This document provides an overview of biotechnology principles and applications. It defines biotechnology as the application of technology to modify biological organisms by adding genes from other organisms. The document discusses how genes are identified, isolated, and manipulated to introduce desired traits. It describes techniques such as homology cloning, complementary genetics, and map-based cloning used to isolate genes. The document explains how genes are introduced into plants using transformation methods like Agrobacterium and biolistics. It provides examples of transgenic crops and their applications in agriculture.
The document discusses biotechnology principles including gene manipulation techniques used to modify genes and introduce them into transgenic organisms. It defines biotechnology as applying technology to modify the biological function of an organism by adding genes from another. Gene manipulation starts at the DNA level by identifying genes that control traits of interest or modifying existing genes. Genes are then introduced into organisms using techniques like transformation to form transgenic organisms that express new traits.
This document discusses several applications of biotechnology including gene cloning, DNA fingerprinting, and genetically modified foods. It provides information on how restriction enzymes can cut DNA at specific sequences to create fragments for gene cloning. DNA fragments can be joined together to form recombinant DNA which is often inserted into plasmids replicated in bacteria. This allows production of human insulin by bacteria. DNA fingerprinting analyzes variable regions of DNA to produce unique patterns that can be used for identification. While genetically modified crops are widely grown, scientists are still examining their potential environmental impacts.
The document discusses biotechnology and its traditional and modern applications. It summarizes that biotechnology has traditionally involved techniques like using yeast to make beer/wine and selective breeding of plants and animals. Modern biotechnology focuses on genetic engineering using recombinant DNA technology to modify genes and achieve goals like understanding disease and improving agriculture. It also discusses techniques like polymerase chain reaction (PCR) and gel electrophoresis that are used in biotechnology and forensics.
Recombinant DNA technology involves recombining DNA segments and allowing recombinant DNA molecules to enter cells and replicate. It was developed in 1973 by scientists Boyer and Cohen. The basic principle is to insert DNA into a vector, introduce it into a host cell where it replicates and produces the gene. Applications include producing human proteins like insulin through genetically engineered bacteria. Safety issues involve ensuring recombinant bacteria do not escape the laboratory and cause epidemics, which is addressed through physical and biological containment methods overseen by regulatory committees.
Recombinant dna technology applicationsRamesh Gupta
Recombinant DNA technology has many applications in medicine including mapping genomes, producing proteins, diagnosing genetic diseases, and gene therapy. The human genome project mapped the entire human genome, finding it contains around 30,000 genes made up of 3.2 billion DNA base pairs. Recombinant DNA techniques allow mass production of human proteins like insulin to treat diseases. Genetic diseases can be diagnosed by analyzing changes in restriction fragment length patterns. DNA fingerprinting using variable tandem repeats is used in forensics and has helped solve criminal and parental identification cases. Gene therapy aims to treat genetic disorders by inserting normal genes to replace defective ones.
The document provides an overview of genetic engineering and biotechnology tools and techniques, including:
- Restriction enzymes, ligase, and reverse transcriptase which are used to cut, splice and reverse DNA/RNA
- Methods for analyzing DNA like gel electrophoresis, hybridization probes, sequencing, and PCR
- Cloning vectors like plasmids and cloning hosts like E. coli that are used to replicate and express recombinant DNA
- Applications of recombinant DNA technology like producing insulin, vaccines, and transgenic organisms
- Genetic engineering techniques like gene therapy, antisense DNA, and triplex DNA for treating diseases
- Genome analysis methods like gene mapping, DNA fingerprinting and microarray analysis.
Recombinant DNA technology uses restriction enzymes and other tools to combine DNA fragments from different sources and insert them into vectors like plasmids. This allows genes to be cloned and mass produced. Key applications include producing human insulin to treat diabetes, vaccines like for hepatitis B, and gene therapy. Plasmids are commonly used vectors that are small, self-replicating DNA molecules found in bacteria. They contain origins of replication, antibiotic resistance genes as selectable markers, and sites for inserting foreign DNA. Recombinant DNA technology has proven important for developing medical treatments and furthering pharmaceutical research.
Genetic engineering principle, tools, techniques, types and applicationTarun Kapoor
Basic principles of genetic engineering.
Study of cloning vectors, restriction endonucleases and DNA ligase.
Recombinant DNA technology. Application of genetic engineering in medicine.
Application of r DNA technology and genetic engineering in the products:
a. Interferon
b. Vaccines- hepatitis- B
c. Hormones- Insulin.
Polymerase chain reaction
Brief introduction to PCR
Basic principles of PCR
The document discusses biotechnology principles including gene manipulation techniques used to modify genes and introduce them into transgenic organisms. It defines biotechnology as applying technology to modify the biological function of an organism by adding genes from another. Gene manipulation starts at the DNA level by identifying genes that control traits of interest or modifying existing genes. Genes are then introduced into organisms using techniques like transformation to form transgenic organisms that express new traits.
This document discusses several applications of biotechnology including gene cloning, DNA fingerprinting, and genetically modified foods. It provides information on how restriction enzymes can cut DNA at specific sequences to create fragments for gene cloning. DNA fragments can be joined together to form recombinant DNA which is often inserted into plasmids replicated in bacteria. This allows production of human insulin by bacteria. DNA fingerprinting analyzes variable regions of DNA to produce unique patterns that can be used for identification. While genetically modified crops are widely grown, scientists are still examining their potential environmental impacts.
The document discusses biotechnology and its traditional and modern applications. It summarizes that biotechnology has traditionally involved techniques like using yeast to make beer/wine and selective breeding of plants and animals. Modern biotechnology focuses on genetic engineering using recombinant DNA technology to modify genes and achieve goals like understanding disease and improving agriculture. It also discusses techniques like polymerase chain reaction (PCR) and gel electrophoresis that are used in biotechnology and forensics.
Recombinant DNA technology involves recombining DNA segments and allowing recombinant DNA molecules to enter cells and replicate. It was developed in 1973 by scientists Boyer and Cohen. The basic principle is to insert DNA into a vector, introduce it into a host cell where it replicates and produces the gene. Applications include producing human proteins like insulin through genetically engineered bacteria. Safety issues involve ensuring recombinant bacteria do not escape the laboratory and cause epidemics, which is addressed through physical and biological containment methods overseen by regulatory committees.
Recombinant dna technology applicationsRamesh Gupta
Recombinant DNA technology has many applications in medicine including mapping genomes, producing proteins, diagnosing genetic diseases, and gene therapy. The human genome project mapped the entire human genome, finding it contains around 30,000 genes made up of 3.2 billion DNA base pairs. Recombinant DNA techniques allow mass production of human proteins like insulin to treat diseases. Genetic diseases can be diagnosed by analyzing changes in restriction fragment length patterns. DNA fingerprinting using variable tandem repeats is used in forensics and has helped solve criminal and parental identification cases. Gene therapy aims to treat genetic disorders by inserting normal genes to replace defective ones.
The document provides an overview of genetic engineering and biotechnology tools and techniques, including:
- Restriction enzymes, ligase, and reverse transcriptase which are used to cut, splice and reverse DNA/RNA
- Methods for analyzing DNA like gel electrophoresis, hybridization probes, sequencing, and PCR
- Cloning vectors like plasmids and cloning hosts like E. coli that are used to replicate and express recombinant DNA
- Applications of recombinant DNA technology like producing insulin, vaccines, and transgenic organisms
- Genetic engineering techniques like gene therapy, antisense DNA, and triplex DNA for treating diseases
- Genome analysis methods like gene mapping, DNA fingerprinting and microarray analysis.
Recombinant DNA technology uses restriction enzymes and other tools to combine DNA fragments from different sources and insert them into vectors like plasmids. This allows genes to be cloned and mass produced. Key applications include producing human insulin to treat diabetes, vaccines like for hepatitis B, and gene therapy. Plasmids are commonly used vectors that are small, self-replicating DNA molecules found in bacteria. They contain origins of replication, antibiotic resistance genes as selectable markers, and sites for inserting foreign DNA. Recombinant DNA technology has proven important for developing medical treatments and furthering pharmaceutical research.
Genetic engineering principle, tools, techniques, types and applicationTarun Kapoor
Basic principles of genetic engineering.
Study of cloning vectors, restriction endonucleases and DNA ligase.
Recombinant DNA technology. Application of genetic engineering in medicine.
Application of r DNA technology and genetic engineering in the products:
a. Interferon
b. Vaccines- hepatitis- B
c. Hormones- Insulin.
Polymerase chain reaction
Brief introduction to PCR
Basic principles of PCR
Biotechnological tools used for diagnosticSunita Jak
This document discusses several biotechnological tools used for diagnostics:
1. DNA isolation, restriction enzyme digestion, polymerase chain reaction (PCR), gel electrophoresis, and DNA sequencing. PCR is described as artificially multiplying genetic material using primers and DNA polymerase. DNA sequencing methods like Sanger sequencing and Maxam-Gilbert are also outlined. The document concludes by briefly discussing gene cloning techniques like recombinant DNA technology and PCR for preparing copies of DNA fragments.
This document discusses recombinant DNA technology and its applications. It begins with an introduction to recombinant DNA technology and its history. It then describes the tools and enzymes used, including restriction enzymes, DNA ligase, reverse transcriptase, and DNA polymerase. Various vectors like bacterial plasmids and bacteriophages are also discussed. The document outlines several applications such as monoclonal antibody production, disease diagnosis, DNA fingerprinting, environmental uses, gene therapy, and xenotransplantation. In summary, the document provides an overview of the key concepts, techniques, and uses of recombinant DNA technology.
Genetic engineering involves direct manipulation of an organism's DNA. It has applications in medicine, agriculture, pollution control and more. Key techniques include isolating genes, manipulating DNA through cloning and PCR, and reintroducing DNA into organisms. This allows transferring genes between species to produce transgenic plants and animals with desired traits. While it promises medical benefits, ethical issues around patenting life and unintended consequences require consideration.
Genetic engineering and Recombinant DNAHala AbuZied
Genetic engineering involves altering the DNA of living organisms using biotechnology. It includes techniques like changing single DNA base pairs, deleting or adding genes, or combining DNA from different species. Recombinant DNA technology is used to create recombinant DNA molecules by manipulating DNA in vitro and introducing them into host organisms. This allows bacteria to be engineered to produce human insulin through inserting the human insulin gene into bacterial plasmids. Genomic libraries can be created by ligating fragmented genomic or cDNA into plasmid vectors to transform bacteria and clone the entire genome.
Recombinant DNA Technology, Forensic DNA Analysis and Human Genome ProjectNateneal Tamerat
Recombinant DNA technology involves joining DNA fragments from different organisms to produce new genetic combinations. It was developed in the 1970s using restriction enzymes, which cut DNA at specific sites. DNA is isolated, cut, and inserted into vectors like plasmids or bacteria, then inserted into host cells. Applications include forensic analysis by matching crime scene DNA to databases, agriculture, diagnosing genetic diseases, and the Human Genome Project, which sequenced the entire human genome in 2001 and revealed insights about human genetics and evolution.
Transfection involves introducing foreign DNA into host cells to produce a new phenotype. There are two main methods of transfection - vector-mediated and non-vector mediated. Vector-mediated transfection uses bacteriophage, retroviral, cosmid, baculovirus, and plasmid vectors to introduce DNA. Non-vector mediated methods include direct techniques like microinjection, electroporation, and particle bombardment, and indirect techniques like calcium phosphate precipitation and DEAE-dextran. Retroviral vectors are modified retroviruses that can introduce foreign DNA into host chromosomal DNA. Microinjection involves injecting DNA directly into cells using a micropipette under a microscope. Electroporation uses electric pulses to create temporary
There are three main methods for isolating genes:
1. Using an automated gene machine to synthesize genes from predetermined nucleotide sequences.
2. Gene cloning, which involves inserting a DNA fragment into a vector that is then transferred into a host cell to produce multiple copies.
3. Polymerase chain reaction (PCR), which amplifies a specific DNA sequence using primers that flank the target sequence.
Recombinant DNA technology allows DNA from different species to be isolated, cut with restriction enzymes, and spliced together to form new recombinant molecules. This involves extracting DNA, cutting it with restriction enzymes to form manageable fragments, inserting fragments into vectors like plasmids, introducing the recombinant vectors into host cells, and amplifying the DNA. Vectors often contain antibiotic resistance genes to select for host cells containing the recombinant DNA. This process allows scientists to isolate and multiply specific genes for study and modification.
Recombinant DNA technology allows for the isolation, alteration, and reinsertion of genes. It involves isolating DNA segments, cutting them using restriction enzymes, joining DNA segments together, and amplifying the resulting recombinant DNA. Vectors like plasmids, lambda phages, and artificial chromosomes are used to carry foreign DNA into host cells. Techniques like PCR, gel electrophoresis, cloning libraries, and nucleic acid hybridization are used in rDNA technology. Applications include producing medicines like insulin, developing pest-resistant crops, and gene therapy to treat genetic diseases.
r-DNA technology allows the manipulation of DNA fragments through restriction endonucleases, cloning techniques, and specific probes. Restriction endonucleases cut DNA into fragments, cloning techniques amplify specific sequences, and probes identify sequences of interest. Real-time PCR and restriction fragment length polymorphism (RFLP) are techniques used to analyze DNA fragments.
The document summarizes viral vectors and their use in gene therapy. It discusses the key properties of viral vectors, including safety, low toxicity, stability, and cell specificity. It then covers specific viral vectors - retroviruses, which can cause diseases but can also be used to transduce genes, and herpesviruses, which allow high transgene capacity and infect dividing and non-dividing cells. Finally, it reviews a research paper that showed baculoviral vectors can efficiently and transiently transduce genes into human neurons derived from embryonic stem cells without integration.
The document provides an overview of DNA technology and biotechnology. It discusses how DNA cloning allows scientists to make multiple copies of genes and study their structure, expression, and function. Key techniques described include recombinant DNA, restriction enzymes, gel electrophoresis, and DNA sequencing. Applications mentioned include genetic engineering of plants, microorganisms, and animals for research, agriculture, medicine, forensics, and environmental cleanup.
1. Recombinant DNA technology involves manipulating DNA from different species and combining them to form new recombinant DNA molecules.
2. Key steps include using restriction enzymes to cut DNA at specific sites, and DNA ligase to join DNA fragments together into vectors like plasmids.
3. The recombinant DNA can then be replicated in host cells like bacteria to produce multiple copies for analysis.
This document summarizes recombinant DNA (rDNA) technology and genetic engineering. It discusses how restriction enzymes and DNA ligases are used to cut and paste DNA from different sources to create recombinant DNA molecules. It also describes genetically modified organisms (GMOs) and provides examples of transgenic organisms. Vectors such as plasmids are used to carry and replicate foreign DNA fragments in bacteria. Selection techniques like antibiotic resistance allow identification of transformed bacteria.
Genetic engineering is the direct manipulation of an organism's genes using biotechnology. The process involves isolating the gene of interest and inserting it into a host organism. New DNA is obtained by isolating and copying genetic material or synthesizing DNA artificially. The inserted gene can be modified for better expression before being combined with regulatory elements and a selectable marker. The gene-construct is then inserted into the host genome using methods like agrobacterium transformation for plants. Genetically modified crops have been developed with traits like increased production, stress tolerance and nutrient content.
Genetic engineering,recombinant DNA technology..ganeshbond
1) In 1996, scientists in Edinburgh announced the creation of Dolly the sheep, the first mammal cloned from an adult cell.
2) Dolly's birth sparked debate around the controversial technique of cloning and its potential application to humans.
3) In 2001, scientists in Texas cloned the world's first kitten, named Cc, using a cell from an adult tortoiseshell cat. The kitten was unveiled in 2002.
Recombinant DNA technology involves intentionally modifying organisms' genomes for practical purposes such as eliminating undesirable traits, combining beneficial traits from different organisms, or creating organisms that synthesize useful products. The key steps involve isolating a gene of interest, inserting it into a plasmid, introducing the plasmid into bacteria, and harvesting copies of the gene or its protein products. Common tools used include mutagens, reverse transcriptase to synthesize cDNA, synthetic nucleic acids, restriction enzymes to cut DNA at specific sites, vectors to deliver genes into cells, and gene libraries containing collections of cloned genes.
This document provides definitions and explanations of key concepts in biotechnology. It begins by defining biotechnology as the application of technology to modify the biological function of an organism by adding genes from another organism. It then discusses why biotechnology is needed, as nature does not contain all the genetic variation desired for traits like improved nutrition. The document explains genetic concepts like genes, alleles, and how they control traits. It describes techniques used in biotechnology like identifying genes of interest, gene manipulation, transformation, and testing transgenic plants. Overall, the summary provides a high-level overview of the basic concepts and processes in biotechnology.
1. Biotechnology relies on restriction enzymes that cut DNA at specific nucleotide sequences. Different enzymes cut DNA in different ways, leaving either blunt or sticky ends. Restriction maps show the lengths of DNA fragments cut by these enzymes.
2. The polymerase chain reaction (PCR) amplifies specific DNA sequences. It uses DNA polymerase to copy short DNA segments billions of times over, by cycling between high and low temperatures.
3. DNA fingerprinting identifies individuals by analyzing variations in noncoding DNA regions containing repeating sequences. The probability that any two individuals will have identical fingerprints across multiple regions is very small.
Biotechnological tools used for diagnosticSunita Jak
This document discusses several biotechnological tools used for diagnostics:
1. DNA isolation, restriction enzyme digestion, polymerase chain reaction (PCR), gel electrophoresis, and DNA sequencing. PCR is described as artificially multiplying genetic material using primers and DNA polymerase. DNA sequencing methods like Sanger sequencing and Maxam-Gilbert are also outlined. The document concludes by briefly discussing gene cloning techniques like recombinant DNA technology and PCR for preparing copies of DNA fragments.
This document discusses recombinant DNA technology and its applications. It begins with an introduction to recombinant DNA technology and its history. It then describes the tools and enzymes used, including restriction enzymes, DNA ligase, reverse transcriptase, and DNA polymerase. Various vectors like bacterial plasmids and bacteriophages are also discussed. The document outlines several applications such as monoclonal antibody production, disease diagnosis, DNA fingerprinting, environmental uses, gene therapy, and xenotransplantation. In summary, the document provides an overview of the key concepts, techniques, and uses of recombinant DNA technology.
Genetic engineering involves direct manipulation of an organism's DNA. It has applications in medicine, agriculture, pollution control and more. Key techniques include isolating genes, manipulating DNA through cloning and PCR, and reintroducing DNA into organisms. This allows transferring genes between species to produce transgenic plants and animals with desired traits. While it promises medical benefits, ethical issues around patenting life and unintended consequences require consideration.
Genetic engineering and Recombinant DNAHala AbuZied
Genetic engineering involves altering the DNA of living organisms using biotechnology. It includes techniques like changing single DNA base pairs, deleting or adding genes, or combining DNA from different species. Recombinant DNA technology is used to create recombinant DNA molecules by manipulating DNA in vitro and introducing them into host organisms. This allows bacteria to be engineered to produce human insulin through inserting the human insulin gene into bacterial plasmids. Genomic libraries can be created by ligating fragmented genomic or cDNA into plasmid vectors to transform bacteria and clone the entire genome.
Recombinant DNA Technology, Forensic DNA Analysis and Human Genome ProjectNateneal Tamerat
Recombinant DNA technology involves joining DNA fragments from different organisms to produce new genetic combinations. It was developed in the 1970s using restriction enzymes, which cut DNA at specific sites. DNA is isolated, cut, and inserted into vectors like plasmids or bacteria, then inserted into host cells. Applications include forensic analysis by matching crime scene DNA to databases, agriculture, diagnosing genetic diseases, and the Human Genome Project, which sequenced the entire human genome in 2001 and revealed insights about human genetics and evolution.
Transfection involves introducing foreign DNA into host cells to produce a new phenotype. There are two main methods of transfection - vector-mediated and non-vector mediated. Vector-mediated transfection uses bacteriophage, retroviral, cosmid, baculovirus, and plasmid vectors to introduce DNA. Non-vector mediated methods include direct techniques like microinjection, electroporation, and particle bombardment, and indirect techniques like calcium phosphate precipitation and DEAE-dextran. Retroviral vectors are modified retroviruses that can introduce foreign DNA into host chromosomal DNA. Microinjection involves injecting DNA directly into cells using a micropipette under a microscope. Electroporation uses electric pulses to create temporary
There are three main methods for isolating genes:
1. Using an automated gene machine to synthesize genes from predetermined nucleotide sequences.
2. Gene cloning, which involves inserting a DNA fragment into a vector that is then transferred into a host cell to produce multiple copies.
3. Polymerase chain reaction (PCR), which amplifies a specific DNA sequence using primers that flank the target sequence.
Recombinant DNA technology allows DNA from different species to be isolated, cut with restriction enzymes, and spliced together to form new recombinant molecules. This involves extracting DNA, cutting it with restriction enzymes to form manageable fragments, inserting fragments into vectors like plasmids, introducing the recombinant vectors into host cells, and amplifying the DNA. Vectors often contain antibiotic resistance genes to select for host cells containing the recombinant DNA. This process allows scientists to isolate and multiply specific genes for study and modification.
Recombinant DNA technology allows for the isolation, alteration, and reinsertion of genes. It involves isolating DNA segments, cutting them using restriction enzymes, joining DNA segments together, and amplifying the resulting recombinant DNA. Vectors like plasmids, lambda phages, and artificial chromosomes are used to carry foreign DNA into host cells. Techniques like PCR, gel electrophoresis, cloning libraries, and nucleic acid hybridization are used in rDNA technology. Applications include producing medicines like insulin, developing pest-resistant crops, and gene therapy to treat genetic diseases.
r-DNA technology allows the manipulation of DNA fragments through restriction endonucleases, cloning techniques, and specific probes. Restriction endonucleases cut DNA into fragments, cloning techniques amplify specific sequences, and probes identify sequences of interest. Real-time PCR and restriction fragment length polymorphism (RFLP) are techniques used to analyze DNA fragments.
The document summarizes viral vectors and their use in gene therapy. It discusses the key properties of viral vectors, including safety, low toxicity, stability, and cell specificity. It then covers specific viral vectors - retroviruses, which can cause diseases but can also be used to transduce genes, and herpesviruses, which allow high transgene capacity and infect dividing and non-dividing cells. Finally, it reviews a research paper that showed baculoviral vectors can efficiently and transiently transduce genes into human neurons derived from embryonic stem cells without integration.
The document provides an overview of DNA technology and biotechnology. It discusses how DNA cloning allows scientists to make multiple copies of genes and study their structure, expression, and function. Key techniques described include recombinant DNA, restriction enzymes, gel electrophoresis, and DNA sequencing. Applications mentioned include genetic engineering of plants, microorganisms, and animals for research, agriculture, medicine, forensics, and environmental cleanup.
1. Recombinant DNA technology involves manipulating DNA from different species and combining them to form new recombinant DNA molecules.
2. Key steps include using restriction enzymes to cut DNA at specific sites, and DNA ligase to join DNA fragments together into vectors like plasmids.
3. The recombinant DNA can then be replicated in host cells like bacteria to produce multiple copies for analysis.
This document summarizes recombinant DNA (rDNA) technology and genetic engineering. It discusses how restriction enzymes and DNA ligases are used to cut and paste DNA from different sources to create recombinant DNA molecules. It also describes genetically modified organisms (GMOs) and provides examples of transgenic organisms. Vectors such as plasmids are used to carry and replicate foreign DNA fragments in bacteria. Selection techniques like antibiotic resistance allow identification of transformed bacteria.
Genetic engineering is the direct manipulation of an organism's genes using biotechnology. The process involves isolating the gene of interest and inserting it into a host organism. New DNA is obtained by isolating and copying genetic material or synthesizing DNA artificially. The inserted gene can be modified for better expression before being combined with regulatory elements and a selectable marker. The gene-construct is then inserted into the host genome using methods like agrobacterium transformation for plants. Genetically modified crops have been developed with traits like increased production, stress tolerance and nutrient content.
Genetic engineering,recombinant DNA technology..ganeshbond
1) In 1996, scientists in Edinburgh announced the creation of Dolly the sheep, the first mammal cloned from an adult cell.
2) Dolly's birth sparked debate around the controversial technique of cloning and its potential application to humans.
3) In 2001, scientists in Texas cloned the world's first kitten, named Cc, using a cell from an adult tortoiseshell cat. The kitten was unveiled in 2002.
Recombinant DNA technology involves intentionally modifying organisms' genomes for practical purposes such as eliminating undesirable traits, combining beneficial traits from different organisms, or creating organisms that synthesize useful products. The key steps involve isolating a gene of interest, inserting it into a plasmid, introducing the plasmid into bacteria, and harvesting copies of the gene or its protein products. Common tools used include mutagens, reverse transcriptase to synthesize cDNA, synthetic nucleic acids, restriction enzymes to cut DNA at specific sites, vectors to deliver genes into cells, and gene libraries containing collections of cloned genes.
This document provides definitions and explanations of key concepts in biotechnology. It begins by defining biotechnology as the application of technology to modify the biological function of an organism by adding genes from another organism. It then discusses why biotechnology is needed, as nature does not contain all the genetic variation desired for traits like improved nutrition. The document explains genetic concepts like genes, alleles, and how they control traits. It describes techniques used in biotechnology like identifying genes of interest, gene manipulation, transformation, and testing transgenic plants. Overall, the summary provides a high-level overview of the basic concepts and processes in biotechnology.
1. Biotechnology relies on restriction enzymes that cut DNA at specific nucleotide sequences. Different enzymes cut DNA in different ways, leaving either blunt or sticky ends. Restriction maps show the lengths of DNA fragments cut by these enzymes.
2. The polymerase chain reaction (PCR) amplifies specific DNA sequences. It uses DNA polymerase to copy short DNA segments billions of times over, by cycling between high and low temperatures.
3. DNA fingerprinting identifies individuals by analyzing variations in noncoding DNA regions containing repeating sequences. The probability that any two individuals will have identical fingerprints across multiple regions is very small.
This document discusses plant biotechnology techniques used to genetically modify organisms. It defines biotechnology as applying technology to modify biological organisms by adding genes from other species. The key techniques discussed are identifying genes from other organisms that control desired traits and introducing those genes into plants through transformation. This allows developing crops with improved traits like herbicide or insect resistance, drought tolerance, or increased nutritional content. The document outlines the process of gene cloning, creation of transformation cassettes containing the gene of interest and selectable marker, and delivery into plants via Agrobacterium or gene gun. Extensive testing of transgenic plants in the lab and field is needed before commercial release to ensure safety and trait expression.
Gene therapy involves delivering genetic material to cells to alter their instructions for a therapeutic purpose. The first human gene therapy trial was conducted in 1990, though no gene therapy products are commercially available yet. Key goals for gene therapy strategies are to administer treatments easily, achieve long-term therapeutic effects from a single application, and target effects specifically to diseased cells with few side effects. Choosing the right viral vector depends on factors like the target cells and desired gene expression level and duration.
This document discusses high-resolution views of the cancer genome using various technologies including DNA microarrays, comparative genomic hybridization, tiling arrays, next-generation sequencing, and DNAse-Seq. It describes how these technologies can be used to analyze gene expression, copy number variation, chromatin structure, and more to better understand cancer at the genomic level. Integrating data from all these sources presents challenges but may help improve individual health outcomes.
Complete Sequencing – Clifford Reid, PhD; CEO, Complete Genomics as presented at the Personalized Health Care Conference at Ohio State. Dr. Reid discussed what complete human sequencing looks like and costs now and in the near future.
Recombinant DNA technology allows scientists to isolate, clone, and manipulate specific genes. DNA from different species can be combined to produce new genetic combinations of value. Genes are cloned by inserting DNA fragments into vectors like plasmids, which are then inserted into host cells where they replicate numerous identical copies of the gene. This cloning process allows genes to be studied, sequenced, and modified in precise ways. Genetically modified organisms can then be produced by adding transgenes to organisms' genomes.
The document discusses gene therapy, including:
1. Defining genes and discussing early milestones in gene isolation and engineering.
2. Explaining the goal of gene therapy to introduce normal genes to compensate for defective genes.
3. Describing various methods of gene delivery including viral and non-viral vectors.
4. Discussing challenges in targeting specific cells/tissues and ensuring safe expression levels.
This document discusses genomic technologies that can be used to observe the human genome and their applications. It covers microarrays, next-generation sequencing, DNA methylation, copy number variation, and more. Challenges include the cost of these technologies and integrating the large amounts of data they produce to improve healthcare.
Recombinant DNA technology involves combining DNA molecules from different sources and introducing them into host organisms. Some key points:
- Recombinant DNA is produced by joining DNA fragments from different sources using restriction enzymes and DNA ligase.
- Plasmids and bacterial cells are commonly used as vectors to replicate and express recombinant DNA. Foreign DNA is inserted into plasmids which are then introduced into bacterial cells.
- Restriction enzymes from bacteria are used to cut DNA at specific sequences. This allows insertion of foreign DNA. DNA ligase joins the DNA fragments back together.
- Applications include production of therapeutic proteins, genetic testing, gene therapy, and genetically modified crops. Recombinant DNA technology
Recombinant DNA technology involves combining DNA molecules from different sources and introducing them into host organisms. Some key points:
- Recombinant DNA is produced by joining DNA fragments from different sources using restriction enzymes and DNA ligase.
- Plasmids and bacterial cells are commonly used as vectors to replicate and express recombinant DNA. Foreign DNA is inserted into plasmids which are then introduced into bacterial cells.
- Restriction enzymes from bacteria are used to cut DNA at specific sequences. This allows insertion of foreign DNA. DNA ligase joins the DNA fragments back together.
- Recombinant DNA technology has many applications including production of medicines, diagnosing genetic diseases, and gene therapy. It
This document provides information about a lecture series on methods in molecular biology. The course is titled "Methods in Molecular Biology" and is worth 3 credit hours. It will be taught by Dr. Sumera Shaheen in the department of biochemistry at Govt. College Women University Faisalabad. The lectures will cover topics such as recombinant DNA technology, vectors, PCR, DNA sequencing, gel electrophoresis, expression of recombinant proteins, antibodies, and blotting techniques. Recommended textbooks for the course are also listed.
Stephen Friend NIH PPP Coordinating Committee Meeting 2012-02-16Sage Base
The document discusses using networked team approaches and integrating omics data to build better disease maps through public-private partnerships like CTCAP and Arch2POCM. It proposes sharing clinical and genomic data from comparator arms of trials to create models and de-risking novel drug targets through developing test compounds in a precompetitive space to accelerate new therapies.
This document outlines the course content for a cell biology course. It covers 10 main topics: introduction to cells, chemical foundations, methods of studying cells, genetic mechanisms, cell signaling, cell membranes and architecture, energetics, cellular traffic, cell birth/lineage/death, and the molecular basis of cancer. The course will involve seminar presentations by students on each topic, along with exams to assess comprehension. Overall, the course provides an introduction to the key concepts and components of cell biology from a biochemical and genetic perspective.
The document discusses epigenetic analysis sequencing, which studies how environmental factors can affect gene expression without changing DNA sequences. It explains that epigenetics plays an important role in gene regulation through two main components: DNA methylation and histone modification. High-resolution sequencing methods like ChIP-seq are important for studying epigenetics and understanding chromatin structure at the single binding site level.
The document discusses genetics and genetic disorders. It provides background on chromosomes, genes, Gregor Mendel's foundational work in genetics, DNA structure, mRNA, tRNA, amino acids, and the genetic code. It also covers topics like genetic engineering, gene therapy, and molecular techniques used to study human diseases.
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.
Genetic engineering is a branch of molecular biology that allows manipulation of an organism's genome. Common practices include eliminating harmful genes, introducing healthy genes, and modifying genes. The process involves isolating the gene of interest using restriction enzymes, inserting the gene into a cloning vector like a bacterial plasmid, locating host cells containing the gene, and cloning those cells to produce the gene product. For example, the human insulin gene can be cloned into bacteria to produce insulin in large quantities at low cost for treatment of diabetes.
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5th LF Energy Power Grid Model Meet-up SlidesDanBrown980551
5th Power Grid Model Meet-up
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Power Grid Model
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The journey concludes with a reflection on the ever-evolving landscape of OS, underscored by the emergence of real-time operating systems (RTOS) and the persistent quest for innovation and efficiency. As technology continues to shape our world, understanding the foundations and evolution of operating systems remains paramount. Join Pravash Chandra Das on this illuminating journey through the heart of computing. 🌟
Deep Dive: Getting Funded with Jason Jason Lemkin Founder & CEO @ SaaStr
Techniques of-biotechnology-mcclean-good
1. Biotechnology:
Principles, Applications,
and Social Implications
From Protein to Product
The techniques used by the biotechnology industry
to modify genes and introduce them into transgenic organisms
Phil McClean
Department of Plant Science
North Dakota State University
NDSU
Extension
2. What is Biotechnology?
How about some definitions
General Definition
The application of technology to improve
a biological organism
Detailed Definition
The application of the technology to modify the
biological function of an organism by adding genes
from another organism
NDSU
Extension
3. These definitions imply biotechnology
is needed because:
•Nature has a rich source of variation
• Here we see bean has many
seedcoat colors and patterns
in nature
But we know nature does not have
all of the traits we need
NDSU
Extension
4. But nature does not contain all the
genetic variation man desires
•Fruits with vaccines
•Grains with improved nutrition
NDSU
Extension
5. What controls this natural variation?
Allelic differences at genes control a specific trait
Definitions are needed for this statement:
Gene - a piece of DNA that controls the
expression of a trait
Allele - the alternate forms of a gene
NDSU
Extension
6. What is the difference between
genes and alleles for Mendel’s Traits?
Mendel’s Genes
Plant height Seed shape
Smooth Wrinkled
Allele
Tall Short
Allele
NDSU
Extension
7. This Implies a
Genetic Continuum
A direct relationship exists between the gene, its alleles,
and the phenotypes (different forms ) of the trait
Alleles must be:
• similar enough to control the same trait
• but different enough to create different phenotypes
NDSU
Extension
8. Allelic Differences for Mendel’s Genes
Plant Height Gene
Gene: gibberellin 3-β-hydroxylase
Function: adds hydoxyl group to GA20 to make GA1
Role of GA1: regulates cell division and elongation
Mutation in short allele: a single nucleotide converts
an alanine to threonine in final protein
Effect of mutation: mutant protein is 1/20 as active
NDSU
Extension
9. Allelic Differences for Mendel’s
Seed Shape Gene
Gene: strach branching enzyme (SBE) isoform 1
Function: adds branch chains to starch
Mutation in short allele: transposon insertion
Effect of mutation: no SBE activity; less starch, more
sucrose, more water; during maturation seed looses
more water and wrinkles
NDSU
Extension
10. Central Dogma of Molecular Genetics
(The guiding principle that controls trait expression)
Protein Trait
(or phenotype)
Translation
Seed shape
DNA RNA
Transcription
(gene)
Plant height
NDSU
Extension
11. In General, Plant Biotechnology Techniques
Fall Into Two Classes
Gene Manipulation
• Identify a gene from another species which controls
a trait of interest
• Or modify an existing gene (create a new allele)
Gene Introduction
• Introduces that gene into an organism
• Technique called transformation
• Forms transgenic organisms
NDSU
Extension
12. Gene Manipulation Starts
At the DNA Level
The nucleus
contains DNA
NDSU
Source: Access Excellence
Extension
15. Genes Are Cloned Based On:
Similarity to known genes
Homology cloning (mouse clone used to obtain human gene)
Protein sequence
Complementary genetics (predicting gene sequence
from protein)
Chromosomal location
Map-based cloning (using genetic approach)
NDSU
Extension
16. Homology Cloning
Clones transferred
to filter
Human clone
Mouse probe
library
added to filter
Hot-spots are human
homologs to mouse gene
NDSU
Extension
17. Complementary Genetics
1. Protein sequence is related to gene sequence
NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COO-
ATG GAT-GCT TGG-AGT-AAA
C C C G
A TCT
G C
A
G
2. The genetic code information is used to design PCR primers
Forward primer: 5’-ATGGAT/CGCN-3’
Reverse primer: 5’-T/CTTNC/GT/ACCA-3’
Notes: T/C = a mixture of T and C at this position;
NDSU
N = a mixture of all four nucleotides
Reverse primer is the reverse complement of the gene sequence
Extension
18. Complementary Genetics
(cont.)
3. Use PCR to amplify gene fragment
a. template DNA is melted (94C)
3’ 5’
5’ 3’
3’ 5’
5’ 3’
b. primers anneal to complementary site in melted DNA (55C)
3’ 5’
5’ 3’
c. two copies of the template DNA made (72C)
3’ 5’
NDSU
5’ 3’
Extension
21. Complementary Genetics
(cont.)
4. Gene fragment used to screen library
Clones transferred
to filter
Human clone
library PCR fragment
probe added to filter
Hot-spots are human gene
NDSU
of interest
Extension
22. Map-based Cloning
Gene Marker
1. Use genetic techniques to
find marker near gene
Gene/Marker
2. Find cosegregating marker
3. Discover overlapping clones
(or contig) that contains the marker Gene/Marker
Gene/Marker
4. Find ORFs on contig
5. Prove one ORF is the gene by Mutant + ORF = Wild type?
NDSU
transformation or mutant analysis Yes? ORF = Gene
Extension
23. Gene Manipulation
• It is now routine to isolate genes
• But the target gene must be carefully chosen
• Target gene is chosen based on desired phenotype
Function:
Glyphosate (RoundUp) resistance
EPSP synthase enzyme
Increased Vitamin A content
Vitamin A biosynthetic pathway enzymes
NDSU
Extension
24. The RoundUp Ready Story
• Glyphosate is a broad-spectrum herbicide
• Active ingredient in RoundUp herbicide
• Kills all plants it come in contact with
• Inhibits a key enzyme (EPSP synthase) in an amino acid pathway
• Plants die because they lack the key amino acids
• A resistant EPSP synthase gene allows crops
to survive spraying
NDSU
Extension
25. RoundUp Sensitive Plants
Shikimic acid + Phosphoenol pyruvate
+ Glyphosate
X
Plant
EPSP synthase
Without amino acids,
X
3-Enolpyruvyl shikimic acid-5-phosphate
(EPSP)
plant dies
X
X
Aromatic
NDSU
amino acids
Extension
26. RoundUp Resistant Plants
Shikimic acid + Phosphoenol pyruvate
+ Glyphosate
RoundUp has no effect;
Bacterial enzyme is resistant to herbicide
EPSP synthase
3-enolpyruvyl shikimic acid-5-phosphate
(EPSP)
With amino acids,
plant lives
Aromatic
NDSU
amino acids
Extension
27. The Golden Rice Story
• Vitamin A deficiency is a major health problem
• Causes blindness
• Influences severity of diarrhea, measles
• >100 million children suffer from the problem
• For many countries, the infrastructure doesn’t exist
to deliver vitamin pills
• Improved vitamin A content in widely consumed crops
an attractive alternative
NDSU
Extension
28. β-Carotene Pathway in Plants
IPP
Geranylgeranyl diphosphate
Phytoene synthase
Phytoene
Problem: Phytoene desaturase
Rice lacks
these enzymes ξ-carotene desaturase
Lycopene
Lycopene-beta-cyclase
Normal
β -carotene
NDSU
Vitamin A
“Deficient” (vitamin A precursor)
Rice Extension
29. The Golden Rice Solution
β-Carotene Pathway Genes Added
IPP
Geranylgeranyl diphosphate
Daffodil gene Phytoene synthase
Phytoene
Vitamin A
Phytoene desaturase
Pathway Single bacterial gene;
is complete performs both functions
and functional ξ-carotene desaturase
Lycopene
Daffodil gene Lycopene-beta-cyclase
β -carotene
NDSU
Golden
Rice (vitamin A precursor)
Extension
30. Metabolic Pathways are Complex
and Interrelated
Understanding pathways
is critical to developing
new products
NDSU
Extension
31. Modifying Pathway Components
Can Produce New Products
Turn On Vitamin Genes =
Relieve Deficiency
Modified Lipids =
New Industrial Oils
Increase amino acids =
Improved Nutrition
NDSU
Extension
32. Trait/Gene Examples
Trait Gene
RoundUp Ready Bacterial EPSP
Golden Rice Complete Pathway
Plant Virus Resistance Viral Coat Protein
Male Sterility Barnase
Plant Bacterial Resistance p35
Salt tolerance AtNHX1
NDSU
Extension
33. Introducing the Gene or
Developing Transgenics
Steps
1. Create transformation cassette
2. Introduce and select for transformants
NDSU
Extension
34. Transformation Cassettes
Contains
1. Gene of interest
• The coding region and its controlling elements
2. Selectable marker
• Distinguishes transformed/untransformed plants
3. Insertion sequences
• Aids Agrobacterium insertion
NDSU
Extension
35. Gene of Interest
Promoter TP Coding Region
Promoter Region
• Controls when, where and how much the gene is expressed
ex.: CaMV35S (constitutive; on always)
Glutelin 1 (only in rice endosperm during seed development)
Transit Peptide
• Targets protein to correct organelle
ex.: RbCS (RUBISCO small subunit; choloroplast target
Coding Region
• Encodes protein product
ex.: EPSP
β-carotene genes
NDSU
Extension
36. Selectable Marker
Promoter Coding Region
Promoter Region
• Normally constitutive
ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter
Coding Region
• Gene that breaks down a toxic compound;
non-transgenic plants die
ex.: nptII [kanamycin (bacterial antibiotic) resistance]
aphIV [hygromycin (bacterial antibiotic) resistance]
Bar [glufosinate (herbicide) resistance]
NDSU
Extension
37. Effect of Selectable Marker
Non-transgenic = Lacks Kan or Bar Gene
Plant dies in presence
of selective compound
X
Transgenic = Has Kan or Bar Gene
Plant grows in presence
of selective compound
NDSU
Extension
38. Insertion Sequences
TL TR
Required for proper gene insertions
• Used for Agrobacterium-transformation
ex.: Right and Left borders of T-DNA
NDSU
Extension
40. Delivering the Gene
to the Plant
• Transformation cassettes are developed in the lab
• They are then introduced into a plant
• Two major delivery methods
• Agrobacterium
Tissue culture
• Gene Gun required to generate
transgenic plants
NDSU
Extension
41. Plant Tissue Culture
A Requirement for Transgenic Development
Callus
grows
A plant part Shoots
Is cultured develop Shoots are rooted;
plant grows to maturity
NDSU
Extension
42. Agrobacterium
A natural DNA delivery system
• A plant pathogen found in nature
• Infects many plant species
• Delivers DNA that encodes for plant hormones
• DNA incorporates into plant chromosome
• Hormone genes expressed and galls form at infection site
Gall on
stem
NDSU
Gall on
leaf
Extension
45. But Nature’s Agrobacterium
Has Problems
Infected tissues cannot be regenerated (via tissue culture)
into new plants
Why?
• Phytohormone balance incorrect regeneration
Solution? Transferred DNA (T-DNA) modified by
• Removing phytohormone genes
• Retaining essential transfer sequences
• Adding cloning site for gene of interest
NDSU
Extension
46. The Gene Gun
• DNA vector is coated onto gold or tungsten particles
• Particles are accelerated at high speeds by the gun
• Particles enter plant tissue
• DNA enters the nucleus and
incorporates into chromosome
• Integration process unknown
NDSU
Extension
47. Transformation Steps
Prepare tissue for transformation
• Tissue must be capable of developing into normal plants
• Leaf, germinating seed, immature embryos
Introduce DNA
• Agrobacterium or gene gun
Culture plant tissue
• Develop shoots
• Root the shoots
Field test the plants
• Multiple sites, multiple years
NDSU
Extension
49. Lab Testing The Transgenics
Insect Resistance Cold Tolerance
Transgene= Transgene=
Bt-toxin protein CBF transcription factors
NDSU
Extension
50. More Modern Examples
Salt Tolerant Mercury Resistance
Transgene= Transgene=
Glyoxylase I Mercuric ion reductase
NDSU
Extension
51. The Next Test Is The Field
Herbicide Resistance
Non-transgenics
Transgenics
NDSU
Extension
52. Final Test
Consumer Acceptance
RoundUp Ready Corn
NDSU
Before After
Extension
53. The Public Controversy
• Should we develop transgenics?
• Should we release transgenics?
• Are transgenics safe?
• Are transgenics a threat to non-transgenic
production systems?
• Are transgenics a threat to natural
eco-systems?
NDSU
Extension
Editor's Notes
Title Page: Biotechnology: Principles, Applications, and Social Implications Part II: From Protein to Product NOTE : In “Slide Show” format, click on the slide. You will notice that on many pages a series of text boxes or images will appear sequentially on the same slide. This is intended to provide you with a progression of concepts. Remember to study the slide notes to get a better understanding of what is being presented in the slides.
The general definition is very broad. Many individuals prefer this definition because they can claim process such as plant breeding or mutagenesis are actually biotechnology. The detailed definition points to the fact that a foreign gene needs to be inserted for a product to be considered a biotech product. Biotechnology involves the modification of a whole range of organisms. This foreign gene can be inserted into plants, animals, fungi, bacteria or viruses. The key for me is that a foreign gene, or an engineered gene from the same species is added back into the organisms. The modified organisms would not have these traits without the intervention of man.
As we are all aware, most species have an abundance of variation. The photograph of bean seeds is a great illustration of the variation in nature. You can notice not only many different colors, but also many different patterns. A large array of interacting genes are responsible for this variation. But man can always dream of a new use for an organism. These dreams often involve asking the species to do something it does not now normally do. Biotechnology involves added new traits to a species.
Two of the most dramatic examples of man’s dreams of improving the utility of plants is shown here. At the top is the picture of bananas. The goal is to express vaccines in these fruits so that individuals who eat them will receive the vaccine and become immune to the disease. The rice photograph illustrates a recent invention called “Golden Rice.” This strain of rice has been engineered to express enzymes required for the vitamin A pathway that don’t normal exist in this species. By the goal is to provide this as crop as a dietary product that will improve the nutrition and health of those who eat it.
Before we can understand who man goes about using biotechnology approaches to modify a species, we must understand basic genetic principles and terminology. All traits are controlled by genes . A gene can have different forms. These forms are called alleles . It is important that you become fluent with these terms and the differences they imply. It is simple as remember that genes have alleles . Or, alleles are alternate forms of a gene .
This slide is intended to help you understand the difference between genes and alleles. Genes control specific traits. Here are two traits of pea that Gregor Mendel, the father of genetics, studied. Plant height is a trait. That trait is controlled by the plant height gene . The plant can be either tall or short. Different alleles of the plant height gene determine if the plant will be tall or short . If you remember from your genetics class, the allele for tall plant height is dominant to the short allele. This means that heterozygous individuals carrying both the tall and short allele will appear to be tall. Using the genetic terminology, this also means that the short allele is recessive to the tall allele. Similarly, seed shape is controlled by a specific gene. The alternate shapes, smooth or wrinkled, are controlled by different allelic forms of the seed shape gene. Similarly, smooth is dominant to the recessive wrinkled phenotype.
As the previous slide demonstrated, each gene has a specific function. The fact that two alleles control alternate forms of a trait means they must also be very similar to each other, but different enough to produce different phenotypes. In modern terminology, each allele will have very similar DNA sequences and will encode the same protein product. But the sequence of the alleles will differ at some point, and that difference is directly responsible for the various phenotypes generated by the different alleles.
So what about the genes encoding the two traits that Mendel studied. This slide provides the essential information regarding the plant height gene. This gene encodes and important enzyme in the gibberllic acid biosynthetic pathway. If you remember, gibberellic acid is responsible for node elongation in plants. If a plant has a longer nodes, it will be taller than a plant with the same number of nodes, but whose nodes are shorter. The specific gene product is gibberellin 3- -hydroxylase. This protein adds a hydroxyl (-OH) group to the gibberellin called GA 20 and this process makes the biologically active GA 1 gibberellin. The dominant allele contains the normal form of this gene, and the result of its expression will be tall plants. The recessive allele is very similar to the dominant allele. In fact, the only difference is a change in a single nucleotide that results in the change of an alanine to a threonine in the final protein. This may seem like a trivial change, but the result of the change is a 20-fold reduction in the active of the protein encoded by this mutant allele. This comparison highlights the type of information that is gathered from these sequence analysis of different alleles.
So what about the smooth vs. wrinkled phenotypes expressed by the seed shape gene? This gene encodes the strach branching enzyme (SBE) isoform 1. The recessive allele has no activity, and the result in the seed is that it contains much more sucrose than starch. The result of higher sucrose levels is a higher water content. As these seeds dry they lose more water and the seed takes on a wrinkled appearance. Unlike the recessive plant height allele that is the result of a single nucleotide change, the recessive seed shape allele is the result of the insertion of a large piece of DNA into the coding region. This large piece of DNA is a transposable element . This element is similar to the elements that Barbara McClintock discovered in corn.
Now that we understand the relationships between alleles at a gene, it is time to place this understanding in a large context. You should become very fluent with this concept: the Central Dogma of Molecular Genetics . This concept is a unifying principle that describes the manner in which the sequence information in the gene is eventually expressed as a trait ( or phenotype ). DNA is the biochemical molecule of all genes. DNA contains the genetic code that will eventually be converted into the protein that will control the phenotype expression. But DNA is not directly used for phenotypic expression. Instead, the information in the DNA molecule is used to create an intermediary molecule ( RNA ). The process to produce RNA is called transcription . RNA is an active molecule in another process called translation that is used to create the protein . The protein itself can have many different functions. It could be an enzyme in a metabolic pathway. Alternatively, it could act as a regulator transcription . Finally, it could serve as a structural component of the cell. Whatever it role is, it will control the final phenotype or the outward appearance of plant.
Remember our definition of biotechnology??? Here is the detailed one again: The application of the technology to modify the biological function of an organism by adding genes from another organism . As the definition implies, we first need to isolate a gene that will be added to our organism of interest. For example, we may wish to add a gene from a bacteria into a plant species. Another approach would be to isolate a gene from our species of interest, modify that gene to change its function, and then reinsert the modified form back into the species. This is the first major biotechnology step, and it is called gene manipulation . Once we have isolated or modified the gene of interest, the next step is gene introduction . This step involves the addition of the gene to our species of interest. In all cases, this introduction must be accompanied by stable integration of that gene into the genome of the target species. Once the gene is stably integrated, it is passed along to all subsequent generation in the same manner as all other genes in the genome. The technique used to integrate the gene into the species is called transformation , and the modified organism is called a transgenic organism .
We will discuss gene manipulation and gene introduction separately. The Central Dogma of Molecular Genetics was introduced several slides back. As a remember, it states that the information stored in DNA is transcribed into RNA, the RNA molecule is used in a process called translation to produce translation to produce a protein, and the protein is involved in some process that actually produces the final phenotype of the organism. The dogma implies we need to manipulate the DNA of gene that encodes for the protein if we are going to develop a transgenic organism. The DNA itself is a double-helix molecule that is stored inside the nucleus of the cell. Every cell inside the organism has exactly the same DNA molecule.
We normally think of the DNA in the form of a chromosome. Chromosomes are the condensed form of DNA . The simplest form of DNA is the double-stranded molecule. These two strands are complementary to each other. This complementarity is based on the fact that if one strand has an adenine at one nucleotide residue, the complementary strand has a thymine residue at the same location. And if one strand has a guanine at a specific residue, the complemenntary strand has a cytosine residue. This is an important concept because it is the basis of an important screening process called hybridization . The double-strand molecule then undergoes a series of condensation steps to produce the chromosome. Each of this different steps are illustrated here in this slide. It is important to remember that throughout the life-cycle of the cell, DNA is in an uncondensed form. The chromosome only appears during the process of cell division.
All of genes reside on a specific location on the chromosome . Any chromosome will contain thousands of genes. To illustrate this point, the human genome consists of about 35,000 genes which are spread over 22 chromosomes. In contrast, the model plant species Arabidopsis thaliana contains about 30,000 genes on just five chromosomes. This illustrates the point that although plants and animals contain about the same number of genes, the have a different number of genes on their chromosomes. The goal of gene manipulation is to isolate that one gene of interest from among the many genes in the genome.
To understand biotechnology, it is important to have a general appreciation of how genes are cloned. These fall into three gene approaches. The first approach is based on the fact that genes between related species have similar DNA sequences. This procedure is called homology cloning . The second procedure is based on the direct relation that exists between the DNA sequence and the final protein sequences. If you have an idea of the protein sequence you can develop a probe to get at the gene. This approach is called complementary genetics . The third procedure relies on experiments that place the gene at a specific genetic location. This procedure is called map-based cloning .
We will first discuss homology cloning. As mentioned earlier, this procedure is based on the fact that the genes between two closely related species have very similar DNA sequences. To take advantage of this procedure, you need a probe from a species similar to the one you are working on. Let's say you know that someone has cloned a gene from mouse that you are interested in as a human geneticist. You would then contact the person and request a copy of that gene. That will be used as a probe in your experiments. The first step is for you to grow out a clone library. This library contains all of the genes from your species. Therefore a human clone library contains all of the genes in the human genome. The clones are then transferred to a special filter where the DNA for each clone binds. They bind at a spot that is directly analogous to their position on the plate in which they are grown. This relationship is illustrated on this slide. The next step is to add the probe to the library. The probe is normally radioactively labeled. The importance of this will be seen shortly. The probe then will bind by base-pair complementarity to a clone to which it is very similar. This is the hybridization step that was mentioned on an earlier slide. Excess probe is washed off, and the washed filter is then exposed to an x-ray film. The film is then read, and any hot-spot is the location of a clone that is similar to the probe you used. It is hot because the probe was radioactievely-labelled. You then go back to the original plate containing your genes and select the clone containing your gene.
The second cloning procedure is called complementary genetics . And as with that procedure, homology cloning requires a probe from some source. The difference with homology cloning though is that we develop our own probe based on the protein sequence. Probe development is based on two simple facts. The first is that the sequence of the gene in its DNA form will predict the protein sequence. This also implies that if you know the protein sequence you can derive the DNA nucleotide sequence. The second fact is that the placement of an amino acid into a growing protein chain is directed by a sequence of three nucleotides. This fact is illustrated in the slide. You should notice that some amino acids are encoded by one triplet sequence, some by two sequences, and others by three, four or six. As you will see this is a minor issue for complementary genetics approaches. The probe is actually synthesized using a procedure called PCR , or p olymerase c hain r eaction. The DNA synthesis procedure requires DNA primers. One primer is designed to a region in the amino (NH3+) part of the gene. As you can see, the sequence of the forward primer is based on the nucleotide sequence that corresponds to the amino acid sequence. When you look at this sequence you will notice the sequence denoted as “A/T”. This means the primer will actually consist of a mixture primers some with an A at that position and some with a T. The symbol “N” means the primer pool will contain a collection of primers each with one of the four nucleotides at that position. Therefore, this primer pool will consist of 8 (2x4) different primers. The reverse primer is synthesized using the same principles, but it is complementary to the genetic code sequence. (It is complementary because of the fact that DNA consists of two complementary strands. Therefore, the PCR synthesis process is designed to produce both strands.) So, as you can see the first nucleotide is complementary to the A/G bases that encode the lysine (Lys) amino acid, and the primer instead consists of T (thymine) or C (cytosine). As with the forward primer, the reverse primer is actually a pool that consists of 32 [2 (T or C) x 4 (A, T, C or G) x 2 (C or G) x 2 (T or A)] primers.
PCR is a DNA synthesis process. The previous slide described the first step in PCR , the development of the primers. The primers satisfy one of the two requirements of DNA synthesis, a primer from which the new chain grows. The second requirement is a DNA template. To generate a DNA template, the double-stranded DNA molecule must be converted into a single-stranded state. This is done by heating the DNA to a high temperature. Step 3a in the slide shows this process. Once the DNA is single stranded, the temperature is lowered so that the primer can anneal. Once annealed, the temperature is raised to the optimum temperature required by a special DNA polymerase enzyme called Tag polymerase. Once the temperature is reached, the DNA is replicated. As you can see in step 3c, now have two strands of DNA.
This is an animation of one step in the PCR process. Take a few minutes and let the animation run through a number of times. It will recycle on its own. This step will show the denaturation (converting the DNA from single- to double-stranded state). The second step is annealing (the binding of the primer to the single-stranded DNA). The final step is extension (the duplication of a strand from the end of the primer).
As the previous two slides illustrated, the many feature of the PCR process is the replication of one double-strand DNA molecule into two. But the PCR process does not involve just a single replication cycle. Rather, the step is repeated many times (35-50 times). This repetition leads to an exponential increase in the amount of DNA. At the end, a large amount of DNA is produced that can be used for a number of purposes. One of those purposes, and the one we are interested with here, is the development of a DNA probe. Remember that we started with a protein sequence of interest, and we used that information to design primers to amplify a DNA fragment that would encode that fragment. Therefore at the end of the PCR replication, we have sufficient DNA to use as a probe for library screening.
The final step in using complementary genetics for cloning involves screening a library. The steps are exactly the same as we described for homology cloning . The only difference is that we now use the PCR synthesized DNA as our probe. The final result will be the isolation of a DNA clone from the library. That clone will contain DNA sequences that will encode the gene for the protein in which we are interested.
The final technique of method of obtaining genes is called map-based cloning . This procedure combines genetic information that locates a gene to a small region of a chromosome. The position of the gene is located to that region by the use of a DNA marker that resides very close to the gene. The first two steps illustrate this point. Typically a marker is discovered at a short distance away from the gene of interest. That marker is then used to discover another marker that is very close that cosegregates with the gene of interest. That cosegregating marker is very close to the gene (closer than the first marker) and is used to isolate a series of overlapping clones or contig . A series of steps is then used to identify ORFs or o pen r eading f rames . An ORF is a DNA sequence that has all the characteristics of a gene. Since the DNA marker (and by association the gene of interest) resides on the contig, one of the ORFs will be the gene. We won’t go into the details, but the ORF that is a gene is eventually identified by one of two procedures. Transformation is one approach. As defined earlier, transformation involves the addition of DNA to an organism and changing that organism’s phenotype. For map-based cloning, the ORF is added to a mutant organism, and if the resulting transgenic plant expresses the wild type phenotype then the ORF is the gene you are trying to clone. For example, if you had and ORF that encoded the gene responsible for plant height in pea, that gene could be added to a short pea plant. If the addition of the ORF is the gene for plant height, that transgenic plant would be tall. An alternative approach is to sequence many mutant phenotypes of a specific ORF. That analysis may provide useful information that would allow you to determine a particular ORF is the gene of interest. This is the approach used in human genetics.
The development of the transgenic organism uses some gene isolated by the procedures that were just outlined. But it is important that the appropriate gene is used to obtain the specific phenotype you wish to develop. We are going to spend a bit of time concentrating on two important phenotypes: glyphosate (RoundUp) resistance and increased vitamin A content . Each of the phenotypes can be achieved by adding one or several genes to a plant.
The RoundUp Ready technology is the most visible plant biotechnology product on the market. To better understand plant biotechnology in general, it is important to understand the development of these transgenic organisms. RoundUp is a brand name herbicide manufactured by Monsanto Corp. The active ingredient in this herbicide is glyphosate. The chemical binds to the active site of the EPSP synthase enzyme. This enzyme is a key to the development of a group of amino acids called the aromatic amino acids. When this enzyme is bound by glyphosate, it can not synthesize those amino acids, and the plants die because protein synthesis is severely disrupted. Glyphosate will not bind the to a particular genetically-engineered version of EPSP synthase. Therefore RoundUp Ready crops with this altered enzyme will survive when sprayed with the herbicide.
This slide shows the actual biochemical pathway that we discussed in the previous slide. EPSP synthase synthesizes 3-enolpyruvly shikimic acid-5-phosphate. This is the essential precursor to aromatic amino acids. When plants are sprayed with a glyphosate-containing herbicide, such as RoundUp, this important precursor is not synthesized, and consequently the plant is starved of aromatic amino acids. The result is plant death.
RoundUp Resistant plants have a very simple solution. An engineered version of EPSP synthase, one that was discovered in a bacteria, is introduced into the plant. This enzyme can not be bound by glphosate. Therefore, if a field is sprayed with the herbicide, the introduced version of the gene produces a functional enzyme. The 3-enolpyruvl shikimic acid-5-phosphate precursor is synthesized normally, and the plant produces enough aromatic amino acids to survive.
The second major plant biotechnology product is more recent and was developed to address the vitamin A deficiency problems prevalent throughout the world. This vitamin deficiency is very critical because it can cause blindness and affects the severity of many diseases including diarrhea and measles. This is a severe problem that affects more than 100 million children worldwide. A simple solution would be to distribute vitamins to the affected children. Unfortunately, many countries where the deficiency is chronic do not have the necessary infrastructure to deliver the vitamin tablets to the most needed. The solution that is currently being promoted is to improve the vitamin content in widely-consumed, and readily available to the consumer. Transgenic rice plants were developed that contain elevated levels of the precursor to vitamin A. This GMO is called “Golden Rice” because of its color: it is yellow rather than white. It is yellow because β -carotene, a yellow precursor to vitamin A is abundant in the seed.
Unlike the single-step RoundUp Ready pathway, the β –carotene synthesis pathway involves multiple enzymes. This important vitamin A precursor cannot be synthsized in rice because it lacks four of the key enzymes. Therefore, the precursor is not made, and the plant contains white kernels.
In a major feat of genetic engineering, scientists inserted a complete functioning -carotene biosynthetic pathway into the rice plant. They did this by inserting genes from daffodil the produce functioniong versions of the first and last enzymes of the pathway. In addition, a single bacterial gene that provides the same function as the second and third enzymes of the pathway, was also introduced. With a functioning pathway, the transgenic rice is able to produce the vitamin A precursor β -carotene. It is this product that gives "Golden Rice" its characteristic yellow color.
The “Golden Rice” story illustrates a key point: it is very important to industry metabolic pathways. These pathways are very important for our understanding of specific products are produced in the organism. Only by understanding this pathways will we be able to create novel new products.
This diagram shows in general the interrelationship between the many different pathways. A key point to understand is that the different sub-pathways interconnect. Therefore modify one component of the pathway may affect the production of a product in a separate sub-pathway. Keeping this in mind, we can now envision how to engineer plants so they produce novel products. We have already seen how modifying a vitamin biosynthetic pathway can positively affect vitamin production. We could also improve nutrition in other ways. For example, if we were to focus our attention on the amino acid pathways we could, for example, increase the lysine content in typically lysine-poor grains. Conversely, someday we might be able to improve legumes by introducing the correct genes necessary to enrich the metionine content. We could also envision new products if we modify other pathways. Oils are a key component to both the food and manufacturing industries. A better understanding of the genes in the complex lipid pathway may allow us to produce better industrial oils.
This slide illustrates the variety of different traits that have been modified in plants. It also shows the particular gene that was introduced into the plant to obtain the specific trait. As you can see, scientists have successfully introduced many different genes and produced many different results. For example, it was discovered that expressing the in the plant a particular protein of a virus, the coat protein, the plant would then become resistant to that virus. This technique has been widely credited with saving the papaya industry in Hawaii, where the papaya ringspot virus nearly eliminated the papaya growing industry. This is a success story that is often overlooked, probably because the problem was to a crop of limited production value.
It is now time to cover the development of transgenic crops in greater depth. The two major steps are creating a transformation cassette that contains the gene of interest, and then successfully introducing the cassette into the plant.
All transformation cassettes contain three regions. The “gene of interest” region contains the actual gene that is being introduced into the plant. This is the gene that provides the new function to the plant. In this diagram, the region is shown in red. Many plant tissues are treated with the transformation cassette during the transformation step. Not all of these tissues actually receive the cassette. To distinguish those that contain the gene from those that don’t, it is necessary to use a selection process. The selectable marker is a gene that provides the ability to distinguish transformed from non-transformed plants. This is shown by green. The most common method to introduce the transformation cassette is by using the plant pathogen Agrobacterium . For this system to work it is necessary that the cassette contain insertion sequences that are used by the bacteria. These are shown by the gray.
All of these components of the transformation cassette contain multiple components. In addition to the coding region that encodes the protein product, the gene of interest region also contains two important controlling regions. The promoter region resides just before the coding region and determines when, where, and to what degree the gene of interest will be expressed. In general, two types of promoter regions are used. A constitutive promoter turns the gene on in all tissues at all times. In general, this leads to a relatively high level of gene expression. The most often used constitutive promoter controls the expression of the 35S RNA of the cauliflower mosaic virus. It is abbreviated as CaMV35S promoter. Other promoters direct a very specific expression pattern. For example, the glutelin 1 promoter directs that the expression of the glutelin storage protein at a specific time of seed development. It also ensures the protein is only expressed in the rice endosperm. If the gene of interest is preceded by the CaMV35S promoter, it will be expressed in all tissues at all times. Conversely, the expression of the target gene could be limited to the endosperm if it is controlled by the glutelin 1 promoter. Some, but not all genes, encode protein that function in the plant organelles. These organelles are the chloroplast and the mitochondria. For example, photosynthesis, and part of the carbon and lipid metabolism pathways are carried out in the organelles. To ensure these protein are delivered to the appropriate organelle, a transit peptide is required. This is a short amino acid sequence that is found directly before the coding region. This sequence is recognized by proteins in the outer membranes of the appropriate organelle. This recognition process leads to the import of the protein into the organelle. Therefore, if you are gene of interest functions in the organelle, an appropriate transit peptide must be included in the transformation cassette
As stated above, the selectable marker is a gene that encodes a protein product. For it to be expressed, it also needs a promoter region. It is typical to use the constitutive CaMV35S RNA promoter. The gene it controls encodes a protein that enables a transformed plant to survive in the presence of normally toxic compound. The most often used selective agents are kanamycin and hygromyin, two bacterial antibiotics, and the herbicide glufosinate. The protein encoded by the selectable marker genes generally renders these selective agents harmless to the transgenic plant.
This slide shows the effect of the selectable marker.
The insertion sequences straddle the gene-of-interest coding region and the selectable marker. These are use by Agrobacteria to create a DNA molecule that is sent out of the bacteria into the plant where it is eventually inserted into the nucleus of a cell in the recipient plant tissue. If the cell follows the proper developmental pathway that leads to a new plant, every cell in that plant will contain the sequences in between the insertion sequences.
This slide demonstrates one of the transformation cassettes used to develop “Golden Rice” was developed. Slowly click through this slide, and you will see each of the components of the cassette.
Two techniques are used to deliver DNA found in the transformation cassette into plant tissues during the plant transformation process. One is a biological system based on the plant pathogen Agrobacterium tumefaciens . The second is a mechanical method where the DNA is “shot” into plant cells using a gene gun. Regardless of the delivery method, the delivery system must use a plant tissue source that can be manipulated to produce new plants.
This slide shows the basic steps of plant tissue culture. Some plant part is placed is on a defined culture media. That media induces the the tissue to develop callus. Callus is an undifferentiated mass of cells. These cells then grow into plant shoots, which are later rooted. The small seedling will then grown into a mature, seed-producing plant. When developing transgenic plants, the transformation cassette is introduced into that plant part that can be induced to grow new plants.
In many ways, Agrobacterium , has been the most successful method of delivering DNA into plants. It is a naturally occurring plant pathogen. It inserts DNA into the nucleus of a plant cell. That DNA contain genes that encode hormones and food products the bacteria uses to support its own growth. Here you can see the gall growth on the plant tissue. This is the natural result of the infection.
This photograph shows exactly how large the gall can grow.
The interaction between the bacteria, Agrobacterium , and the host plant is very complex. It has been studied in great depth and many of the major details of the interaction have been described. It is this understanding that allowed other scientists to convert Agrobacterium into a plant transformation vector that delivers the cassette. It works this way. An Agrobacterium strain in which the phytohormones have been removed is used. The transformation cassette is then introduced into that Agrobacterium strain. The source tissue for plant transformation is infected with the strain. Then the steps described previously are applied. The tissue is grown in the presence of the selective agent. That tissue culture media also contains the hormones necessary to allow the plant tissue develop new plants. Because of the selection pressure placed on that tissue, those new shoots will contain the important genes of interest found in the transformation cassette. For simplicity sake, many details are being left out. But these are many steps.
Early on it was known that tissues infected with Agrobacterium could not be coaxed to regenerate new plants. Soon it was realized that the plant hormone balance was not correct. To over come this effect, the genes encoding the phytohormones were removed. Once removed, plant tissues infected with the modified Agrobacterium could produce the regenerated plant. With this realization, it was a simple step to envision how to deliver genes of interest into a plant: include these genes in the cassette.
The second method currently in use is the gene gun. The principle is very simple. The transformation cassette DNA is coated onto a particle. That particle is then accelerated (using ballistics or an air stream). The particle then enters the plant cell. At that point, the transformation cassette DNA is eluted off the particle, and by a process that is not known, the DNA becomes integrated into the nucleus of the cell. The same basic principles guiding plant transformation with Agrobacterium is used with the gene gun. A tissue source that is capable of being manipulated to produce new plants is treated; in this case it is shot with particles containing the transformation cassette. The tissue is then placed under selection, and those shoots that develop contain the gene of interest.
This slide summarizes the steps necessary for plant transformation.
And this slide illustrates those steps.
This and the next slide illustrate the types of traits that can be obtain using genetic engineering of plants. Notice the particular types of genes that were used to obtain these traits. Some genes encode a protein that directly provides the trait. This is illustrated by the gene that encodes the Bt-toxin protein that is harmful to plant insect pests. Other genes encode protein that regulate the expression of a trait. Cold tolerance can be added to a crop by introducing gene that encode a transcription factor. These factors interact with other genes to turn on their expression.
More recently, such varied traits as salt tolerance and mercury resistance have been introduced into plants transferring genes for specific proteins.
The last step in plant genetic engineering is field testing. This slide shows a field that contains herbicide resistant and tolerant plants.
What is needed is for the public to accept these crops. Examples such as these, were a corn crop is freed of weed pressure make a compelling case for acceptance of these new agricultural products. But, it should be noted that these traits are all producer orientated.
The public, in general, is interested in the consumer perspective. Here are the general questions that drive the controversy.