GENETIC ENGINEERING

  • Genetic Engineering 

    • Process in which pieces of DNA are transferred from one organism to another

      (ex. Gene taken from firefly to make tobacco plant glow in the dark)

    • Directly changing,adding, or subtracting, of an organism's genes.

  • DNA Recombination

    • Process of modifying the genes of organisms for practical purposes

    • Done when a piece of DNA is combined with another DNA from another source

  • Recombinant DNA

    • The product of recombination 

    • Organisms get to have traits not normally found in the their species: known as GMO “Genetically Modified Organisms”

History of Recombinant DNA Technology

  • (Late 1973) 

    • Stewart Lim and Werner Arber discovered restrictions enzymes (endonuclease) in E.Coli

    • Every virus has a specific restriction enzyme

    • Endonuclease : Cuts DNA at a specific site where there are adjacent base sequences to create sticky ends on the cut DNA site allowing DNA fragments to join together.

  • (1973)

    • Herbert Boyer and Stanley Cohen formed a successful experiment

      • First one : Involves the recombination of plasmids in the DNA of E.Coli

      • Next : Involves recombination of plasmid DNA with frog DNA (resulted in the production of an extra protein)

5 Steps in Genetic Engineering

  1. Isolation of the Genes

  2. Insertion of those genes into a transfer vector (a virus/plasmid used as a conduit)

  3. Transfer of the vector to the organism to be modified

  4. Transformation of the organisms cells

  5. Separation of the genetically modified organisms (GMO) from organisms that have not been successfully modified

Modification of Traits may Involve:

  1. Introduction of new traits into an organism

  2. Enhancement of a present trait by increasing the expression of the desired gene

  3. Enhancement of a present trait by disrupting the inhibition of the desired genes’ expression.

Modification of Traits may Involve:

  1. Cutting/Cleavage of DNA by restriction enzymes (RE’s)

  2. Selection of an appropriate vector or vehicle which would propagate the recombinant DNA

    (ex. Circular plasmid in bacteria with a foreign gene of interest)

  3. Ligation (join together) of the gene of interest with the vector

  4. Transfer of the recombinant plasmid into a host cell (that would carry out replication to make copies of the recombinant plasmid)

  5. Selection process to screen which cells actually contain the gene of interest

  6. Sequencing of the gene to find out the primary structure of the protein

DNA Sequencing

  • The process of working out the order of the building blocks/bases in a strand of DNA

  • Before sequencing it must be cut into smaller pieces inserted into plasmid DNA and put into bacterial cells (to make multiple copies of it as bacteria multiplies)

  • DNA is  isolated from the bacteria and transferred to a plate where sequencing will take place

  • Ingredients include: DNA primer, Free DNA bases (ACGT), Modified DNA bases (“terminator bases”), DNA polymerase.

  • To start sequencing everything is heated to 96c to separate the DNA into two single strands

  • Then lower the temperature to 50c so the DNA primers can bind to the plasmid DNA

  • Increase temperature to 60c so the DNA Polymerase binds to the DNA primer

  • DNA Polymerase then makes a new strand of DNA by adding unlabeled DNA bases to the target DNA 

  • It continues to add DNA bases until a Terminator base is added which makes the DNA polymerase stop and fall away from the strand

  • Heat again to 90c to separate the new strand from the original

  • To read the sequence of DNA the process of Electrophoresis is used.

  • Towards the end of the capillary tube a laser lights up the terminator bases and is recorded via camera

  • Every terminator base is a different color

    ( A = Green, C = Blue, G = Yellow, T = Red )

  • The sequencing machine records the colors to get the letters of the sequence of the DNA

Technologies and tools used in Recombinant DNA Technology

  1. Gel Electrophoresis

    • A method used to separate DNA fragments based on their size.

    • The movement of charged molecules occuring in an electrical field that occurs on a Gel Medium.

    • Requirements : Casting Tray (to contain the gel), Gel, Comb, Electric supply, DNA sample.

    • Comb is used to form a well structure to load the DNA mixture. Placed near the negative terminal of the gel. 

    • Coloured loading dye is used on the DNA samples so it can be tracked

    • Buffer solution is used for better conductivity of electricity.

    • Loading dye must reach the Anode faster than the DNA to indicate when the power supply will be turned off

    • A mixture of DNA fragments is placed at one end of a porous gel (Agarose gel obtained from seaweed), and an electric voltage is applied to the gel. Using a capillary tube.

    • The negatively charged DNA molecules (due to phosphate groups) move toward the positive end of the gel. (Smaller fragments move faster)

    • As a result the fragments are automatically arranged from shortest to longest

    • Ethidium Bromide (binds to DNA molecules) is added to the Agarose Gel so we can observe the DNA molecules. The DNA molecules will become bright orange bands under UV light

    • DNA Ladder : Standard chart used to measure the length of DNA fragments

    • Elution : Extraction of desired DNA fragment from Agarose Gel. Used for downstreaming processing.

    • Important for characterizing DNA fragments, fingerprinting, comparing the genome of different organisms, and locating and identifying one particular gene out of the millions of genes in an individual’s genome.

  2. DNA Splicing

    • A method used to provide the identity and order of the nucleotides in a DNA strand. Small and single-stranded pieces of DNA are placed in test tubes with an enzyme that can make a complementary DNA strand by using the original DNA strand as a template

    • A supply of the four nucleotide bases found in DNA are then added, along with a small amount of one of the bases that has been labeled with fluorescent dyes

  3. Polymerase Chain Reactions

    • Its goal is to amplify specific DNA sequences

    • Important in detecting diseases/infectious agents

    • To make copies of a piece of DNA

    • DNA is heated to separate its two strands then cooled to allow the primers to bind to the single-stranded DNA.

    • Primers : short DNA strands that provide a place for the DNA polymerase to start working. As the polymerase starts working, new strands of the separated DNA are formed. Continuous heating and cooling allows further separation of DNA and formation of new DNA strands.

    3 main steps

    1. Denaturation

      • Separating the two strands of a starting DNA sample (template DNA)

      • By heating it up to 95c can last 10 sec but mostly 30-60 seconds

    2. Annealing

      • Specify the region of DNA to be amplified using Primers (Forward and Reverse) M 20 bases long

      • Forward primer will match the sequence of DNA at the beginning

      • Reverse primer will match the sequence of DNA at the end on the other strand

      • Primers will anneal to the complementary regions on the template DNA when temperature cools to 50-65c (temp. Depends on the strand) 

      • Usually anneals between 5-30 seconds

      • Scientist add more primers to increase the chance of template DNA sticking to primers instead of each other 

    3. Extension

      • Heat the reaction to approximately 72c to extend the annealed areas using DNA polymerase that will create a new strand of DNA

      • Lasts between 10 seconds to a few minutes

      • TAQ Polymerase : Special polymerase that was found in thermophilic bacteria

      • Thermal Cycler : machine used for the entire process

    Ingredients:

    1. Template DNA

    2. DNA Primers

    3. Taq Polymerase

    4. Nucleotides (building blocks of DNA)

    5. Buffer (Salts and Ions that allows polymerase to function)

Process used in Recombinant DNA Technology

  1. Transformation using Vector

    • Recombinant DNA may be created through transformation with the help of a vector such as bacteria cells

    • Restriction endonuclease is used to cut the piece of the door DNA

    • Sticky ends : Areas in the DNA where the bases are ready to pair. Restriction enzymes cut the DNA only at specific nucleotides sequence. (like a key and lock)

    • An enzyme known as DNA Ligase is used to insert the donor DNA into the vector. It seals the sticky ends by joining the phosphate and the sugar bonds in the DNA. The inserted DNA also contains a genetic marker for identification.

    • The recombinant DNA is then inserted into a bacterial cell, such as E.Coli

    Plasmid

    • A circular piece of DNA in a bacterium that replicate independently from the host DNA

    • Small, Stable, and Easy to Manipulate

    • Found in the 1940’s and had many names (i.e Bioblasts, Plasmagenes, Episome, and Cytogenes)

    • (1952) nobel laureate Joshua Letterberg coined “Plasmid” (Cytoplasm + Id)

    • Contains genes that give its host an ability that it didn't have before (ex. Antibiotic resistance)

    • Constructs/Vectors: name of plasmids created in the lab

      Parts of a Plasmid

      1. Origin of Replication “ori”

        • Tells the plasmid where to begin replication

      2. Antibiotic Resistance Gene

        • Allows scientist to separate cells that have plasmids from those that don't

      3. Restriction Sites

        • Site wherein genes can be removed and replaced

        • Located in a multiple cloning site

      4. Promoter Site

        • Acts as a green light that allows gene transcription

        • RNA polymerase binds to the promoter moving along the strand, as it moves along the strand it creates a new strand of mRNA expressing the gene

      Why are plasmids used?

      • Reason 1: Contains a gene sequence that serves as a bacteria origin or replication. This is where the foreign DNA can be inserted into the bacteria cell.

      • Reason 2: Contains a genetic marker, which makes it possible to distinguish bacteria that carry plasmids-containing foreign DNA. 

      (Some of these markers code for antibiotic resistance)

  2. Vectorless Gene Transfer

    • Similar to transformation but doesn’t include vectors

    Types of Vectorless Transfer

    1. Electroporation

      • A physical transfection method to artificially introduce nucleic acid into cells

      • Nucleic acids and host cell are placed in a conductive solution

      • An electric circle is enclosed around solution

      • Temporary holes are formed in the plasma membrane of host cell (phospholipid bilayer) by applying a significant amount of electricity in the culture medium.

      • The nucleic acids are forced into the solution due to the difference between the negative charge of the solution and the positive charge of the host cell

      • The cell then regenerates is phospholipid bilayer

        Pros and Cons

        • Easy, Rapid, Stable, Applicable for all cell types

        • High Cell mortality

    2. Protoplast Fusion

      • Cells are treated with chemicals to initiate recombination. In this process, bacteria cell walls are digested, turning the cells into protoplasts

    3. Microinjection

      • The host cell is immobilized by applying a mild suction with blunt pipette. The foreign gene is then injected with a microinjection needle, thus creating recombinant DNA

    4. Using a Particle Gun

      • The host cell is bombarded with tungsten particles coated with foreign DNA. This process is used in the field of agriculture

  3. Transduction

    • The process wherein genetically engineered bacteriophages (viruses that parasitize bacteria) are introduced into the cell to create the desired recombinant DNA

    • A phage enzyme is produced when inserted into the host cell that causes the cells DNA to break down into small fragments.

    • The Phage DNA is then replicated and phage code proteins are produced (bacterial DNA may then be surrounded as well)

    • Bacterial DNA is then transported to a new cell where it can be integrated thereby transferring genes to the recipient.

Application of Recombinant DNA

- Genetic Advances in Agriculture - - - - -

  • Transgenic plants

    • Plants that contain genes from other organisms 

    • Important part in the field of Agriculture

    • Using recombinant DNA technology, plants can be grown with genes responsible for producing natural insecticides

Some bacterium used in recombinant DNA technology:

  1. Pseudomonas Syringae

    • Recombinant variant of this bacterium called “ice-minus bacterium”

    • Lacks the gene responsible for ice formation which prevents frost crystals from forming on plants

  2. Pseudomonas Flourescens

    • A nonpathogenic bacterium that has the ability to produce proteins rapidly

    • Advantageous in developing biotherapeutics and vaccines

  3. Agrobacterium Tumefaciens

    • Has a tumor-inducing (Ti) plasmid that causes crown gall disease in plants

    • The Ti plasmid in this can be replaced with a recombinant plasmid allowing the modified bacterium to introduce beneficial genes to plants

Improvements in Plants

  • Enhanced potential for more vigorous growth and increases yields (hybrid vigor-heterosis)

  • Increase resistance to natural predators and pests, including insects, disease-causing microorganisms

  • Production of hybrids with a combination of superior traits derived from two different strains or species

    (ex. Pluot - plum+apricot, Tayberry - blackberry+raspberry)

Selection of Genetic Variation with desirable qualities

  • Increased protein value and content of limiting amino acids

  • Smaller plant size, reducing vulnerability to adverse weather conditions

    Examples: 

    • Herbicide,Insect, and Virus Resistance

    • Nutritional Enhancement

    • Altered Oil Content

    • Delayed Ripening

Improvements in Animals

  • Development of superior breeds in livestock

    Examples:

    • Chickens : Grow faster, Produce higher quality meat, Lay more eggs

    • Pigs and Cows : Artificial insemination ( Sperm from a male with superior genetic traits used to fertilize thousands of females )

- Genetic Advances in Medicine - - - - -

Advances in Cancer Research

  • Effective early detection and more effective approaches to treatment

Genetic Counseling

  • Provides couple with objective information on which they can base rational decisions about child-bearing

Immunogenetics

  • Compatible blood transfusions and organ transplants

Recombinant DNA Techniques

  • Gene Therapy

    • Applies genetic engineering to the treatment of human diseases

    • Treats genetic disorders by inserting normal copies of genes into cells of afflicted individuals

  • DNA biotechnology

    • Manipulating and cloning a variety of genes

      (ex. Insulin, Blood clotting factors, Growth hormones)

  • Human Genome Project

    • The entire genetic complement (genome) of several species is being sequenced.