Lecture-2.3 Biotechnology Techniques, DNA Cloning

Lesson 2: Techniques in Biotechnology DNA CLONING AND RECOMBINANT DNA TECHNOLOGY Presented by: Trishia Jade Acilo Instructor I Department of Plant Breeding and Genetics Visayas State University Visca, Baybay City, Leyte HOW ARE YOU FEELING TODAY? Topic Outline DNA Cloning Restriction Endonucleases...

Lesson 2: Techniques in Biotechnology DNA CLONING AND RECOMBINANT DNA TECHNOLOGY Presented by: Trishia Jade Acilo Instructor I Department of Plant Breeding and Genetics Visayas State University Visca, Baybay City, Leyte HOW ARE YOU FEELING TODAY? Topic Outline DNA Cloning Restriction Endonucleases Important Steps in DNA Cloning DNA Vectors Cloning Vectors Recombinant DNA Technology DNA Cloning To clone means to make identical copies. DNA cloning is a molecular biology technique which is used for the creation of exact copies or clones of a particular gene or DNA Involves separating a specific gene or DNA segment from a larger chromosome, attaching it to a small carrier DNA. a target gene is inserted into a suitable cloning vector e.g. plasmid. restriction enzymes - “cut and paste” the DNA Ex. restriction endonuclease DNA ligase DNA Cloning The resultant hybrid DNA is called recombinant DNA, which is transferred to a proper host (bacteria, virus or yeast, plants, animals) and replicated to make multiple copy of the selected gene. When cloned under an appropriate expression vector, a gene can be expressed (I.e. transcribed and translated), at desired level to produce recombinant proteins. DNA Cloning This technology has made it possible to isolate, clone and produce DNA for all the genes in appropriate quantity so that they can be sequenced and characterized. Similarly, some of the genes which are expressed at very low level, can be cloned and desired amount of recombinant proteins can be produced This technique is the first stage of most of the genetic engineering experiments: production of DNA libraries PCR DNA sequencing DNA Cloning: Process 1. Isolating the donor DNA gene 2. Selecting specific vector 3. Incorporating the donor gene in the chosen vector The vector is split by the same RE enzyme that has been used for isolating of the DNA gene. The mixture of the vector and donor gene is combined and in the presence of the DNA ligase, a recombinant vector is formed 4. Transforming the recombinant vector to a host cell. 5. Isolating the recombinant cell The host cell is then allowed to grow in culture media. However, this might contain both, recombinant and non-recombinant cells. This mixture needs to be separated for which the marker gene is used. Restriction Endonucleases Restriction endonucleases are enzymes that recognize a specific DNA sequence, called a restriction site, and cleave the DNA within or adjacent to that site by cutting the phosphodiester bonds between the nucleotides. Example: EcoRI, recognizes the following sequence: 5’-GAATTC-3’ 3’-CTTAAG-5’ It cleaves the DNA between the G and A on each strand, leaving two pieces and producing 5′ overhangs of four nucleotides, as shown here: 5’-G-----3’ 5’-AATTC-3’ 3’-CTTAA-3’ 3’-----G- 5’ Restriction Endonucleases The termini produced by EcoRI, since they are complementary at their single-stranded overhangs, are said to be cohesive or “sticky.” Recognition sites for specific enzymes range in size from 4 to 13 base pairs. A number of restriction enzymes have been isolated from a variety of microbial sources Most restriction enzymes used in gene cloning, are palindromes; sequences that read in the 5’-to-3’ direction on one strand, are the same as those in the 5’-to-3’ direction on the opposite strand. 5’-GAATTC-3’ 3’-CTTAAG-5’ Overhanging, sticky ends on both sides  Restriction Endonucleases Some Examples: Recognition sites for BamHI: 5’-GGATCC-3’ specific enzymes range in size from 4 to 13 base 3’-CCTAGG-5’ Bacillus amyloliquefaciens H pairs HindIII: 5’-AAGCTT-3’ 3’-TTCGAA-5’ Haemaphilus influenzae Rd Most restriction enzymes KpnI: 5’-GGTACC-3’ used in gene cloning, are palindromes 3’-CCATGG-5’ Klebsiella pneumoniae Ex: 5’-GAATTC-3’ SacI: 5’-GAGCTC-3’ 3’-CTTAAG-5’ 3’-CTCGAG-5’ Streptomyces achromogenes Important steps in DNA Cloning I. Isolation of DNA (Gene of Interest) Fragments to be Cloned gene of our interest (GI) A gene of interest is a fragment of gene whose product (a protein, enzyme or a hormone) interests us. Example: gene encoding for the hormone insulin, drought and salt tolerance, herbicide Restriction Endonucleases enzyme – recognize restriction site and cleave DNA sequences II. Selecting a specific vector Cloning vectors - DNA molecules that transfers the foreign DNA molecule into the host cell. Types: Plasmids , bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), mammalian artificial chromosomes (MACs). The most widely used vectors are plasmids. Plasmids - are circular DNA pieces found within bacterial cells among the bacterial chromosomes. Ti-Plasmids -Agrobacterium tumefaciens -causing tumor formation in many plants It is not vitally important like chromosomal DNA of the host However the genes they carry may provide additional characteristics for the host cell and increase its competitiveness in the environment. Their replication is regulated in two ways: stringent or relaxed.  stringent control of replication - plasmids are dependent on the presence of initiation proteins synthesized by the host cell in order to start their own replication. tend to be low copy number relaxed control of replication- plasmids can initiate DNA replication independently of the host's initiation proteins only require the host's replication machinery for elongation and termination. tend to be high copy number III. Incorporating the donor gene in the chosen vector The vector is split by the same Restriction enzyme enzyme that has been used for isolating of the DNA gene. The mixture of the vector and donor gene is combined and in the presence of the DNA ligase, a recombinant vector/DNA is formed. recombinant DNA technology IV. Transforming the recombinant vector to a host cell: Transformation Genetic Transformation: It is the process information which carried out to transfer the recombinant codes for protein of interest. DNA molecule into the host cell. to get the multiple copies of GI To let GI get express and produce the Restriction Enzyme protein needed This is also called competency: ability of a cell to take up foreign DNA. 1. Chemical Method 2. Electroporation 3. Micro-injection: 4. Gene Gun Method 5. Disarmed Pathogen Vectors  V. Detection of Recombinant Clone and Isolating the recombinant cell The transformation process generates a mixed population of transformed and non-trans- formed host cells by reporter genes, colony hybridization technique, marker gene, PCR, antibiotic resistance, etc. A colony containing the right plasmid is grown in Example: A plasmid typically contains an antibiotic bulk and used for plasmid resistance gene or protein production Selection: bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic DNA Vectors Bacterial pathogens of plants either have the pathogenic genes in their genomes or carry plasmids enabling them infect the plants. Ex: Agrobacterium tumefaciens carry the Ti plasmid, causing tumor formation in many plants. Agrobacterium tumefaciens is a soil-borne bacterium Causes Crown gall disease Agrobacterium is a natural genetic engineer This bacterium has a natural ability to transfer the so- called T-DNA in its Ti plasmid into plant cells By replacing its T-DNA with desired gene/s, Agrobacterium is able to transfer the desired genes into the genome of target plants Agrobacterium Ti Plasmid The Ti-plasmid Ti-plasmid from different strains of A. turmefaciens has some features in common: One or more regions of T- DNA a vir-region an origin of replication a region enabling conjugative transfer genes for catabolism of opines Cloning Vectors A vector is a DNA molecule that is used to carry a foreign DNA into the host cell. It has the ability to self replicate and integrate into the host cell. Vectors can be a plasmid from the bacterium, a cell from the higher organism or DNA from a virus. The target DNA is inserted into the specific sites of the vector and ligated by DNA ligase. The vector is then transformed into the host cell for replication Features of Cloning Vectors 1. Origin of Replication (ori) A specific set/ sequence of nucleotides where replication initiates. For autonomous replication inside the host cell. Foreign DNA attached to ori also begins to replicate. 2. Cloning Site Point of entry or analysis for genetic engineering. Vector DNA at this site is digested and foreign DNA is inserted with the aid of restriction enzymes. Features of Cloning Vectors 3. Selectable Marker Gene that confers resistance to particular antibiotics or selective agent which, under normal conditions, is fatal for the host organism. Confers the host cell the property to survive and propagate in culture medium containing the particular antibiotics. 4. Marker or Reporter Gene Permits the screening of successful clones or recombinant cells. Cloning Vectors: General Characteristics Small in size Must have an origin of replication. Must also be compatible with the host organism. Possess a restriction site. Introduction of donor fragment must not intervene with the self-replicating property of the cloning vector. A selectable marker, possibly an antibiotic resistance gene, must be present to screen the recombinant cells. Capable of working under the prokaryotic as well as the eukaryotic system. Multiple cloning sites should be present Factors affecting the choice of the cloning vector DNA insert size Size of the vector Restriction Size Efficiency of cloning Major Types of Cloning Vectors A. Plasmids Plasmids were the first vectors to be used in gene cloning. They are naturally occurring and autonomously replicating extra-chromosomal double-stranded circular DNA molecules. They are present in bacteria, archaea, and eukaryotes. The size of plasmids ranges from 1.0 kb to 250 kb. DNA insert of up to 10 kb can be cloned in the plasmids. The plasmids have high copy number which is useful for production of greater yield of recombinant plasmid for subsequent experiments. Examples: pBR322, pUC18, F plasmid, Col plasmid. Major Types of Cloning Vectors A. Plasmids Advantages of using Plasmids as vectors: Easy to manipulate and isolate because of small size. More stable because of circular configuration. Replicate independent of the host. High copy number. Detection easy because of antibiotic-resistant genes. Disadvantages: p= plasmid Large fragments cannot be cloned. B= Bolivar (name of the scientist) R= Rodriguez (name of the scientist) Size range is only 0 to 10kb. 322= number of plasmid discovered in the same lab Standard methods of transformation are inefficient. Major Types of Cloning Vectors B. Bacteriophage Bacteriophages or phages are viruses which infect bacterial cells. It can be replaced with foreign DNA without disrupting its life cycle A maximum of 15 kb DNA can be packaged into the phage. If the vector DNA is too small, it cannot be packaged properly into the phage. Examples: Phage Lambda, M13 Phage, etc. Major Types of Cloning Vectors C. Bacterial Artificial Chromosomes (BAC) They are capable of accommodating large sequences without any risk of rearrangement. BACs are frequently used for studies of genetic or infectious disorders. High yield of DNA clones is obtained. They are present in low copy number. The eukaryotic DNA inserts with repetitive sequences are structurally unstable in BACs often resulting in deletion or rearrangement. Major Types of Cloning Vectors D. YAC Vectors YACs are yeast expression vectors. A very large DNA fragments whose sizes ranging from 100 kb to 3000 kb can be cloned using YACs. Mostly YACs are used for cloning very large DNA fragments and for the physical mapping of complex genomes. YACs have an advantage over BACs in expressing eukaryotic proteins that require post translational modifications. But, YACs are known to produce chimeric effects which make them less stable compared to BACs. Major Types of Cloning Vectors E. Human artificial chromosomes (HACs) Human artificial chromosomes (HACs) or mammalian artificial chromosomes (MACs) are still under development. HACs are microchromosomes that can act as a new chromosome in a population of human cells. HACs range in size from 6 to 10 Mb that carry new genes introduced by human researchers. HACs can be used as vectors in transfer of new genes, studying their expression and mammalian chromosomal function can also be elucidated using these microchrosomes in mammalian system. Types of Cloning 1. Natural Cloning - bacteria can produce identical offspring through asexual reproduction. Binary fission, Budding, fragmentation, and parthenogenesis 2. Artificial Cloning Involves a complex technique of collecting genes from one organism called the foreign DNA and inserting it into a vector. different types of artificial cloning include; Gene or DNA cloning Reproductive cloning Therapeutic cloning Types of artificial Cloning A. Gene Cloning Clones any DNA fragment, sequence, or entire gene sequence Extraction of gene or DNA fragment from the genome. Restriction enzymes are used for DNA fragmentation and extraction. Cloning vectors or the Polymerase Chain Reaction used to generate multiple copies of the DNA. This method is used in the field of healthcare and medicine. Biopharmaceuticals like insulin, growth hormones are created using this artificial method of cloning. Types of artificial Cloning B. Reproductive Cloning An artificial cloning method that produces identical copies of the entire organism. produce identical clones or copies of the parent through asexual reproduction. The most common method used for reproductive cloning is Somatic Cell Nuclear Transfer (SCNT). nucleus is obtained from the egg cell. Types of artificial Cloning The nucleus is replaced with somatic cells (body cells that are not sperm cells or egg cells) that are either grown in culture, obtained from an individual, or preserved as frozen tissue. The somatic cell is transferred through fusion or direct injection. The egg is stimulated for division. It is transferred into the uterus of an animal (surrogate mother) The cell develops inside the uterus and culminates with birth. The animal produced is the clone of the donor cell. Ex: Dolly the sheep. Animal Cloning Animals are cloned in one of two ways. A. Embryo twinning- split an embryo in half. Those two halves are then placed in a mother’s uterus. Each part of the embryo develops into a unique animal share the same genes. B. Somatic cell nuclear transfer The nucleus from egg cell is replaced with somatic cells (donor) Reproductive cloning Animals A. Embryo twinning Egg is collected from female. Egg is fertilized through in vitro fertilization Embryo is grown to 8-16 cells. Then the embryo is split into half The embryos are finally planted to surrogate mothers Each part of the embryo develops into a unique animal, and the two animals share the same genes. The method may be used to clone any mammalian embryos including humans. Reproductive cloning in Animals B. Somatic cell nuclear transfer (SCNT) The DNA from an animal’s somatic cell is transferred into an egg cell that has had its nucleus and DNA removed. The egg develops into an embryo that contains the same genes as the cell donor. Then the embryo is implanted into an adult female’s uterus to grow. In 1996, Scottish scientists cloned the first animal, a sheep they named Dolly. She was cloned using an udder cell taken from an adult sheep. Since then, scientists have cloned cows, cats, deer, horses, and rabbits. Types of artificial Cloning C. Therapeutic Cloning Production of embryonic stem cells for use in replacing or repairing damaged tissues or organ Achieved by transferring a diploid nucleus from a body cell into an egg whose nucleus ha s been removed and by harvesting cells from the resulting blastocyst. This procedure has proved to be a breakthrough in the field of therapy and medicine. Therapeutic cloning is used for producing skin, tissue, cartilage, and bone in accident and burn victims. Recombinant DNA Technology The technique of altering the genetic makeup of cells or organisms by deliberately inserting, removing, or altering individual genes Recombinant DNA - A DNA sequence produced artificially by joining pieces of DNA from different organisms Created by “cutting and pasting” different DNA fragments restriction enzymes DNA ligase Vector Host Organism Recombinant DNA: The Ingredients Foreign/Inserted gene restriction enzymes DNA ligase Vector Host Organism Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA Technology  Uses/Applications of Recombinant DNA Technology I. Medicine a. Production of vaccines Ex: Influenza vaccine, Hepatitis B vaccine, etc b. Production of human proteins and Hormones Ex: Insulin c. Treating infectious diseases Ex: Tuberculosis, Cancer, AIDS d. Diagnosis of diseases Ex: hepatitis virus, and HIV detection  Uses/Applications of Recombinant DNA Technology II. Agriculture genetically-modified organisms (GMO) Flavr Savr tomatoes, golden rice, Bt-cotton BGH (Bovine growth hormone) hormone allows cattle to gain weight more rapidly and also to produce meat with lower fat content and produce 10% more milk. Production of novel plants with high yielding, better qualities and pest resistant abilities. Development of Root Nodules in Cereal Crops (wheat, rice, maize, barley)- nitrogen fixation  Uses/Applications of Recombinant DNA Technology III. Industry Fermentation: improve strains of microbes which play important role in fermentation. Production of chemical compounds and enzymes which are commercially and industrially important.  Uses/Applications of Recombinant DNA Technology IV. Biotechnology: Gene therapy: helps scientists to replace the defective genes in the genome of the organism. Creating transgenic animal: Example: Transgenic cattle, transgenic chicken, Dolion, Lozebra. Production of monoclonal antibodies Bioremediation QUESTIONS??