Genetic Engineering

genetic engineering

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

• 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

“GMO” stands for?

stewart lim and werner arber

discovered restriction enzymes (endonuclease) in E.Coli.

(Late 1973)

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.

herbert boyer and stanley cohen (1973)

formed successful experiments:

• 1st: the recombination of plasmids in the DNA of E.Coli

• 2nd: 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 in Genes may Involve

1. Introduction

2. Enhancement by increasing expression

3. Enhancement by disrupting expression

Process of Modifying Genes (Steps)

1. Cutting/Cleavage

2. Selection of an appropriate vector

3. Ligation

4. Transfer

5. Selection process

6. Sequencing

DNA Sequencing

Process of working out the order of the building blocks/bases in a strand of DNA

Ingredients in DNA Sequencing

DNA Primer, Free DNA bases (ACGT), Modified DNA bases “Terminator Bases”, DNA Polymerase

Process of DNA Sequencing

• 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

Corresponding Colors of Terminator Bases

A is Green, C is Blue, G is Yellow, T is Red.

1. gel electrophoresis

2. dna splicing

3. polymerase chain reactions

what are the 3 common technologies and tools used in recombinant dna technology?

gel electrophoresis

• is 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.

• smaller fragments move faster, larger fragments move slower. Thus they are arranged from shortest to longest

• a mixture of dna fragments is places at one end of a porous gel, and an electric voltage is applied to the gel.

• this is important for characterizing dna fragments, fingerprinting, comparing the genome of different organisms, etc.

Requirements to Conduct Electrophoresis

Casting Tray, Gel, Comb, Electric supply, DNA Sample

Comb in Electrophoresis

Used to form a well structure to load the DNA mixture

Coloured Loading Dye

Used on the DNA samples so it can be tracked

Buffer solution

Used for better conductivity of electricity

agarose gel

it is a gel used in electrophoresis that is obtained from seaweed.

Phosphate Groups

Reason why DNA molecules are negatively charged

Ethidium Bromide

• 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 fragments from Agarose Gel

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.

polymerase chain reactions (PCR)

• 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.

1. denaturation

2. annealing

3. extension

what are the 3 main steps of polymerase chain reactions (PCR)?

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

Annealing

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

• 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

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

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.

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 in PCR

Template DNA, DNA Primers, TAQ Polymerase, Nucleotides, Buffer

1. transformation using vector

2. vectorless gene transfer

3. transduction

what are the 3 process used in recombinant dna technology?

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)

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

(1952) Joshua Letterberg

A nobel laureate that coined the term Plasmid (Cytoplasm+Id)

Constructs/Vectors

Name of plasmids created in the lab

1. origin of replication (ori)

2. antibiotic resistance gene

3. restriction sites

4. promoter site

what are the 4 parts of a plasmid?

Origin of Replication “Ori”

Tells the plasmid where to begin replication

Antibiotic Resistance Gene

Allows scientists to separate cells that have plasmids from those that do not

Restriction Sites

Site wherein genes can be removed and replaced

Located in a multiple cloning site

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.

Why are Plasmids used? Reason 2.

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

vectorless gene transfer

• similar to transformation but doesn’t include vectors

1. electroporation

2. protoplast fusion

3. microinjection

4. using a particle gun

what are the 4 types of vectorless gene therapy

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

protoplast fusion

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

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.

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.

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 cell’s 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.

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

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

Pseudomonas Flourescens

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

• Advantageous in developing biotherapeutics and vaccines

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

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 )

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

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.