Molecular methods such as PCR and LAMP have revolutionized infectious disease diagnosis by allowing rapid and sensitive detection of pathogens. PCR amplifies specific DNA sequences, and real-time PCR with fluorescent probes like TaqMan or molecular beacons allows quantification during amplification. LAMP is an inexpensive isothermal method that amplifies DNA with high sensitivity and specificity using multiple primers and strand displacement. Both PCR and LAMP have advanced diagnosis by detecting pathogens earlier and multiplexing the detection of multiple targets in a single sample.
Probe labeling is defined as sequence use to search the mixture of nucleic acid for molecule containing complementary sequence.In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA
The document discusses various types of bacteriophage vectors that can be used for cloning genomic DNA, including their structure and applications. Phage derivatives like lambda phages and M13 phages have been developed as cloning vectors since they allow large DNA fragments to be cloned and can package millions of recombinant phage particles. The document describes different types of phage vectors like insertion vectors containing a single cloning site, replacement vectors where the insert substitutes phage DNA sequences, and hybrid plasmid-phage vectors. It provides details about various vector systems including cosmids, which combine phage and plasmid properties to clone large DNA fragments.
Bacterial Identification by 16s rRNA Sequencing.pptRakesh Kumar
Bacteria are the most abundant life forms on Earth, with a single gram of soil containing 40 million bacterial cells. Most bacterial species have yet to be identified due to their abundance. DNA sequencing of the 16s rRNA gene is a common technique used to identify bacterial species. The process involves isolating bacteria from a sample, extracting DNA, amplifying and sequencing the 16s rRNA gene, and comparing the sequence to databases to identify matches. 16s rRNA gene sequencing provides a more accurate identification of bacteria than phenotypic methods.
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.
DNA microarrays contain thousands of DNA probes attached to a solid surface that allow for the simultaneous analysis of gene expression across many genes. The core principle is based on DNA hybridization, where fluorescently labeled cDNA or RNA samples are hybridized to complementary probes on the array. By detecting which probes light up after hybridization and washing, researchers can determine which genes are expressed or detect genetic variations in the sample. Microarrays have numerous applications, including gene expression analysis, disease diagnosis, drug discovery, and toxicology research. They provide a fast way to study thousands of genes but results require further validation and correlations do not necessarily indicate causation.
Lectut btn-202-ppt-l28. variants of pcr-iiRishabh Jain
Reverse transcriptase PCR (RT-PCR) is used to amplify cDNA copies of RNA. It involves reverse transcribing RNA to cDNA then amplifying the cDNA with PCR. RT-PCR can be used to study gene expression and diagnose genetic diseases. Variations include band-stab PCR which reamplifies low yield fragments, degenerate PCR which uses mixed primers for related gene families, and anchored PCR which attaches a known sequence to amplify unknown 5' sequences. Real-time PCR monitors fluorescence during amplification to quantify templates in each cycle, allowing visualization of reactions in real-time. It is commonly used to measure changes in gene expression.
Probe labeling is defined as sequence use to search the mixture of nucleic acid for molecule containing complementary sequence.In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA
The document discusses various types of bacteriophage vectors that can be used for cloning genomic DNA, including their structure and applications. Phage derivatives like lambda phages and M13 phages have been developed as cloning vectors since they allow large DNA fragments to be cloned and can package millions of recombinant phage particles. The document describes different types of phage vectors like insertion vectors containing a single cloning site, replacement vectors where the insert substitutes phage DNA sequences, and hybrid plasmid-phage vectors. It provides details about various vector systems including cosmids, which combine phage and plasmid properties to clone large DNA fragments.
Bacterial Identification by 16s rRNA Sequencing.pptRakesh Kumar
Bacteria are the most abundant life forms on Earth, with a single gram of soil containing 40 million bacterial cells. Most bacterial species have yet to be identified due to their abundance. DNA sequencing of the 16s rRNA gene is a common technique used to identify bacterial species. The process involves isolating bacteria from a sample, extracting DNA, amplifying and sequencing the 16s rRNA gene, and comparing the sequence to databases to identify matches. 16s rRNA gene sequencing provides a more accurate identification of bacteria than phenotypic methods.
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.
DNA microarrays contain thousands of DNA probes attached to a solid surface that allow for the simultaneous analysis of gene expression across many genes. The core principle is based on DNA hybridization, where fluorescently labeled cDNA or RNA samples are hybridized to complementary probes on the array. By detecting which probes light up after hybridization and washing, researchers can determine which genes are expressed or detect genetic variations in the sample. Microarrays have numerous applications, including gene expression analysis, disease diagnosis, drug discovery, and toxicology research. They provide a fast way to study thousands of genes but results require further validation and correlations do not necessarily indicate causation.
Lectut btn-202-ppt-l28. variants of pcr-iiRishabh Jain
Reverse transcriptase PCR (RT-PCR) is used to amplify cDNA copies of RNA. It involves reverse transcribing RNA to cDNA then amplifying the cDNA with PCR. RT-PCR can be used to study gene expression and diagnose genetic diseases. Variations include band-stab PCR which reamplifies low yield fragments, degenerate PCR which uses mixed primers for related gene families, and anchored PCR which attaches a known sequence to amplify unknown 5' sequences. Real-time PCR monitors fluorescence during amplification to quantify templates in each cycle, allowing visualization of reactions in real-time. It is commonly used to measure changes in gene expression.
If a microbiologist is studying bacteria that premeditate, or break down, toxic wastes and wants to know which specific genes are active when that bacterium is degrading, say, PCBs, he would likely use a tool called the DNA microarray.
Microarrays enable scientists to monitor the activities of hundreds or thousands of genes at once. All microarrays (also called DNA chips or gene chips) work on the basic principle that complementary nucleotide sequences in DNA (and RNA) match up like the two halves of a piece of Velcro coming together.
Pattern of gene activity on a microarray chip.
A microarray consists of an orderly arrangement of bits of genetic material in super-tiny spots laid down in a grid on a suitable surface, often a glass slide with a specially chemically treated surface.
This document discusses commonly used protein expression systems. It begins by explaining how recombinant DNA technology has enabled the cloning and expression of mammalian genes in different systems to produce therapeutic and vaccine proteins. It notes that the suitable expression system depends on factors like productivity, bioactivity, purpose and protein characteristics. The document then examines various prokaryotic (bacterial) and eukaryotic expression systems in detail, including E. coli, Bacillus subtilis, Lactococcus lactis, Pseudomonas, Corynebacterium, yeast, mammalian cells and baculovirus cells. It highlights advantages and disadvantages of each system for recombinant protein expression.
The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips.
Yeast artificial chromosomes (YACs) are engineered DNA molecules that can clone and replicate large DNA sequences in yeast cells. YACs contain essential yeast elements like a centromere and telomeres that allow them to behave like natural yeast chromosomes. YACs can clone very large inserts of up to 10 megabases of foreign DNA, making them useful for generating whole genome libraries.
This document discusses the history and properties of plasmids. Plasmids are extrachromosomal DNA molecules that are able to replicate independently of a cell's chromosomal DNA. They were first observed in bacteria in the early 1950s and play important roles in processes like antibiotic resistance and virulence. The document outlines the early studies that helped characterize plasmids and describes some of their key properties, such as their circular structure and ability to exist in different conformations. It also discusses how plasmids are used as cloning vectors to amplify genes and produce proteins for applications in research, medicine, and agriculture.
Artificial chromosomes are synthetic chromosomes introduced into host cells to propagate and transfect DNA fragments larger than plasmids can hold. Yeast artificial chromosomes (YACs) specifically are engineered chromosomes derived from yeast DNA ligated into bacterial plasmids, allowing insertion of 100-1000kb DNA fragments. YACs contain elements for yeast and bacterial replication and selection, and are useful for cloning large genomic fragments like whole human genes for mapping the genome.
This document discusses biosafety guidelines for recombinant DNA research. It defines biosafety as applying safety principles to potentially hazardous biological materials or organisms. Guidelines have been developed by organizations like the National Institutes of Health and Department of Biotechnology in India. There are four biosafety levels depending on the risk posed by the organisms and experiments, with increasing safety requirements at higher levels. Risk assessment involves evaluating characteristics of the organisms and modifications to determine the biosafety level needed. Risk management aims to minimize risks to human health and the environment through prevention measures and policies.
Lectut btn-202-ppt-l27. variants of pcr-iRishabh Jain
This document describes various types of PCR including inverse PCR, colony PCR, hot start PCR, multiplex PCR, in situ PCR, long PCR, nested PCR, touchdown PCR and their applications. Inverse PCR is used to amplify unknown flanking sequences. Colony PCR screens bacterial colonies without isolating DNA. Hot start PCR prevents nonspecific amplification. Multiplex PCR amplifies multiple targets simultaneously. Nested PCR increases specificity with two primer sets. Touchdown PCR optimizes annealing temperatures.
This document discusses the construction and screening of genomic libraries. It explains that a genomic library contains DNA fragments that represent an organism's entire genome. The library is constructed by isolating, purifying, and fragmenting genomic DNA, then cloning the fragments into suitable vectors. Common vectors for large DNA fragments include lambda phage, YACs, and BACs. The library can then be screened to identify clones containing genes of interest using various methods like hybridization, PCR, or screening for gene expression and complementation. Hybridization methods for screening include colony hybridization and plaque hybridization.
DNA ligation is the joining of two nucleic acid fragments through the action of an enzyme. Several factors can affect the ligation reaction, including the concentration of enzyme, DNA, and cofactors like ATP or NAD+. The DNA concentration is particularly important, as higher concentrations favor intermolecular ligation between separate DNA molecules, while lower concentrations favor intramolecular ligation where a DNA molecule joins its own ends. DNA ligase carries out ligation through a three-step catalytic mechanism involving adenylation of the enzyme and two phosphoryl transfers.
1. A DNA microarray contains thousands of DNA probes attached to a solid surface in defined locations. Each probe represents a single gene.
2. Sample mRNA is converted to fluorescently labeled cDNA and hybridized to the DNA microarray. The level of fluorescence indicates the expression level of each gene.
3. After washing, the microarray is scanned and analyzed to determine changes in gene expression between control and test samples. This allows high-throughput analysis of gene expression profiles.
in this presentation, what are the steps and strategies involved the gene cloning and i was focused only on the 1st two steps of gene cloning.they are generation of foreign DNA molecules and selection of suitable vectors.
Lectut btn-202-ppt-l25. introduction of dna into host cellsRishabh Jain
This document discusses various methods for introducing DNA into host cells, which is an important step in genetic engineering. It describes transformation, which is introducing DNA into living cells, and transfection, which is introducing viral DNA into living cells. The document then outlines several biological, chemical, and physical methods for transformation and transfection in bacteria, plants, insects, and animal cells. These include techniques like bacterial infection with bacteriophage, Agrobacterium-mediated plant transformation, microprojectile bombardment, electroporation, microinjection, and calcium phosphate transfection. It also discusses factors that affect transformation efficiency.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
This document discusses the use of 16S ribosomal RNA (rRNA) gene sequencing for bacterial identification and phylogenetic analysis. It explains that the 16S rRNA gene is highly conserved, making it useful for comparing distantly related organisms. The document outlines the process of 16S rRNA gene sequencing, including PCR amplification using conserved primer regions and sequencing of variable regions. It also discusses various methods that have been developed using 16S rRNA, such as TRFLP profiling and ribotyping, to study microbial communities.
Restriction enzymes are molecular scissors found in bacteria that cut DNA at specific recognition sequences. They break the covalent bonds within a DNA strand and the hydrogen bonds between the strands. Bacteria use these enzymes to protect themselves from foreign DNA. Restriction enzymes are now used widely in laboratories to cut DNA in recombinant DNA techniques and other applications like DNA fingerprinting. They produce sticky or blunt ends that allow targeted joining of DNA fragments.
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
Ribosomes are complex structures found in all living cells which functions in protein synthesis machinery. Basically ribosome’s consists of two subunits, each of which is composed of protein and a type of RNA, known as ribosomal RNA (rRNA). Prokaryotic ribosomes consist of 30S subunit (small sub unit) and 50S subunit (large sub unit) which together make up the complete 70S ribosome, where S stands for Svedberg unit non-SI unit for sedimentation rate. 30S subunit is composed of 16S ribosomal RNA and 21 polynucleotide chains while 50S subunit is composed of two rRNA species, the 5S and 23S rRNAs. The presence of hyper variable regions in the 16S rRNA gene provides a species specific signature sequence which is useful for bacterial identification process. 16S Ribosomal RNA sequencing is widely used in microbiology studies to identify the diversities in prokaryotic organisms as well as other organisms and thereby studying the phylogenetic relationships between them. The advantages of using ribosomal RNA in molecular techniques are as follows
Ribosomes and ribosomal RNA are present in all cells.
RNA genes are highly conserved in nature.
Culturing of microbial cells is absent in the sequencing techniques.
A gene library is a large collection of DNA fragments cloned from an organism. It contains genomic DNA or cDNA sequences. Gene libraries are constructed using molecular tools like restriction enzymes and ligases to cut and paste DNA fragments into vectors such as plasmids, phages, or artificial chromosomes. The choice of vector depends on the size of the genome being cloned. Libraries allow screening to identify genes of interest through techniques like hybridization or expression screening. cDNA libraries contain only expressed sequences without introns, making them preferable for cloning eukaryotic genes in prokaryotes.
Polymerase Chain Reaction: Principles, Applications, and Advancements | The L...The Lifesciences Magazine
Polymerase Chain Reaction, often abbreviated as PCR, is a laboratory technique used to amplify specific segments of DNA through a series of temperature-controlled cycles.
Polymerase chain reaction (PCR) is a technique used to amplify DNA sequences. It involves using short DNA sequences called primers and DNA polymerase to replicate the target DNA segment. During PCR, the target DNA is denatured, primers anneal to the single strands, and DNA polymerase extends the primers to make copies of the DNA. This cycling process allows exponential amplification of the target sequence, generating millions of copies. PCR is widely used in medical research and forensic analysis to detect pathogens and identify DNA profiles.
If a microbiologist is studying bacteria that premeditate, or break down, toxic wastes and wants to know which specific genes are active when that bacterium is degrading, say, PCBs, he would likely use a tool called the DNA microarray.
Microarrays enable scientists to monitor the activities of hundreds or thousands of genes at once. All microarrays (also called DNA chips or gene chips) work on the basic principle that complementary nucleotide sequences in DNA (and RNA) match up like the two halves of a piece of Velcro coming together.
Pattern of gene activity on a microarray chip.
A microarray consists of an orderly arrangement of bits of genetic material in super-tiny spots laid down in a grid on a suitable surface, often a glass slide with a specially chemically treated surface.
This document discusses commonly used protein expression systems. It begins by explaining how recombinant DNA technology has enabled the cloning and expression of mammalian genes in different systems to produce therapeutic and vaccine proteins. It notes that the suitable expression system depends on factors like productivity, bioactivity, purpose and protein characteristics. The document then examines various prokaryotic (bacterial) and eukaryotic expression systems in detail, including E. coli, Bacillus subtilis, Lactococcus lactis, Pseudomonas, Corynebacterium, yeast, mammalian cells and baculovirus cells. It highlights advantages and disadvantages of each system for recombinant protein expression.
The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips.
Yeast artificial chromosomes (YACs) are engineered DNA molecules that can clone and replicate large DNA sequences in yeast cells. YACs contain essential yeast elements like a centromere and telomeres that allow them to behave like natural yeast chromosomes. YACs can clone very large inserts of up to 10 megabases of foreign DNA, making them useful for generating whole genome libraries.
This document discusses the history and properties of plasmids. Plasmids are extrachromosomal DNA molecules that are able to replicate independently of a cell's chromosomal DNA. They were first observed in bacteria in the early 1950s and play important roles in processes like antibiotic resistance and virulence. The document outlines the early studies that helped characterize plasmids and describes some of their key properties, such as their circular structure and ability to exist in different conformations. It also discusses how plasmids are used as cloning vectors to amplify genes and produce proteins for applications in research, medicine, and agriculture.
Artificial chromosomes are synthetic chromosomes introduced into host cells to propagate and transfect DNA fragments larger than plasmids can hold. Yeast artificial chromosomes (YACs) specifically are engineered chromosomes derived from yeast DNA ligated into bacterial plasmids, allowing insertion of 100-1000kb DNA fragments. YACs contain elements for yeast and bacterial replication and selection, and are useful for cloning large genomic fragments like whole human genes for mapping the genome.
This document discusses biosafety guidelines for recombinant DNA research. It defines biosafety as applying safety principles to potentially hazardous biological materials or organisms. Guidelines have been developed by organizations like the National Institutes of Health and Department of Biotechnology in India. There are four biosafety levels depending on the risk posed by the organisms and experiments, with increasing safety requirements at higher levels. Risk assessment involves evaluating characteristics of the organisms and modifications to determine the biosafety level needed. Risk management aims to minimize risks to human health and the environment through prevention measures and policies.
Lectut btn-202-ppt-l27. variants of pcr-iRishabh Jain
This document describes various types of PCR including inverse PCR, colony PCR, hot start PCR, multiplex PCR, in situ PCR, long PCR, nested PCR, touchdown PCR and their applications. Inverse PCR is used to amplify unknown flanking sequences. Colony PCR screens bacterial colonies without isolating DNA. Hot start PCR prevents nonspecific amplification. Multiplex PCR amplifies multiple targets simultaneously. Nested PCR increases specificity with two primer sets. Touchdown PCR optimizes annealing temperatures.
This document discusses the construction and screening of genomic libraries. It explains that a genomic library contains DNA fragments that represent an organism's entire genome. The library is constructed by isolating, purifying, and fragmenting genomic DNA, then cloning the fragments into suitable vectors. Common vectors for large DNA fragments include lambda phage, YACs, and BACs. The library can then be screened to identify clones containing genes of interest using various methods like hybridization, PCR, or screening for gene expression and complementation. Hybridization methods for screening include colony hybridization and plaque hybridization.
DNA ligation is the joining of two nucleic acid fragments through the action of an enzyme. Several factors can affect the ligation reaction, including the concentration of enzyme, DNA, and cofactors like ATP or NAD+. The DNA concentration is particularly important, as higher concentrations favor intermolecular ligation between separate DNA molecules, while lower concentrations favor intramolecular ligation where a DNA molecule joins its own ends. DNA ligase carries out ligation through a three-step catalytic mechanism involving adenylation of the enzyme and two phosphoryl transfers.
1. A DNA microarray contains thousands of DNA probes attached to a solid surface in defined locations. Each probe represents a single gene.
2. Sample mRNA is converted to fluorescently labeled cDNA and hybridized to the DNA microarray. The level of fluorescence indicates the expression level of each gene.
3. After washing, the microarray is scanned and analyzed to determine changes in gene expression between control and test samples. This allows high-throughput analysis of gene expression profiles.
in this presentation, what are the steps and strategies involved the gene cloning and i was focused only on the 1st two steps of gene cloning.they are generation of foreign DNA molecules and selection of suitable vectors.
Lectut btn-202-ppt-l25. introduction of dna into host cellsRishabh Jain
This document discusses various methods for introducing DNA into host cells, which is an important step in genetic engineering. It describes transformation, which is introducing DNA into living cells, and transfection, which is introducing viral DNA into living cells. The document then outlines several biological, chemical, and physical methods for transformation and transfection in bacteria, plants, insects, and animal cells. These include techniques like bacterial infection with bacteriophage, Agrobacterium-mediated plant transformation, microprojectile bombardment, electroporation, microinjection, and calcium phosphate transfection. It also discusses factors that affect transformation efficiency.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
This document discusses the use of 16S ribosomal RNA (rRNA) gene sequencing for bacterial identification and phylogenetic analysis. It explains that the 16S rRNA gene is highly conserved, making it useful for comparing distantly related organisms. The document outlines the process of 16S rRNA gene sequencing, including PCR amplification using conserved primer regions and sequencing of variable regions. It also discusses various methods that have been developed using 16S rRNA, such as TRFLP profiling and ribotyping, to study microbial communities.
Restriction enzymes are molecular scissors found in bacteria that cut DNA at specific recognition sequences. They break the covalent bonds within a DNA strand and the hydrogen bonds between the strands. Bacteria use these enzymes to protect themselves from foreign DNA. Restriction enzymes are now used widely in laboratories to cut DNA in recombinant DNA techniques and other applications like DNA fingerprinting. They produce sticky or blunt ends that allow targeted joining of DNA fragments.
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
Ribosomes are complex structures found in all living cells which functions in protein synthesis machinery. Basically ribosome’s consists of two subunits, each of which is composed of protein and a type of RNA, known as ribosomal RNA (rRNA). Prokaryotic ribosomes consist of 30S subunit (small sub unit) and 50S subunit (large sub unit) which together make up the complete 70S ribosome, where S stands for Svedberg unit non-SI unit for sedimentation rate. 30S subunit is composed of 16S ribosomal RNA and 21 polynucleotide chains while 50S subunit is composed of two rRNA species, the 5S and 23S rRNAs. The presence of hyper variable regions in the 16S rRNA gene provides a species specific signature sequence which is useful for bacterial identification process. 16S Ribosomal RNA sequencing is widely used in microbiology studies to identify the diversities in prokaryotic organisms as well as other organisms and thereby studying the phylogenetic relationships between them. The advantages of using ribosomal RNA in molecular techniques are as follows
Ribosomes and ribosomal RNA are present in all cells.
RNA genes are highly conserved in nature.
Culturing of microbial cells is absent in the sequencing techniques.
A gene library is a large collection of DNA fragments cloned from an organism. It contains genomic DNA or cDNA sequences. Gene libraries are constructed using molecular tools like restriction enzymes and ligases to cut and paste DNA fragments into vectors such as plasmids, phages, or artificial chromosomes. The choice of vector depends on the size of the genome being cloned. Libraries allow screening to identify genes of interest through techniques like hybridization or expression screening. cDNA libraries contain only expressed sequences without introns, making them preferable for cloning eukaryotic genes in prokaryotes.
Polymerase Chain Reaction: Principles, Applications, and Advancements | The L...The Lifesciences Magazine
Polymerase Chain Reaction, often abbreviated as PCR, is a laboratory technique used to amplify specific segments of DNA through a series of temperature-controlled cycles.
Polymerase chain reaction (PCR) is a technique used to amplify DNA sequences. It involves using short DNA sequences called primers and DNA polymerase to replicate the target DNA segment. During PCR, the target DNA is denatured, primers anneal to the single strands, and DNA polymerase extends the primers to make copies of the DNA. This cycling process allows exponential amplification of the target sequence, generating millions of copies. PCR is widely used in medical research and forensic analysis to detect pathogens and identify DNA profiles.
Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA. It allows scientists to make millions to billions of copies of the target DNA sequence. Real-time quantitative PCR (qPCR) allows quantification of the amount of target DNA or RNA present. In situ hybridization is a technique that uses labeled nucleic acid probes to localize specific DNA or RNA sequences within cells in preserved tissue samples.
PCR can be used for cloning, DNA fingerprinting, gene expression analysis, DNA sequencing, and more. It works by amplifying a specific region of DNA through repeated heating and cooling cycles. This allows very small amounts of DNA to be exponentially multiplied, enabling various applications. PCR was invented in 1983 by Kary Mullis and revolutionized molecular biology.
SLIDE CONTAIN BREIF NOTE ON PCR. IT CONTAINS 21 SLIDES INCLUDING, WHAT IS PCR? COMPONENTS, WORKING MECHANISM, APPLICATIONS, CONCLUSION, AND SOME REFRENCES, HISTORY ALSO
This document provides an overview of polymerase chain reaction (PCR) including its history, principles, components, cycle, limitations, advantages, disadvantages, types, and applications. PCR is described as an in vitro method for enzymatically synthesizing specific DNA sequences using two oligonucleotide primers that flank the target region. It allows for exponential amplification of minute amounts of DNA. Real-time PCR is highlighted as an advancement that provides ease of quantification, greater sensitivity, reproducibility, and precision compared to traditional PCR. The document also reviews various applications of PCR in fields such as medicine, forensics, and research.
Next generation sequencing has revolutionized DNA sequencing by allowing millions of DNA fragments to be sequenced in parallel. This has increased sequencing speed and reduced costs. NGS is now used for applications like genome sequencing, transcriptome analysis, metagenomics, and studying genetic variation. It has provided insights into molecular biology and genomics that were not possible with previous sequencing methods.
It contains information about- DNA Sequencing; History and Era sequencing; Next Generation Sequencing- Introduction, Workflow, Illumina/Solexa sequencing, Roche/454 sequencing, Ion Torrent sequencing, ABI-SOLiD sequencing; Comparison between NGS & Sangers and NGS Platforms; Advantages and Applications of NGS; Future Applications of NGS.
This document provides an overview of polymerase chain reaction (PCR). It describes how PCR is used to amplify specific DNA sequences in vitro. Key steps involve denaturing the DNA, annealing primers to the DNA, and extending the primers using a DNA polymerase. The document outlines the components of PCR including DNA template, primers, nucleotides, DNA polymerase, and buffers. It also describes different types of PCR like quantitative PCR and their applications in fields like disease diagnosis, genetic engineering, and forensics.
Molecular diagnostic techniques involve manipulating and analyzing DNA, RNA, and proteins. These techniques are used in areas like neoplastic disorders, infectious diseases, and inherited conditions. Common techniques include amplification methods like PCR, blotting techniques such as Southern blot and Western blot, and hybridization methods like in situ hybridization and microarrays. Molecular diagnostics provides advantages like automation, speed, reliability, and sensitivity, though limitations include potential errors and sensitivity to inhibitors. These techniques have revolutionized fields like medicine, agriculture, forensics, and more by enabling abundant production of DNA and detection of genetic disorders.
The document describes a microarray study to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. Specimens will be obtained from human carotid/coronary arteries and atherosclerotic plaques in mouse models. Gene expression will be profiled using microarrays and correlated with histopathology, pH, temperature, spectroscopy and other variables. The goal is to identify genes associated with vulnerable plaques and rupture. Plaques from influenza-infected and drug-treated mice will also be analyzed to study effects on gene expression and plaque structure.
132 gene expression in atherosclerotic plaquesSHAPE Society
This document discusses microarray studies to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. It begins with background on the history and applications of DNA microarrays. Key steps discussed include probe design, sample preparation including tissue collection, labeling RNA samples, hybridizing samples to a microarray chip, scanning and analyzing image data. The document outlines creating a custom microarray based on selected genes and correlating gene expression with temperature, pH, spectroscopy and histopathology of plaques. It will also analyze gene expression in influenza-infected mice and mice where plaques are induced to rupture with drugs. Human carotid artery specimens from surgery will be analyzed from symptomatic and asymptomatic patients.
The document describes a microarray study to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. Specimens will be obtained from human carotid/coronary arteries and atherosclerotic plaques in mouse models. Gene expression will be profiled using microarrays and correlated with histopathology, pH, temperature, spectroscopy and other variables. Plaques from influenza-infected and drug-treated mice will also be analyzed to identify genes associated with plaque rupture. The goal is to better understand plaque vulnerability and identify potential drug targets.
Polymerase chain reaction (PCR) is a technique used to amplify small segments of DNA. During PCR, millions of copies of a DNA segment are made in just a few hours by separating the DNA strands, annealing primers, and extending new DNA strands using DNA polymerase. The key components of PCR are the target DNA, primers, DNA polymerase, nucleotides, and buffer solution. The PCR process involves denaturation, annealing, and extension steps that are repeated for multiple cycles to exponentially amplify the target DNA segment. PCR has many applications in basic research, applied research, and medical diagnosis.
This document discusses molecular diagnostics techniques. It begins by introducing molecular diagnostics and its significance in detecting pathogens, genetic mutations, and biomarkers. It then describes several key techniques used in molecular diagnostics, including nucleic acid amplification methods like PCR, isothermal amplification, and hybridization techniques. It also discusses methods like microarrays, genotyping, and mass spectrometry. The document provides examples of how these techniques are applied to detect various infectious diseases and genetic conditions.
This document summarizes rapid detection methods for foodborne pathogen bacteria. It discusses how foodborne illnesses are a major public health problem and rapid detection of pathogens is needed. Several detection methods are outlined, including traditional culturing as well as newer techniques like PCR, real-time PCR, LAMP, and immunoassays. LAMP is highlighted as a new method that can rapidly detect pathogens under isothermal conditions. The document concludes that LAMP is a promising technique for pathogen detection due to its speed, simplicity and accuracy.
A biochemical technique used in Molecular Biology to amplify a specific fragment of target DNA.
PCR is used in medical and biological research, including cloning, genetic analysis, genetic fingerprinting, diagnostics, pathogen detection and genetic fingerprinting
Polymerase chain reaction (PCR) is a technique used to amplify specific DNA sequences. It allows targeted DNA sequences to be selectively amplified millions of fold in a few hours. PCR consists of repeated cycles of heating and cooling of the DNA sample to denature and replicate the targeted sequence using DNA polymerase and primers. The amplified DNA can then be analyzed using gel electrophoresis. PCR has many applications including DNA cloning, gene expression analysis, DNA fingerprinting, paternity testing, and detecting infectious diseases and genetic mutations. The researcher aims to identify novel single nucleotide polymorphisms (SNPs) in the UGT1A7 gene in Circassian and Chechen subpopulations compared to Jordanians which may impact the metabolism of ir
Avs molecular diagnostic techniques for detection of plant pathogensAMOL SHITOLE
PCR is a technique used to detect plant pathogens through amplification of DNA. It involves denaturing DNA, annealing primers, and polymerizing new strands of DNA. This process is repeated to exponentially increase the amount of target DNA. Nested PCR improves sensitivity by adding a second round of amplification. Other techniques like RT-PCR, IC-PCR, bio-PCR, and co-operational PCR have also been used to detect pathogens through nucleic acid amplification and analysis. PCR provides an efficient way to diagnose and study plant pathogens.
Similar to Molecular methods of diagnosing infectious disease (20)
share - Lions, tigers, AI and health misinformation, oh my!.pptxTina Purnat
• Pitfalls and pivots needed to use AI effectively in public health
• Evidence-based strategies to address health misinformation effectively
• Building trust with communities online and offline
• Equipping health professionals to address questions, concerns and health misinformation
• Assessing risk and mitigating harm from adverse health narratives in communities, health workforce and health system
Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
- 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
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2. Diagnostic methods inDiagnostic methods in
MMicrobiologyicrobiology
Task of the method – to make the
microorganism “visible” and
“measureable”
Cultivation
Bio-testing
Immunological methods
Biochemical methods
Microscopy
Molecular methods
3. Watson and CrickWatson and Crick ’s Discovery of’s Discovery of
DNA -Path to Molecular MedicineDNA -Path to Molecular Medicine
4. Genotyping MethodsGenotyping Methods
Genetic methodsGenetic methods
generally seek togenerally seek to
detect polymorphisimdetect polymorphisim
at the level of nucleicat the level of nucleic
acidacid
Genotypes are moreGenotypes are more
specific, more easilyspecific, more easily
quantified andquantified and
standardized amongstandardized among
the differentthe different
organismsorganisms
5. Basic requirementBasic requirement
The genome isThe genome is
unique in eachunique in each
individualindividual
UltimateUltimate
discriminatory stepdiscriminatory step
would be to sequencewould be to sequence
the entire genome ofthe entire genome of
every organism, butevery organism, but
not practical ornot practical or
economicaleconomical
6. What is PracticableWhat is Practicable
Several methods inSeveral methods in
detecting nuclei aciddetecting nuclei acid
polymorphisim in apolymorphisim in a
chosen geneticchosen genetic
marker are commonlymarker are commonly
used to target theused to target the
genome or organism.genome or organism.
7. Kary Mullis - Nobel prize inKary Mullis - Nobel prize in
19931993
Kary Mullis sharedKary Mullis shared
the 1993 Nobel Prizethe 1993 Nobel Prize
in Chemistry within Chemistry with
Michael Smith. MullisMichael Smith. Mullis
received the prize forreceived the prize for
his development ofhis development of
the Polymerase Chainthe Polymerase Chain
Reaction (PCR)Reaction (PCR)
8. PCR a Revolution inPCR a Revolution in
ScienceScience
A process first describedA process first described
by Kjell Kleppe and 1968by Kjell Kleppe and 1968
Nobel laureate H. GobindNobel laureate H. Gobind
Khorana that allows theKhorana that allows the
amplification of specificamplification of specific
DNA sequences. TheDNA sequences. The
improvements providedimprovements provided
by Mullis have made PCRby Mullis have made PCR
a central technique ina central technique in
biochemistry andbiochemistry and
molecular biologymolecular biology
9. Polymerase ChainPolymerase Chain
ReactionReaction
Polymerase chain reactionPolymerase chain reaction ((PCRPCR) is a) is a
technique to amplify a single or few copiestechnique to amplify a single or few copies
of a piece of DNA across several orders ofof a piece of DNA across several orders of
magnitude, generating millions or moremagnitude, generating millions or more
copies of a particular DNA sequence. Thecopies of a particular DNA sequence. The
method relies on thermal cycling,method relies on thermal cycling,
consisting of cycles of repeated heatingconsisting of cycles of repeated heating
and cooling of the reaction for DNAand cooling of the reaction for DNA
melting and enzymatic replication of themelting and enzymatic replication of the
DNA.DNA.
10. Taq polymerase - backboneTaq polymerase - backbone
of PCR technologyof PCR technology
Almost all PCRAlmost all PCR
applications employ aapplications employ a
heat-stable DNAheat-stable DNA
polymerase, such aspolymerase, such as
Taq polymeraseTaq polymerase ,,
an enzyme originallyan enzyme originally
isolated from theisolated from the
bacteriumbacterium ThermusThermus
aquaticusaquaticus..
11. Oligonucleotide – DNAOligonucleotide – DNA
PrimersPrimers
This DNA polymeraseThis DNA polymerase
enzymatically assemblesenzymatically assembles
a new DNA strand froma new DNA strand from
DNA building blocks, theDNA building blocks, the
nucleotides, by usingnucleotides, by using
single-stranded DNA as asingle-stranded DNA as a
template and DNAtemplate and DNA
Oligonucleotide (alsoOligonucleotide (also
called DNA primers),called DNA primers),
which are required forwhich are required for
initiation of DNAinitiation of DNA
synthesis.synthesis.
14. PCR in ClinicalPCR in Clinical
MicrobiologyMicrobiology
Molecular detectionMolecular detection
has mostly come tohas mostly come to
thethe clinicalclinical
microbiologymicrobiology
laboratory in the formlaboratory in the form
of PCR technology,of PCR technology,
initially involvinginitially involving
single round orsingle round or
nested proceduresnested procedures
with detection by gelwith detection by gel
electrophoresis.electrophoresis.
15. Helps Rapid DetectionHelps Rapid Detection
Polymerase chainPolymerase chain
reaction (PCR)reaction (PCR)
techniques have led thetechniques have led the
way into this new era byway into this new era by
allowing rapid detectionallowing rapid detection
of microorganisms thatof microorganisms that
were previously difficult orwere previously difficult or
impossible to detect byimpossible to detect by
traditional microbiologicaltraditional microbiological
methods.methods.
16. Automation and MultiplexAutomation and Multiplex
PCRPCR
With the advent ofWith the advent of
multiplex PCR, real-multiplex PCR, real-
time PCR andtime PCR and
improvements inimprovements in
efficiency throughefficiency through
automation, the costsautomation, the costs
of molecular methodsof molecular methods
are decreasing suchare decreasing such
that the role ofthat the role of
molecular methodsmolecular methods
will further increase.will further increase.
17. Progress in MolecularProgress in Molecular
MethodsMethods
Molecular methodsMolecular methods
have now progressedhave now progressed
beyond identificationbeyond identification
to detect antimicrobialto detect antimicrobial
resistance genes andresistance genes and
provide public healthprovide public health
information such asinformation such as
strain characterisationstrain characterisation
byby genotyping.genotyping.
18. Molecular based TestsMolecular based Tests
needneed
Nucleic acid-based tests used inNucleic acid-based tests used in
diagnosing infectious diseases usediagnosing infectious diseases use
standard methods for isolating nucleicstandard methods for isolating nucleic
acids from organisms and clinical materialacids from organisms and clinical material
and restriction endonulease enzymes, geland restriction endonulease enzymes, gel
electrophoresis, and nucleic acidelectrophoresis, and nucleic acid
hybridization techniques to analyze DNAhybridization techniques to analyze DNA
or RNAor RNA
19. Moleculardiagnostics is a set of methods
to study primary structure (sequence) of DNA
•Hybridization with complementary sequences
•Amplification (synthesis) of species specific sequences
PCR – polymerase chain reaction
-A-A-T-T-C-G-C-G-A-T-G-
- T-T-A-A-G-C-G-C-T-A-C-
-A-A-T-T-C-G-C-G-A-T-G-
-A-A-T-T-C-G-C-G-A-T-G-
-A-A-T-T-C-G-C-G-A-T-G-
-A-A-T-T-C-G-C-G-A-T-G-
-A-A-T-T-C-G-C-G-A-T-G-
20. Advances on PCR MethodsAdvances on PCR Methods
Fairly recently, a newFairly recently, a new
method of PCRmethod of PCR
quantification hasquantification has
been invented. This isbeen invented. This is
calledcalled ““real-timereal-time
PCR”PCR” because itbecause it
allows the scientist toallows the scientist to
actually view theactually view the
increase in theincrease in the
amount of DNA as itamount of DNA as it
is amplified.is amplified.
22. New Technologies – RealNew Technologies – Real
Time AssaysTime Assays
The Real Time assays are proving to betterThe Real Time assays are proving to better
technologiestechnologies
1 Rapid1 Rapid
2 Quantitative measurement2 Quantitative measurement
3 Lower contamination rate3 Lower contamination rate
4 Higher sensitivity4 Higher sensitivity
5 Higher specificity5 Higher specificity
6 Easy standardization6 Easy standardization
Now a new gold standard for rapid diagnosis ofNow a new gold standard for rapid diagnosis of
virus infection in the acute phase samples.virus infection in the acute phase samples.
23. RT - PCRRT - PCR
Proving to beProving to be
AccurateAccurate
PrecisePrecise
Easy to performEasy to perform
RT PCR technologiesRT PCR technologies
are easy to transferare easy to transfer
research Laboratoryresearch Laboratory
protocols toprotocols to
DiagnosticDiagnostic
LaboratoriesLaboratories
24. OVERVIEW of RT - PCROVERVIEW of RT - PCR
tissue
extract RNA
copy into cDNA
(reverse transciptase)
do real-time PCR
analyze results
25. Real Time ReportersReal Time Reporters
All real time PCR systems rely upon theAll real time PCR systems rely upon the
detection and quantization of fluorescentdetection and quantization of fluorescent
reporter, the signal of which increases inreporter, the signal of which increases in
direct proportion of the amount of PCRdirect proportion of the amount of PCR
product in a reaction.product in a reaction.
26. REAL TIME PCRREAL TIME PCR
Cyber GreenCyber Green
USINGUSING
SYBER®SYBER®
GREENGREEN
The simplest andThe simplest and
economical format theeconomical format the
reporter is the doublereporter is the double
strand DNA specificstrand DNA specific
dye SYBR ® Greendye SYBR ® Green
Called as MolecularCalled as Molecular
Probe.Probe.
27. How SYBR Green worksHow SYBR Green works
SYBR green binds toSYBR green binds to
double stranded DNAdouble stranded DNA
and upon excitationand upon excitation
emits lightemits light
Thus as PCR productThus as PCR product
accumulates theaccumulates the
fluoresce increasesfluoresce increases
29. Documentation ofDocumentation of
AmplificationAmplification
The light emitted fromThe light emitted from
the dye in the excitedthe dye in the excited
state is received by astate is received by a
computer and shown oncomputer and shown on
a graph display, such asa graph display, such as
this, showing PCRthis, showing PCR
cycles on the X-axis andcycles on the X-axis and
a logarithmic indicationa logarithmic indication
of intensity on the Y-of intensity on the Y-
axis.axis.
30. Molecular Beacons
Molecular Beacons
Uses FRET
Fluorescence Resonance Energy Transfer
Uses two sequence specific
Oligonucleotide labelled with fluorescent
dyes
31. Molecular Beacons – RTMolecular Beacons – RT
PCRPCR
Molecular beacons areMolecular beacons are
designed to adopt adesigned to adopt a
hairpin structure whilehairpin structure while
free in solution, briningfree in solution, brining
the fluorescent dye andthe fluorescent dye and
quencher in closequencher in close
proximity. When aproximity. When a
molecular beaconmolecular beacon
hybridizes to a target thehybridizes to a target the
fluorescent dye emitsfluorescent dye emits
light upon irradiation, andlight upon irradiation, and
rebind to target in everyrebind to target in every
cycle for signalcycle for signal
measurement.measurement.
32. Loop Mediated IsothermalLoop Mediated Isothermal
Amplification (LAMP)Amplification (LAMP)
Loop mediated isothermal amplification isLoop mediated isothermal amplification is
a simple, rapid, specific and cost effectivea simple, rapid, specific and cost effective
nucleic acid amplification methodnucleic acid amplification method
characterized by use of 8 distinct regionscharacterized by use of 8 distinct regions
on the target gene.on the target gene.
The amplification proceeds at a constantThe amplification proceeds at a constant
temperature using strand displacementtemperature using strand displacement
reaction.reaction.
33. LAMPLAMP
Amplification andAmplification and
detection of gene can bedetection of gene can be
completed in a singlecompleted in a single
step, by incubating thestep, by incubating the
mixture of samples,mixture of samples,
primers DNA polymeraseprimers DNA polymerase
with strand displacementwith strand displacement
activity and substrates atactivity and substrates at
a constant temperature ofa constant temperature of
636300
c.c.
34. LAMPLAMP
Compared with PCR, andCompared with PCR, and
real time PCR, the LAMPreal time PCR, the LAMP
has advantages ofhas advantages of
reaction simplicity andreaction simplicity and
detection sensitivity.detection sensitivity.
The higher sensitivity andThe higher sensitivity and
specificity of LAMPspecificity of LAMP
reaction is attributed toreaction is attributed to
continuous amplificationcontinuous amplification
under isothermalunder isothermal
condition employing sixcondition employing six
primers recognizing eightprimers recognizing eight
distinct regions of thedistinct regions of the
target.target.
35. Advantages of LAMPAdvantages of LAMP
LAMP functions on isothermalLAMP functions on isothermal
amplification.amplification.
LAMP does not require any thermal cyclerLAMP does not require any thermal cycler
and thus can be performed even withand thus can be performed even with
water bath/heating blockwater bath/heating block
LAMP method do not require sophisticatedLAMP method do not require sophisticated
temperature control devicestemperature control devices
Cost effectiveCost effective
36. Lesser False Positives inLesser False Positives in
LAMPLAMP
In LAMP bothIn LAMP both
amplification andamplification and
detection occurdetection occur
simultaneously during thesimultaneously during the
exponential phaseexponential phase
without going through thewithout going through the
plateau phase where theplateau phase where the
non spuriousnon spurious
amplification leads toamplification leads to
lower sensitivity and falselower sensitivity and false
positivity.positivity.
37. Loop Mediated IsothermalLoop Mediated Isothermal
Amplification in Clinical DiagnosisAmplification in Clinical Diagnosis
LAMP technology proving to be ideal inLAMP technology proving to be ideal in
detection of DNA or RNA of the pathogenicdetection of DNA or RNA of the pathogenic
organismsorganisms
Proving to be highly efficient in diagnosis of ViralProving to be highly efficient in diagnosis of Viral
and Bacterial infections,and Bacterial infections,
LAMP is capable of detecting the presence ofLAMP is capable of detecting the presence of
pathogenic agents earlier than PCRpathogenic agents earlier than PCR
38. LAMP proving an efficientLAMP proving an efficient
TechnologyTechnology
A one step single tubeA one step single tube
real time acceleratedreal time accelerated
reverse transcription loopreverse transcription loop
mediated isothermalmediated isothermal
amplificationamplification (RT-LAMP(RT-LAMP))
assays for rapid detectionassays for rapid detection
of some recentlyof some recently
emerged viral pathogenemerged viral pathogen
eg West Nile, SARS,eg West Nile, SARS,
Dengue, JapaneseDengue, Japanese
encephalitis Chikungunyaencephalitis Chikungunya
Norwalk, H5N1 highlyNorwalk, H5N1 highly
pathogenic avianpathogenic avian
influenza., andinfluenza., and
CMV,HPV,VZVCMV,HPV,VZV
39. Multiplex PCRMultiplex PCR
TaqMan probes andTaqMan probes and
Molecular beacons allowMolecular beacons allow
multiple DNA species tomultiple DNA species to
be measured in the samebe measured in the same
sample ( Multiplex PCR)sample ( Multiplex PCR)
since fluorescent dyessince fluorescent dyes
with different emissionwith different emission
spectra may be attachedspectra may be attached
to different probesto different probes
40. Uses of Automated RT -Uses of Automated RT -
PCRPCR
Several viral infections can be detected in acuteSeveral viral infections can be detected in acute
phase serum samples.phase serum samples.
Increasingly used in for early and accurateIncreasingly used in for early and accurate
detection of almost all human viruses includingdetection of almost all human viruses including
Measles, Mumps, Herpes simplex viruses,Measles, Mumps, Herpes simplex viruses,
Rota viruses Noro virus, Influenzae virusRota viruses Noro virus, Influenzae virus
type A and B, Respiratory Synciticaltype A and B, Respiratory Syncitical
virus, SARS, Dengue Japanesevirus, SARS, Dengue Japanese
Encephalitis, Hepatitis B and C, WestEncephalitis, Hepatitis B and C, West
Nile, Chikungunya,HIV, Avian flu virus.Nile, Chikungunya,HIV, Avian flu virus.
41. Multiplex PCR in RealMultiplex PCR in Real
TimeTime
Multiplex real timeMultiplex real time
quantitative RT-PCRquantitative RT-PCR
assays have beenassays have been
developed fordeveloped for
simultaneoussimultaneous
detectiondetection
identification andidentification and
quantification of HBV,quantification of HBV,
HCV and HIV-! InHCV and HIV-! In
plasma and Serumplasma and Serum
samples.samples.
42. Real-Time PCR MethodReal-Time PCR Method
Molecular BeaconsMolecular Beacons
Molecular beaconsMolecular beacons
are short segments ofare short segments of
single-stranded DNAsingle-stranded DNA
(Figure 1). The(Figure 1). The
sequence of eachsequence of each
molecular beaconmolecular beacon
must be customizedmust be customized
to detect the PCRto detect the PCR
product of interest.product of interest.
43. StandardsStandards
Same copy number in all cellsSame copy number in all cells
Expressed in all cellsExpressed in all cells
Medium copy number advantageousMedium copy number advantageous
– correction more accuratecorrection more accurate
Reasonably large intronsReasonably large introns
No pseudo geneNo pseudo gene
No alternate splicing in region you wantNo alternate splicing in region you want
to PCRto PCR
44. Real-time PCRReal-time PCR
applicationsapplications
Quantitation of gene expressionQuantitation of gene expression
Pathogen detectionPathogen detection
Viral quantitationViral quantitation
Array verificationArray verification
Drug therapy efficacyDrug therapy efficacy
DNA damage measurementDNA damage measurement
Quality control and assay validationQuality control and assay validation
GenotypingGenotyping
45. Establishing PCRlaboratory
Sample handling
DNA preparation
Clean room
Stocksolutions
Laboratory
Mixing site
Thermocycler
Amplification
Detection
Documentation
QC & QA
Quality control & assurance
R & D
(Research and development)
Alternatives: - commercial kits
- robots + kits
No alternative
47. QIAGEN One Step RT-PCRQIAGEN One Step RT-PCR
KitKit
The QIAGEN One StepThe QIAGEN One Step
RT-PCR Kit is designedRT-PCR Kit is designed
for easy and sensitivefor easy and sensitive
one-step RT-PCR usingone-step RT-PCR using
any RNA template. Aany RNA template. A
unique enzymeunique enzyme
combination and speciallycombination and specially
developed reaction bufferdeveloped reaction buffer
ensure efficient reverseensure efficient reverse
transcription and PCR intranscription and PCR in
one tube.one tube.
48. RT-PCR in one stepRT-PCR in one step
The Robus™ T I Kit is baseThe Robus™ T I Kit is base
RobusT RT-PCRRobusT RT-PCR
Kits perform cDNAKits perform cDNA
synthesis andsynthesis and
PCR amplificationPCR amplification
of cDNAof cDNA
successively in asuccessively in a
single tube duringsingle tube during
a continuousa continuous
thermal cyclingthermal cycling
49. Uses and Advantages inUses and Advantages in
Testing by PCR MethodsTesting by PCR Methods
Clinical diagnostics: detection and quantificationClinical diagnostics: detection and quantification
of infectious microorganisms, cancer cells andof infectious microorganisms, cancer cells and
genetic disordersgenetic disorders
Capable of amplifying long targets, up to 6.0 kbCapable of amplifying long targets, up to 6.0 kb
One-tube system allows rapid, sensitive andOne-tube system allows rapid, sensitive and
reproducible analysis of RNA with minimal risk ofreproducible analysis of RNA with minimal risk of
sample contaminationsample contamination
Amplifies products from a wide variety of totalAmplifies products from a wide variety of total
RNA or mRNA sourcesRNA or mRNA sources
50. Advantages
Molecularmethods
•High sensitivity and specificity
•Detects pathogen, not immune response
•Quick results
•High transport toleration
In-house (home-brew) PCR methods
•Cost effective
•High sensitivity
•High quality
•Fast implementation of scientific discoveries
•Customer friendly
R&Dis absolutely necessary
51. Avoiding contaminationAvoiding contamination
The single most important source of PCR
product contamination is the generation of
aerosols of PCR amplicons that is associated
with the post-PCR analysis. Methods for
eliminating this aerosol range from physical
design of laboratories and use of specific
pipettes to chemical and enzymatic approaches.
The choice of method is often dependent on the
frequency of amplification of a target amplicon
and the relative amounts and concentrations of
the amplicons created by the PCR.
52. LIGASE CHAIN REACTIONLIGASE CHAIN REACTION
A method of DNA amplification similar to PCR.A method of DNA amplification similar to PCR.
LCR amplifies the probe molecule rather thanLCR amplifies the probe molecule rather than
producing amplicon through polymerization ofproducing amplicon through polymerization of
nucleotides.nucleotides.
Two probes are used per each DNA strand andTwo probes are used per each DNA strand and
are ligated together to form a single probe.are ligated together to form a single probe.
LCR uses both a DNA polymerase enzyme andLCR uses both a DNA polymerase enzyme and
a DNA ligase enzyme to drive the reaction.a DNA ligase enzyme to drive the reaction.
53. STEPSSTEPS
DenaturationDenaturation:: Heat double-strandedHeat double-stranded
DNA to denature it usually at 95DNA to denature it usually at 9500
C forC for
several minutes.several minutes.
Annealing:Annealing: Annealing of probes toAnnealing of probes to
target DNA ( at 60target DNA ( at 6000
C).C).
Ligation:Ligation: Joining of the probes byJoining of the probes by
thermostable DNA ligase. ( at 60thermostable DNA ligase. ( at 6000
C).C).
54.
55.
56. Nucleic acid sequence basedNucleic acid sequence based
amplification(NASBA)amplification(NASBA)
Does not require thermal cycling.Does not require thermal cycling.
In this presentation, we will be using Sybr green to monitor DNA synthesis. Sybr green is a dye which binds to double stranded DNA but not to single-stranded DNA and is frequently used to monitor the synthesis of DNA during real-time PCR reactions. When it is bound to double stranded DNA it fluoresces very brightly (much more brightly than ethidium bromide does, which is why we use Sybr Green rather than ethidium bromide; we also use Sybr green because the ratio of fluorescence in the presence of double-stranded DNA to the fluorescence in the presence of single-stranded DNA is much higher that the ratio for ethidium bromide). Other methods can also be used to detect the product during real-time PCR, but will not be discussed here. However, many of the principles discussed below apply to any real-time PCR reaction.