Unit-IV GENE Unit-IV GENE SEQUENCINGSEQUENCING

Gene Sequencing and the Human Gene Sequencing and the Human Genome ProjectGenome Project

Gene SequencingGene Sequencing

DNA sequencing uses electrophoresis to DNA sequencing uses electrophoresis to separate DNA fragments of different lengths. A separate DNA fragments of different lengths. A DNA synthesis reaction is set up that includes DNA synthesis reaction is set up that includes DNA from the region being investigated.DNA from the region being investigated.The reaction also includes:The reaction also includes:DNA Polymerase DNA Polymerase A specific DNA primerA specific DNA primerAll DNA nucleotidesAll DNA nucleotidesA small amount of 4 kinds of chemically altered A small amount of 4 kinds of chemically altered DNA nucleotides.DNA nucleotides.

As a group , the DNA molecules that are As a group , the DNA molecules that are created by this technique have the created by this technique have the following properties:following properties:They all start at the same point, because They all start at the same point, because they all started with the same primer.they all started with the same primer.There are copies of DNA molecules that There are copies of DNA molecules that had their replication halted at each had their replication halted at each nucleotide in the sequence of the sample nucleotide in the sequence of the sample DNA when a dideoxyribose nucleotide was DNA when a dideoxyribose nucleotide was incorporated.incorporated.DNA molecules of the same length are DNA molecules of the same length are labeled with the same color of fluorescent labeled with the same color of fluorescent dye.dye.

Gene Sequencing and the Human Gene Sequencing and the Human Genome ProjectGenome Project

The The Human Genome ProjectHuman Genome Project ( (HGPHGP) is an international scientific ) is an international scientific research project with a primary goal of determining the sequence of research project with a primary goal of determining the sequence of chemical base pairs which make up DNA, and of identifying and chemical base pairs which make up DNA, and of identifying and mapping the approximately 20,000–25,000 genes of the human mapping the approximately 20,000–25,000 genes of the human genome from both a physical and functional standpoint.genome from both a physical and functional standpoint.

HGP was a 13 year effort to determine the human DNA sequence. Work HGP was a 13 year effort to determine the human DNA sequence. Work began in 1990.began in 1990.It was first proposed in 1986 by U.S department of Energy (DOE) .It was first proposed in 1986 by U.S department of Energy (DOE) .Many countries like-Australia, Brazil, Canada, China, Denmark, France, Many countries like-Australia, Brazil, Canada, China, Denmark, France, Germany, Mexico, Sweden etc. contributed both funds and labour resources Germany, Mexico, Sweden etc. contributed both funds and labour resources to the Human Genome Project.to the Human Genome Project.While the objective of the Human Genome Project is to understand the While the objective of the Human Genome Project is to understand the genetic makeup of the human species, the project has also focused on genetic makeup of the human species, the project has also focused on several other nonhuman organisms such as several other nonhuman organisms such as E. coli,E. coli, the fruit fly, and the the fruit fly, and the laboratory mouse. laboratory mouse. HGP was one of the most ambitious projects ever undertaken in the HGP was one of the most ambitious projects ever undertaken in the biological sciences.biological sciences.

Human Genome Project GoalsHuman Genome Project Goals

The specific goals of the HGP are to::The specific goals of the HGP are to::Identify all the estimated 80,000 genes in Identify all the estimated 80,000 genes in the human DNA, the human DNA, Determine the sequences of the 3 billion Determine the sequences of the 3 billion DNA chemical bases. DNA chemical bases. Store this information in databases, Store this information in databases, Develop tools for data analysis, and Develop tools for data analysis, and Address the possible ethical, legal, and Address the possible ethical, legal, and social issues.social issues.

Advantages of Human Genome Advantages of Human Genome Project:Project:

Knowledge of the effects of variation of Knowledge of the effects of variation of DNA among individuals can revolutionize DNA among individuals can revolutionize the ways to diagnose, treat and even the ways to diagnose, treat and even prevent a number of diseases that affects prevent a number of diseases that affects the human beings. the human beings.

It provides clues to the understanding of It provides clues to the understanding of human biology. human biology.

Human Genome Project TechniqueHuman Genome Project Technique

Two steps are followed-Two steps are followed-First, physical maps were constructed by First, physical maps were constructed by determining the location of specific “markers” determining the location of specific “markers” and the proximity of these markers to genes.and the proximity of these markers to genes.The markers were known sequences of DNA The markers were known sequences of DNA that could be located on the last chromosome.that could be located on the last chromosome.This physical map was used to organize the vast This physical map was used to organize the vast amount of data produced by the second amount of data produced by the second technique, which was for the labs to determine technique, which was for the labs to determine the exact order of nitrogenous bases of the DNA the exact order of nitrogenous bases of the DNA for each chromosome.for each chromosome.

Human Genome Project Human Genome Project ApplicationsApplications

The information provided by the human The information provided by the human genome project is extremely useful in genome project is extremely useful in diagnosing diseases. diagnosing diseases.

Providing genetic counselling to those Providing genetic counselling to those considering having children.considering having children.

This info can identify human genes and This info can identify human genes and proteins that can targets for drugs and proteins that can targets for drugs and new gene therapies.new gene therapies.

GENE CLONINGGENE CLONING

Cloning a gene means isolating an exact copy of Cloning a gene means isolating an exact copy of a single gene from the entire genome of an a single gene from the entire genome of an organism. organism. Usually this involves copying the DNA sequence Usually this involves copying the DNA sequence of that gene into a smaller, more accessible of that gene into a smaller, more accessible piece of DNA, such as a plasmid. This makes it piece of DNA, such as a plasmid. This makes it easier to study the function of the individual easier to study the function of the individual gene in the laboratory.gene in the laboratory.Cloning gene means cutting the source DNA into Cloning gene means cutting the source DNA into smaller, manageable pieces with restriction smaller, manageable pieces with restriction enzymes.enzymes.

PlasmidPlasmid

A plasmid is one example of a vector that is A plasmid is one example of a vector that is used to carry DNA into bacterial cells.used to carry DNA into bacterial cells.It is a circular piece of DNA that is found free in It is a circular piece of DNA that is found free in the cytoplasm of some bacteria.the cytoplasm of some bacteria.The Plasmid can be cut with a restriction The Plasmid can be cut with a restriction enzyme, so that the plasmid DNA will have enzyme, so that the plasmid DNA will have sticky ends, which can attach to the source sticky ends, which can attach to the source DNA. DNA. The enzyme ligase creates the bond between The enzyme ligase creates the bond between the plasmid DNA and the source DNA.the plasmid DNA and the source DNA.The plasmid and its inserted source DNA is The plasmid and its inserted source DNA is recombinant DNA. recombinant DNA.

Genetically Modified OrganismsGenetically Modified Organisms

The GMO organisms are genetically engineered The GMO organisms are genetically engineered to contain genes at least from one other species.to contain genes at least from one other species.

They contain recombinant DNA They contain recombinant DNA

Viruses, bacteria, fungi, plants, and animals are Viruses, bacteria, fungi, plants, and animals are the examples of organisms that have been the examples of organisms that have been engineered so that they contain genes from engineered so that they contain genes from atleast one unrelated genes.atleast one unrelated genes.

GMO’s are capable of expressing the protein-GMO’s are capable of expressing the protein-coding regions found on recombinant DNA.coding regions found on recombinant DNA.

Applications of GM technologyApplications of GM technology

The primary application is to put herbicide-The primary application is to put herbicide-resistance or pest-resistance genes into crop resistance or pest-resistance genes into crop plants.plants.Edible GM crops are used for animal feed.Edible GM crops are used for animal feed.Insertion of genes from a specific kind of Insertion of genes from a specific kind of bacterium called bacterium called Bacillus Thuringiensis Bacillus Thuringiensis israeliensis (Bti) israeliensis (Bti) produces a protein that cause produces a protein that cause destruction to the insects and pest.destruction to the insects and pest.GMO’s are used with a great success to clean GMO’s are used with a great success to clean up oil spills and toxic waste dumps, the process up oil spills and toxic waste dumps, the process known as BIOREMEDIATION.known as BIOREMEDIATION.

Recombinant DNA procedures are Recombinant DNA procedures are also responsible for-also responsible for-

Human Insulin, used in the control of diabetes.Human Insulin, used in the control of diabetes.

Nutritionally enrched “golden rice”, capable of Nutritionally enrched “golden rice”, capable of supplying poor people in less developed supplying poor people in less developed countries with beta- carotene which is missing countries with beta- carotene which is missing from normal rice.from normal rice.

Interferon , used as an antiviral agent.Interferon , used as an antiviral agent.

Human growth hormone , used to stimulate Human growth hormone , used to stimulate growth in children lacking this hormone.growth in children lacking this hormone.

Genetically Modified FoodsGenetically Modified Foods

Crops such as corn, soyabean, sugar beet, rice, cotton Crops such as corn, soyabean, sugar beet, rice, cotton are short list of GM crops being grown and sold.are short list of GM crops being grown and sold.Such crop have the potential of supplying the essential Such crop have the potential of supplying the essential amino acids, fatty acids and other nutrients.amino acids, fatty acids and other nutrients.Turnip can produce interferon, an antiviarl agent.Turnip can produce interferon, an antiviarl agent.Tobacco can create antibodies to fight human diseases.Tobacco can create antibodies to fight human diseases.Oil seed plants can serve as a source of human brain Oil seed plants can serve as a source of human brain hormones.hormones.As the population is growing, GM food may be an As the population is growing, GM food may be an important part of meeting the need for food. important part of meeting the need for food.

Basic ReviewBasic Review

What is the purpose of PCR?What is the purpose of PCR?What benefits does human genome What benefits does human genome project offer?project offer?What role does Electrophoresis play in What role does Electrophoresis play in DNA comparisons?DNA comparisons?Describe how viruses are used in gene Describe how viruses are used in gene therapy.therapy.What are the advantages of creating GM What are the advantages of creating GM foods? What are some of the concerns?foods? What are some of the concerns?

GENE THERAPYGENE THERAPYThe field of biotechnology allows scientists and The field of biotechnology allows scientists and medical doctors to work together and potentially medical doctors to work together and potentially cure genetic disorders.cure genetic disorders.Unlike contagious diseases, genetic diseases Unlike contagious diseases, genetic diseases cannot be transmitted, because they are caused cannot be transmitted, because they are caused by a genetic means for a particular disorder- not by a genetic means for a particular disorder- not separate, disease causing organisms such as separate, disease causing organisms such as bacteria and viruses.bacteria and viruses.Gene therapy involves inserting genes, deleting Gene therapy involves inserting genes, deleting genes and manipulating the action of genes in genes and manipulating the action of genes in order to cure or lessen the effects of genetic order to cure or lessen the effects of genetic diseases.diseases.

STEM CELLSSTEM CELLSStem cells are cells that are self-renewing and Stem cells are cells that are self-renewing and have not yet completed determination or have not yet completed determination or differentiation, so that they have the potential to differentiation, so that they have the potential to develop into many different cell types.develop into many different cell types.

Scientists can generate stem cells by nuclear Scientists can generate stem cells by nuclear transfer techniques; they also occur naturally transfer techniques; they also occur naturally through out the body.through out the body.

They are involved in many activities including They are involved in many activities including tissue regeneration, wound healing and cancer tissue regeneration, wound healing and cancer treatment. treatment.

Embryonic and Adult Stem CellsEmbryonic and Adult Stem Cells

Embryonic stem cells have not undergone Embryonic stem cells have not undergone determination and differentiation and have the determination and differentiation and have the ability to become any tissue in the body, they are ability to become any tissue in the body, they are of great interest to scientists.of great interest to scientists.

As an embryo develops, its stem cells go through As an embryo develops, its stem cells go through the process of determination and differentiation to the process of determination and differentiation to create all the necessary tissues.create all the necessary tissues.

To study the embryonic stem cells, scientists must To study the embryonic stem cells, scientists must remove the embryos, destroying the embryos.remove the embryos, destroying the embryos.

Adult Stem CellsAdult Stem Cells

Adult stem cells are “tissue-specific”. These are Adult stem cells are “tissue-specific”. These are found in the red bone marrow, where they found in the red bone marrow, where they divide.divide.Example is hematopoietic stem cells as these Example is hematopoietic stem cells as these cells are able to become many different types of cells are able to become many different types of cells found in blood-red blood cells, white blood cells found in blood-red blood cells, white blood cells and platelets.cells and platelets.The disadvantage of using these types of stem The disadvantage of using these types of stem cells is that they have already become partially cells is that they have already become partially determined and do not have potential to become determined and do not have potential to become every cell type.every cell type.

Concept ReviewConcept Review

What benefits does stem cell research What benefits does stem cell research offer?offer?

What are some of the concerns with What are some of the concerns with research on stem cells?research on stem cells?

Embryonic stem cells are found in Embryonic stem cells are found in embryos and adult stem cells are found in embryos and adult stem cells are found in adults. In what other way they are adults. In what other way they are different?different?