water and
dried. This cycle repeats as the print head returns to wet the tips
in the next set of targets, continuing until all targets of a 96 well
microwell plate have been printed. An autoloading mechanism
removes spent microwell plates and can serve up new plates. By
this method, microarray slides can be printed with as many as
15000 precise and discrete cDNA targets
38.
39.
40. Microarray hybridization. This pseudocolored image
represents a portion of a microarray with the reference
probe (normal fibroblasts) in green and
rhabdomyosarcoma in red. The up (red) and down
(green) regulation of several diseased genes are
illustrated. Representative genes of interest are boxed.
Cancer Res. 58(1998)5009-5013
42. • The impact of molecular biology and genetic
research on the discovery of the root causes of a
wide variety of hereditary and acquired diseases
has long been self-evident.
• Now growing research in this field has shown that
conditions like hypertension, arryhtmias, certain
cardiomyopathies, Hyperlipidemia, aortic
aneurysms, myocardial infarction are all
genetically determined.
Molecular Genetics and Its Application
to Understanding Cardiovascular Disease
43. Gene Therapy
• Gene therapy, i.e; to introduce genes in selected cells to
treat genetic or acquired diseases .Recombinant DNA
technology has revolutionized this field.
• Clinical trials are underway to treat a variety of
conditions like cancer, AIDS, familial
hypercholesterolemia, cystic fibrosis, and many other
diseases with this therapy.
• The growing acceptance of gene therapy for
cardiovascular diseases is due to its potential to treat
common , multifactorial disorders like cancer,
atherogenesis, and inflammation.
44. Our Aim
• We intend to induce rupture of atherosclerotic plaque in
apo-e mice by injecting several drugs, which we hope will
cause rupture of the the hemodynamic system and raising
the plaque by altering oxidative stress and alter the
composition of the plaque.
• After achieving this goal we will perform gene- array
study on these specimens and will try to find out which
genes are expressed more in plaques which are ruptured or
are prone to rupture, by comparing our results at the
histopathology lab which will define the structural details
of such vulnerable plaques.
45. • Drugs being used to
induce rupture of
atherosclerotic plaque
in apo-e mice
47. L-NAME
• L-NAME (N-nitro-L-arginine methyl ester), is a negative
effector of nitric oxide synthase.
• Action:By virtue of this action, it blocks the action of
nitric oxide and interferes with the arterial dilatation,
causing more load on the heart and the circulation
• The production of NO is a major contributor to the
endothelium-dependent relaxation in large isolated
arteries, including coronary, mesenteric, pulmonary,
systemic, and cerebral arteries.
48. • All the actions of nitric-oxide are inhibited by L-NAME,
as well as it promotes adhesion of platelets and leukocytes
to the vascular lumen..
• Also the inhibitory effect of NO towards inhibiting platelet
aggregation action of prostacyclin and inhibition of growth
of vascular smooth cells is abolished by L-NAME.
49. Interleukin-1 beta
• Class: Cytokine
• These cytokines are polypeptides produced
by many cell types(but principally activated
lymphocytes and macrophages).
• Their secretion can be stimulated by
endotoxin, immune complexes, toxins,
physical injury, and a variety of
inflammatory processes.
50. • Monocytes are the main source of
secreted IL-1. They express
predominantly IL-1-beta while
human keratinocytes express
large amounts of IL-1-alpha .
Murine macrophages display a
transition from IL-1-beta to IL-1-
alpha production during
maturation of monocytes into
inflammatory macrophages .
51. Action
• Their most important actions in inflammation are
the local effects on endothelium, the systemic
acute-phase reactions, and the effect on
fibroblasts.
• In particular they induce the synthesis and surface
expression of the endothelial adhesion molecules
that mediate leukocyte sticking and increase
surface thrombogenicity of the endothelium.
52. Adrenalin
• Class: Sympathomimetic
• Mech: At low doses, beta effects on the vascular
system predominate, whereas at high doses, alpha
effects are strongest.
• Action: Major actions are on the cardiovascular
system, causes tachycardia, increases
myocardium’s oxygen demand, constricts
arterioles in the skin, mucous membrane and
viscera.
53. • Also causes broncodilation, hyperglycemia,
lipolysis.
• Adverse effects: causes CNS disturbances
including tremor, anxiety, and marked elevation of
blood pressure, and arrythmias.
54. Methionine
• Class: Amino acid
• Metabolism of the amino acid methionine, a
limiting amino acid in the synthesis of many
proteins, affects several biochemical pathways
involving the production of nutrients which are
essential to the optimal functioning of the
cardiovascular, skeletal, and nervous system
Homocysteine is an intermediate product of
methionine metabolism.
55. Action
• Homocysteine has direct cytotoxic effects on
endothelial cells in vitro
• Homocysteine has a direct procoagulant effect on
factors V and X and inhibits protein C activation.
• Additionally, there is exciting preliminary data
linking a flux in the homocysteine pathway with
liver disease, leukemia, psoriasis and other
relatively common disorders. Research is being
conducted currently in many of these areas.
56. • In the past 20+ years, research has shown that
elevated homocysteine, or
hyperhomocysteinaemia, is linked to
atherosclerosis, pregnancies complicated by neural
tube defects, early pregnancy loss and venous
thrombosis.
• Patients with chronic renal disease have a two to
three fold increase in homocysteine levels .
57. Buthionine Sulfoximine
• Class: selective glutathione (GSH) synthase
inhibitor.
• Due to their action causing depletion of
GSH, which is a necessary component of
the natural antioxidant system, BSO causes
oxidative stress, which might cause
hypertension by inactivation and
sequestration of NO(mediated by ROS).
58. Cocaine
• Class: Sympathomimetic
• Mech: Blocks neuronal re-uptake of
norepinephrine, serotonin, and dopamine.
• Action: Stimulates CNS, causes euphoria,
increases motor activity.
• Potentiates the action of cathecholamines,i.e;
tachycardia, hypertension, pupillary dilation and
peripheral vasoconstriction.
59. • Adverse effects:
• Anxiety, causing increased blood pressure and
heart rate, sweating.
• Depression, Like all stimulant drugs, cocaine
stimulation of CNS is followed by a period of
mental depression.
60. Xanthine, Xanthine-Oxidase
• Xanthine oxidase (XO) is a complex enzyme
containing flavins, molybdenum, iron and sulfide
cofactors. The reaction catalyzed by Xanthine
oxidase is shown below:
• XO
• Xanthine + O2 + H2O ------> Uric Acid + H2O2
• XO is thought to be the principal source of free
radical generation via degradation of nucleotides
to the end product, uric acid.
61. • Xanthine can exert lethal effects by
generating free radicals, this has been
proved by several studies, and the effcet of
these free radicals is to depress myocardial
contractility, by virtue of robbing the
myocytes of high energy phospahtes, also it
causes the LDLs to be more prone to be
oxidized.
62. Doses of drugs used
Based on references from articles
from Pub-Med
63. • . Substance: LNAME
Route of Administration: IP
Dose: 20 mg/kg/day for 7 days
Frequency: 7
References: Nitric oxide 2000Apr;4(2):85-93
Life Sci 1995;57(21):1949-61,
Br J Pharmacology:1994oct;113(2):345-8
64. • 2. Substance: adrenalin
Route of Administration: IP
Dose: 1 microgram/kg
Frequency: 7
References: Auton Pharmacol 1998 jun;18(3):149-
155
Life Sci 1982 Apr 26;30(170:1465-72
Can J Cardiol 1990 Mar;6(2):71-4
.
66. • 4. Substance: xanthine
Route of Administration: IP
Dose: 0.0012 mM/kg/day for 7 days
Frequency: 7
References: Naunyn Schmiedebergs Arch Pharmacol
1994 Sp;350 (3):227-83
Naunyn Schmiedebergs Arch Pharmacol 1992
Oct;346(4):457-61
67. • 5. Substance: xanthine oxidase
Route of Administration: IP
Dose: 0.035 U/ml/day for 7 days
Frequency: 7
References: Cardiovasc Res 1986 Aug;20(8):597-
603
Mol Cell Biochem 1995 Apr 26;145(2):103-10
Naunyn Schmiedebergs Arch Pharmacol 1994
Sp;350 (3):227-83
Naunyn Schmiedebergs Arch Pharmacol 1992
Oct;346(4):457-61
68. • 6. Substance: IL-1 beta
Route of Administration: IP
Dose: 1ug/mouse/day for 7 days
Frequency:7
Reference: Cancer Biother Radiopharm 1997
Apr:12(2):101-9
Am J Physiol 1995 Apr;268(4 Pt 1):E551-7
Brain Res 1999 Jan 9;815(2):337-48
Pediatr Res 1995 Jun;37(6):714-9
J Neuroendocrinol 1994 Apr;6(2):217-24
69. • Substance: methionine
Route of Administration: IP
Dose: 1280 mg/kg/day for 7 days
Frequency: 7
Reference: Pharmacol Biochem Behav 1999
Sep;64(1):89-93
70. • Substance: Buthionine sulfoximine
Route of Administration: IP
Dose:1 mmol/kg
Frequency: 7
References:
In previous studies doses of 2.5mmol, 7.2mmolI/V,
And 5mmol/kg, and 3mmol/kghave been used, the dose
we are going to use is much lower than that.
Hepatology 2000 Aug;32(2):321-33
J Pharmacol Exp Ther 1998Nov;287(2):515-20
72. 9/11/00 42
Samples:
A. Human carotid atherosclerotic plaque from St.
Luke’s hospital OR: ( endarterectomy results)
B. Ruptured human coronary atherosclerotic
plaque from sudden cardiac death victims with
plaque rupture from Dr Kolgogi (Dr Virmani’s
Lab)
C. Atherosclerotic plaque of Apo-E K/O old mice
(18 months) fed with high fat diet that have been
prompted to rupture using several interventions
( modified Falk procedures).
D. Atherosclerotic plaque of Apo-E K/O 18 months
old mice without being prompted to rupture.
E. Normal C57BL mice artery.
73. Results
• We will perform gene-array on the RNAs
obtained form the ruptures palque to see
which genes are more expressed in the
palque
• We will also compare our results in the
histochemical laboratory
74. 9/10/00 43
Gene array method
We will use mRNA for probe. RNA will be extracted
according the protocol and then cDNA is made by
RT-PCR. Hybridize with specific probes and
scanned.
We will use Affymetrix gene array materials.
The chips for samples As & Bs contain 12000
human genes.
The chips for samples Cs and Ds contain 19000
mouse genes.
We will scan the chips with Affymetrix scanner.
75. If we do not find
anything
Very pleasant, at least
we shalll find
something new