ChIP-seq is a technique to identify where proteins bind to DNA in the genome. It involves cross-linking proteins to DNA in cells, fragmenting the DNA, immunoprecipitating the protein-DNA complexes using an antibody for the protein of interest, and then sequencing the retrieved DNA. This allows mapping of the genomic binding sites for the protein. The document discusses experimental design considerations for ChIP-seq, such as antibody choice and controls. It also reviews data analysis steps including read mapping, peak calling to identify enriched regions, and downstream analyses like motif finding. Higher resolution techniques like ChIP-exo are also introduced that can identify protein binding sites at base pair level.
This document provides an outline for a presentation on directed evolution. It discusses the process of directed evolution, which involves randomly introducing mutations at the genetic level followed by selection of variants with desired protein characteristics. The document also covers types of mutations, naturally evolutionary processes like random mutagenesis and gene recombination that directed evolution mimics, library size, selection and screening strategies, applications, and advantages of directed evolution over rational design.
The document discusses how normal cells are transformed into cancer cells through multiple genetic changes over many years. It describes several key changes that occur during this process, including immortalization where cells can divide indefinitely, transformation where cells grow independently of external signals, and metastasis where cancer cells invade other tissues. Several types of genetic alterations are also discussed that can activate oncogenes or inactivate tumor suppressor genes, such as mutations, amplification, insertion, or translocation events. This leads to deregulated cell growth and proliferation by disrupting normal cell signaling pathways.
This document discusses recombinant DNA technology and its applications. It summarizes that Herbert Boyer and Stanley Cohen developed recombinant DNA technology in the 1970s, showing that genetically engineered DNA molecules can be cloned in foreign cells. It then provides examples of how recombinant DNA technology is used in agriculture, medicine, and industry for purposes such as producing important proteins and antibiotics, developing disease-resistant crops, and diagnosing diseases.
description of functional genomics and structural genomics and the techniques involved in it and also decribing the models of forward genetics and techniques involved in it and reverse genetics and techniques involved in it
Directed enzyme evolution is a technique that mimics natural selection to engineer proteins. It involves introducing random mutations into genes and screening proteins for modified activity. The key steps are selecting a gene, creating a library of mutant genes through error-prone PCR or other mutagenesis methods, expressing the proteins, and selecting variants with improved properties. Examples where directed evolution has been applied include improving the activity of enzymes used in producing the antibiotic cephalosporin and in the cholesterol-lowering drug atorvastatin. The goal is to leverage natural selection to develop enzymes with desired industrial applications like increased stability, activity, or substrate specificity.
This document discusses signal transduction and how it relates to cancer. It describes how growth factors and receptors contribute to normal signal transduction and how this process is deregulated in cancer. It explains that growth factors regulate growth, proliferation and survival, which are all altered in cancer. Several growth factors and receptors that can contribute to oncogenesis are identified. It also summarizes several key intracellular signaling pathways, like MAPK pathways, that are activated by growth factors and can result in the cancer phenotype if altered.
In this presentation, i have described how defects in DNA repair results in cancer and various DNA repair genes which are involved in the repair of damaged DN
ChIP-seq is a technique to identify where proteins bind to DNA in the genome. It involves cross-linking proteins to DNA in cells, fragmenting the DNA, immunoprecipitating the protein-DNA complexes using an antibody for the protein of interest, and then sequencing the retrieved DNA. This allows mapping of the genomic binding sites for the protein. The document discusses experimental design considerations for ChIP-seq, such as antibody choice and controls. It also reviews data analysis steps including read mapping, peak calling to identify enriched regions, and downstream analyses like motif finding. Higher resolution techniques like ChIP-exo are also introduced that can identify protein binding sites at base pair level.
This document provides an outline for a presentation on directed evolution. It discusses the process of directed evolution, which involves randomly introducing mutations at the genetic level followed by selection of variants with desired protein characteristics. The document also covers types of mutations, naturally evolutionary processes like random mutagenesis and gene recombination that directed evolution mimics, library size, selection and screening strategies, applications, and advantages of directed evolution over rational design.
The document discusses how normal cells are transformed into cancer cells through multiple genetic changes over many years. It describes several key changes that occur during this process, including immortalization where cells can divide indefinitely, transformation where cells grow independently of external signals, and metastasis where cancer cells invade other tissues. Several types of genetic alterations are also discussed that can activate oncogenes or inactivate tumor suppressor genes, such as mutations, amplification, insertion, or translocation events. This leads to deregulated cell growth and proliferation by disrupting normal cell signaling pathways.
This document discusses recombinant DNA technology and its applications. It summarizes that Herbert Boyer and Stanley Cohen developed recombinant DNA technology in the 1970s, showing that genetically engineered DNA molecules can be cloned in foreign cells. It then provides examples of how recombinant DNA technology is used in agriculture, medicine, and industry for purposes such as producing important proteins and antibiotics, developing disease-resistant crops, and diagnosing diseases.
description of functional genomics and structural genomics and the techniques involved in it and also decribing the models of forward genetics and techniques involved in it and reverse genetics and techniques involved in it
Directed enzyme evolution is a technique that mimics natural selection to engineer proteins. It involves introducing random mutations into genes and screening proteins for modified activity. The key steps are selecting a gene, creating a library of mutant genes through error-prone PCR or other mutagenesis methods, expressing the proteins, and selecting variants with improved properties. Examples where directed evolution has been applied include improving the activity of enzymes used in producing the antibiotic cephalosporin and in the cholesterol-lowering drug atorvastatin. The goal is to leverage natural selection to develop enzymes with desired industrial applications like increased stability, activity, or substrate specificity.
This document discusses signal transduction and how it relates to cancer. It describes how growth factors and receptors contribute to normal signal transduction and how this process is deregulated in cancer. It explains that growth factors regulate growth, proliferation and survival, which are all altered in cancer. Several growth factors and receptors that can contribute to oncogenesis are identified. It also summarizes several key intracellular signaling pathways, like MAPK pathways, that are activated by growth factors and can result in the cancer phenotype if altered.
In this presentation, i have described how defects in DNA repair results in cancer and various DNA repair genes which are involved in the repair of damaged DN
Tumor suppressor genes normally inhibit cell growth but can be inactivated through mutations, leading to cancer. The retinoblastoma (RB) gene was the first tumor suppressor gene discovered. According to Knudson's two-hit hypothesis, both copies of the RB gene must be inactivated for retinoblastoma to develop, either through two spontaneous mutations or one inherited mutation plus another acquired mutation. The RB protein regulates the cell cycle by binding to the E2F transcription factor and preventing cell cycle progression. RB can be inactivated through mutations in the gene, overexpression of cyclin-dependent kinases, or viral oncoproteins like HPV E7 binding RB instead of E2F. Cancers associated with
Ch11 lecture regulation of gene expressionTia Hohler
1) Gene expression in eukaryotes is regulated at multiple levels, including transcription, epigenetic modifications to DNA and histones, alternative splicing of mRNA, and microRNAs inhibiting translation.
2) Transcription is regulated through the binding of transcription factors to enhancer and silencer regions near gene promoters. DNA methylation and histone modifications can alter chromatin structure and gene activity.
3) Alternative splicing of pre-mRNA and the actions of microRNAs introduce additional regulatory mechanisms by generating different protein isoforms from a single gene or inhibiting specific mRNAs post-transcriptionally.
Genetically engineered animals are created through genetic manipulation techniques like DNA microinjection or cloning to introduce new traits. They are being engineered for agriculture and medicine, such as producing human proteins and antibodies in their milk or blood for treating diseases. While this technology aims to increase productivity and quality, it raises ethical concerns about interfering with animal integrity and welfare, as well as potential environmental impacts. Regulations require ensuring modified animals and their products are safe for animal and human consumption.
Gene expression and transcript profiling involves determining the pattern of genes expressed at the transcriptional level under specific circumstances by measuring the expression of thousands of genes simultaneously. This allows one to understand cellular function. Common techniques for profiling include DNA microarrays, RNA sequencing, and EST tags. DNA microarrays involve hybridizing cDNA or cRNA samples to probes on a chip to determine relative abundance of sequences. RNA sequencing uses next-generation sequencing to reveal presence and quantity of RNA in a sample.
This presentation details the definition of cell cytotoxicity and cell viability, the difference between the two term and methods of assessment of cells in culture for presence and absence of cytotoxic chemicals or metabolites.
DNA microarrays, also known as DNA chips, allow simultaneous measurement of gene expression levels for every gene in a genome. They detect mRNA levels by hybridizing cDNA to arrays of gene probes spotted on glass slides or other surfaces. Differences in gene expression between cell types or conditions can be measured and analyzed to answer biological questions.
Recombinant human erythropoietin was developed to treat anemia. It was first isolated from urine in 1977 and its gene was cloned in 1985, allowing industrial production. This paved the way for FDA approval in 1989 for use in chronic renal patients on dialysis to increase red blood cell production. It is now used to treat anemia associated with renal failure, cancer, prematurity, and HIV. It can also support erythropoiesis after chemotherapy or transplants and increase hemoglobin levels before surgery or for athletes. Current research continues to explore new applications of recombinant human erythropoietin.
DNA methylation in lung cancer 1 (1) (2).pptx9596276530AMIN
This document discusses epigenetics and DNA methylation in lung cancer. It notes that lung cancer is the second most common malignancy characterized by uncontrolled cell growth in lung tissue. Epigenetics involves reversible changes to gene expression through DNA methylation and histone modification without changing DNA sequence. DNA methylation involves adding a methyl group to cytosine bases and is regulated by DNA methyltransferases (DNMTs) that are dysregulated in lung cancer, leading to hypermethylation of tumor suppressor genes. Inhibitors of DNMTs can re-express silenced genes. Smoking is highly associated with lung cancer risk and impacts DNA methylation patterns through increased DNMT1 expression. Determining methylation patterns of multiple genes
Histone proteins package DNA into nucleosomes and higher order chromatin structures. There are core histones including H2A, H2B, H3, and H4, and linker histone H1. Histone modifications such as acetylation and methylation can alter gene expression without changing DNA. Genomic imprinting differentially expresses genes based on parental origin through epigenetic mechanisms like DNA methylation and histone modification. Imprinted genes are also found in plants and play a role in hybridization.
Proteomics is the study of the proteome, which is the entire set of proteins expressed by a genome, cell, tissue or organism. This document discusses several techniques used in proteomics including 2D gel electrophoresis, mass spectrometry, and protein databases. It provides examples of applications such as biomarker identification for disease diagnosis and drug target discovery. Limitations include the complexity of proteomes and no single technique being adequate for complete analysis. Overall, proteomics techniques help further our understanding of protein structure, function and interactions to gain insights into biological processes and diseases.
This document discusses tumor suppressor genes. It begins by defining a tumor suppressor gene as a gene that protects cells from cancer progression by normally functioning to inhibit cell division or promote cell death. It describes the "two-hit hypothesis" whereby both copies of a tumor suppressor gene must be mutated for full cancer development. Examples are given of important tumor suppressor genes like retinoblastoma protein (pRb) and p53, which are commonly mutated in many cancer types.
What is in situ hybridization
Radioactive ISH
Fluorescent ISH
Colorimetric ISH
ISH: three variables
The sample
The probe
Optimizing ISH Detection
ISH controls
Data Analysis
Gene knockout is a genetic technique where researchers engineer organisms by disabling specific genes. This is done by inserting foreign DNA into the target gene, interrupting its sequence and making the gene nonfunctional. Organisms with a knocked out gene, called knockout organisms, are used to study the function of genes and differences compared to normal organisms. Knockout is commonly used in mice and other animals by modifying embryonic stem cells and moss by transforming protoplasts. The process allows researchers to learn about genes with unknown functions.
A single-nucleotide polymorphism (SNP) is a variation in a single DNA building block (nucleotide) that differs between members of a species. SNPs are the most common type of genetic variation among humans, with around 0.1% of bases differing between individuals. They can occur in coding regions, where they may alter the resulting protein, or non-coding regions. SNPs are significant for mapping genes and studying an individual's predisposition to diseases like cancer or response to medications. They can be identified by comparing DNA sequences from many individuals or through laboratory techniques like SNP genotyping.
The document discusses transgenesis, which is introducing an exogenous gene into an organism so it exhibits a new property transmittable to offspring. Methods described include retrovirus-mediated gene transfer, microinjection of DNA into fertilized eggs, and embryonic stem cell-mediated gene transfer. Transgenesis has advantages like being more specific and faster than traditional breeding. However, it also carries risks of unpredictability if defense mechanisms silence or inactivate foreign genes.
complete Single Nucleotide Polymorphiitsm Detection methods with Advance techniques with its applications
Single nucleotide polymorphisms are single base variations between genomes within a species.
There are at least 10 million polymorphic sites in the human genome.
SNPs can distinguish individuals from one another
Denaturing Gradient Gel Electrophoresis
Chemical Cleavage Of Mismatch
Single-stranded Conformation Polymorphism (SSCP)
MutS Protein-binding Assays
Mismatch Repair Detection (MRD)
Heteroduplex Analysis (HA)
Denaturing High Performance Liquid Chromatography (DHPLC)
UNG-Mediated T-Sequencing
RNA-Mediated Finger printing with MALDI MS Detection
Sequencing by Hybridization
Direct DNA Sequencing
Single-feature polymorphism (SFP)
Invader probe
Allele-specific oligonucleotide probes
PCR-based methods
Allele specific primers
Sequence Polymorphism-Derived (SPD) markers
Targeting induced local lesions in genomes (TILLinG)
Minisequencing primers
Allele-specific ligation probes
dbSNP is a public archive of genetic polymorphisms including SNPs, insertions/deletions, and repeats. It contains contextual sequence information, frequency data, and experimental methods. dbSNP supports research areas like physical mapping, functional analysis, pharmacogenomics, and evolution. Variations are used as positional markers similar to STSs. Submitted SNPs are assigned IDs and aligned to reference genomes, clustering shared positions into reference SNP clusters. BLAST and FASTA tools allow searching. Null results of invariant sequences are also submitted.
Tumor suppressor genes normally inhibit cell growth but can be inactivated through mutations, leading to cancer. The retinoblastoma (RB) gene was the first tumor suppressor gene discovered. According to Knudson's two-hit hypothesis, both copies of the RB gene must be inactivated for retinoblastoma to develop, either through two spontaneous mutations or one inherited mutation plus another acquired mutation. The RB protein regulates the cell cycle by binding to the E2F transcription factor and preventing cell cycle progression. RB can be inactivated through mutations in the gene, overexpression of cyclin-dependent kinases, or viral oncoproteins like HPV E7 binding RB instead of E2F. Cancers associated with
Ch11 lecture regulation of gene expressionTia Hohler
1) Gene expression in eukaryotes is regulated at multiple levels, including transcription, epigenetic modifications to DNA and histones, alternative splicing of mRNA, and microRNAs inhibiting translation.
2) Transcription is regulated through the binding of transcription factors to enhancer and silencer regions near gene promoters. DNA methylation and histone modifications can alter chromatin structure and gene activity.
3) Alternative splicing of pre-mRNA and the actions of microRNAs introduce additional regulatory mechanisms by generating different protein isoforms from a single gene or inhibiting specific mRNAs post-transcriptionally.
Genetically engineered animals are created through genetic manipulation techniques like DNA microinjection or cloning to introduce new traits. They are being engineered for agriculture and medicine, such as producing human proteins and antibodies in their milk or blood for treating diseases. While this technology aims to increase productivity and quality, it raises ethical concerns about interfering with animal integrity and welfare, as well as potential environmental impacts. Regulations require ensuring modified animals and their products are safe for animal and human consumption.
Gene expression and transcript profiling involves determining the pattern of genes expressed at the transcriptional level under specific circumstances by measuring the expression of thousands of genes simultaneously. This allows one to understand cellular function. Common techniques for profiling include DNA microarrays, RNA sequencing, and EST tags. DNA microarrays involve hybridizing cDNA or cRNA samples to probes on a chip to determine relative abundance of sequences. RNA sequencing uses next-generation sequencing to reveal presence and quantity of RNA in a sample.
This presentation details the definition of cell cytotoxicity and cell viability, the difference between the two term and methods of assessment of cells in culture for presence and absence of cytotoxic chemicals or metabolites.
DNA microarrays, also known as DNA chips, allow simultaneous measurement of gene expression levels for every gene in a genome. They detect mRNA levels by hybridizing cDNA to arrays of gene probes spotted on glass slides or other surfaces. Differences in gene expression between cell types or conditions can be measured and analyzed to answer biological questions.
Recombinant human erythropoietin was developed to treat anemia. It was first isolated from urine in 1977 and its gene was cloned in 1985, allowing industrial production. This paved the way for FDA approval in 1989 for use in chronic renal patients on dialysis to increase red blood cell production. It is now used to treat anemia associated with renal failure, cancer, prematurity, and HIV. It can also support erythropoiesis after chemotherapy or transplants and increase hemoglobin levels before surgery or for athletes. Current research continues to explore new applications of recombinant human erythropoietin.
DNA methylation in lung cancer 1 (1) (2).pptx9596276530AMIN
This document discusses epigenetics and DNA methylation in lung cancer. It notes that lung cancer is the second most common malignancy characterized by uncontrolled cell growth in lung tissue. Epigenetics involves reversible changes to gene expression through DNA methylation and histone modification without changing DNA sequence. DNA methylation involves adding a methyl group to cytosine bases and is regulated by DNA methyltransferases (DNMTs) that are dysregulated in lung cancer, leading to hypermethylation of tumor suppressor genes. Inhibitors of DNMTs can re-express silenced genes. Smoking is highly associated with lung cancer risk and impacts DNA methylation patterns through increased DNMT1 expression. Determining methylation patterns of multiple genes
Histone proteins package DNA into nucleosomes and higher order chromatin structures. There are core histones including H2A, H2B, H3, and H4, and linker histone H1. Histone modifications such as acetylation and methylation can alter gene expression without changing DNA. Genomic imprinting differentially expresses genes based on parental origin through epigenetic mechanisms like DNA methylation and histone modification. Imprinted genes are also found in plants and play a role in hybridization.
Proteomics is the study of the proteome, which is the entire set of proteins expressed by a genome, cell, tissue or organism. This document discusses several techniques used in proteomics including 2D gel electrophoresis, mass spectrometry, and protein databases. It provides examples of applications such as biomarker identification for disease diagnosis and drug target discovery. Limitations include the complexity of proteomes and no single technique being adequate for complete analysis. Overall, proteomics techniques help further our understanding of protein structure, function and interactions to gain insights into biological processes and diseases.
This document discusses tumor suppressor genes. It begins by defining a tumor suppressor gene as a gene that protects cells from cancer progression by normally functioning to inhibit cell division or promote cell death. It describes the "two-hit hypothesis" whereby both copies of a tumor suppressor gene must be mutated for full cancer development. Examples are given of important tumor suppressor genes like retinoblastoma protein (pRb) and p53, which are commonly mutated in many cancer types.
What is in situ hybridization
Radioactive ISH
Fluorescent ISH
Colorimetric ISH
ISH: three variables
The sample
The probe
Optimizing ISH Detection
ISH controls
Data Analysis
Gene knockout is a genetic technique where researchers engineer organisms by disabling specific genes. This is done by inserting foreign DNA into the target gene, interrupting its sequence and making the gene nonfunctional. Organisms with a knocked out gene, called knockout organisms, are used to study the function of genes and differences compared to normal organisms. Knockout is commonly used in mice and other animals by modifying embryonic stem cells and moss by transforming protoplasts. The process allows researchers to learn about genes with unknown functions.
A single-nucleotide polymorphism (SNP) is a variation in a single DNA building block (nucleotide) that differs between members of a species. SNPs are the most common type of genetic variation among humans, with around 0.1% of bases differing between individuals. They can occur in coding regions, where they may alter the resulting protein, or non-coding regions. SNPs are significant for mapping genes and studying an individual's predisposition to diseases like cancer or response to medications. They can be identified by comparing DNA sequences from many individuals or through laboratory techniques like SNP genotyping.
The document discusses transgenesis, which is introducing an exogenous gene into an organism so it exhibits a new property transmittable to offspring. Methods described include retrovirus-mediated gene transfer, microinjection of DNA into fertilized eggs, and embryonic stem cell-mediated gene transfer. Transgenesis has advantages like being more specific and faster than traditional breeding. However, it also carries risks of unpredictability if defense mechanisms silence or inactivate foreign genes.
complete Single Nucleotide Polymorphiitsm Detection methods with Advance techniques with its applications
Single nucleotide polymorphisms are single base variations between genomes within a species.
There are at least 10 million polymorphic sites in the human genome.
SNPs can distinguish individuals from one another
Denaturing Gradient Gel Electrophoresis
Chemical Cleavage Of Mismatch
Single-stranded Conformation Polymorphism (SSCP)
MutS Protein-binding Assays
Mismatch Repair Detection (MRD)
Heteroduplex Analysis (HA)
Denaturing High Performance Liquid Chromatography (DHPLC)
UNG-Mediated T-Sequencing
RNA-Mediated Finger printing with MALDI MS Detection
Sequencing by Hybridization
Direct DNA Sequencing
Single-feature polymorphism (SFP)
Invader probe
Allele-specific oligonucleotide probes
PCR-based methods
Allele specific primers
Sequence Polymorphism-Derived (SPD) markers
Targeting induced local lesions in genomes (TILLinG)
Minisequencing primers
Allele-specific ligation probes
dbSNP is a public archive of genetic polymorphisms including SNPs, insertions/deletions, and repeats. It contains contextual sequence information, frequency data, and experimental methods. dbSNP supports research areas like physical mapping, functional analysis, pharmacogenomics, and evolution. Variations are used as positional markers similar to STSs. Submitted SNPs are assigned IDs and aligned to reference genomes, clustering shared positions into reference SNP clusters. BLAST and FASTA tools allow searching. Null results of invariant sequences are also submitted.