DNA Manipulation & Genetic Engineering

• DNA manipulation, or genetic engineering, uses biotechnology to directly alter an organism's genome.
• Alteration is achieved by introducing new DNA or editing existing DNA.
• Aims to produce organisms with improved or novel traits.
• Organisms that have had their DNA altered are called genetically modified organisms (GMOs).

DNA manipulation techniques

• Enzymes such as polymerase, ligase, and endonucleases are used to manipulate DNA.
• CRISPR-Cas9 functions in bacteria and is used to edit an organism's genome.
• Polymerase chain reaction amplifies DNA.
• Gel electrophoresis sorts DNA fragments for DNA profiling.
• Recombinant plasmids are used as vectors to transform bacterial cells, shown by the production of insulin.
• Genetically modified and transgenic organisms are used in agriculture to increase crop productivity and disease resistance.

Tool Kit For DNA Manipulation

• Restriction enzymes cut DNA into precise fragments.
• Electrophoresis is for separating fragments by size.
• Probes used to locate particular DNA fragments.
• Ligase enzyme used to Join DNA fragments.
• DNA synthesizer & reverse transcriptase make specific DNA fragments.
• Polymerase chain reaction (PCR) used to amplify DNA.

Cutting Tool: Restriction Enzymes

• Restriction enzymes are essential for genetic engineering because purified forms of naturally occurring bacterial enzymes are molecular scalpels, allowing genetic engineers to cut DNA in a controlled manner.
• Used to cut DNA molecules at precise sequences of 4 to 8 base pairs called recognition sites.
• Using a tool kit of over 400 restriction enzymes is how genetic engineers isolate, sequence DNA, and manipulate individual genes from any type of organism.
• Restriction enzymes are named according to the organism and the order in which they were identified.
• Restriction enzymes can be made by biotech laboratories, providing researchers with a pure sample.

Restriction Enzymes: Recognition

• For cutting, DNA is dissolved and a particular restriction enzyme is added.
• DNA molecules will be cut into two or more fragments if there is a recognition site present.
• Fragment lengths depend on the relative positions of the recognition sites.
• In some cases, two different restriction enzymes may be added to a DNA sample.
• Blunt ends are formed when the restriction enzyme cuts both DNA strands directly opposite.
• When cuts are not directly opposite, overhanging ends of two or three bases result; these cuts are called ‘sticky ends'
• Cut location determines whether the DNA fragments will have blunt or sticky ends.

Sticky Ends vs. Blunt Ends

• Restriction enzymes cut DNA and leave an overhang that are called "sticky ends".
• The sticky ends may only be joined to other sticky ends with a complementary base sequence.
• Cutting DNA to produce blunt ends DNA can be joined to any other blunt end fragment.
• The joining tends to be non-specific because there are no sticky ends to act as recognition sites.

Joining Tool: Ligation

• Ligation is a process through which DNA fragments produced using restriction enzymes may be reassembled or joined.
• DNA ligase assist with DNA fragments joining.
• DNA of different origins are joined, creating recombinant DNA because it has been recombined from different sources.
• Recombinant DNA plasmid creation involves ligation.
• DNA ligase is responsible for catalyzing the joining of DNA fragments at the sugar-phosphate backbone.

Annealing

• "Sticky ends" come together and join by base pairing, which is called annealing.
• This allows DNA fragments from a different source, perhaps a plasmid, to be joined to the DNA fragment.
• The joined fragments will usually form either a linear or circular molecule.
• Sticky ends must have been cut by the same restriction enzyme, so that base pairs in overhang are complementary.

CRISPR-Cas9

• CRISPR-Cas9 technology is an advanced gene manipulation tool with more precision than currently used, and promises greater benefits in the future.

• The combination of CRISPR with the enzyme (endonuclease) Cas9, can cut a specific region of DNA

• CRISPR gene-editing technique requires molecular 'scissors' to cut the target DNA and a 'guide' to direct the 'scissors' to where the cuts will be made in the DNA.

• Two tools are 'scissors' that are a Cas9 nuclease, a bacterial enzyme, and a guide that's a segment of designed single strand of RNA.

• The Cas9 nuclease can unwind double-stranded DNA and cut both strands of double-stranded DNA at a precise location.

• The guide RNA is designed to include a 20-base sequence that is complementary to part of the target DNA.

• CRISPR and Cas9 genes form an adaptive immune system helping bacteria to remember specific viral sequences and fight back upon reinfection.

• Humans produce use antibodies to help defend against a threat.

Technique: CRISPR

• CRISPR-Cas9 technology is an efficient, easy-to-use, and inexpensive tool for precise gene editing.
• Applied to every eukaryotic species, humans included.
• Key components of CRISPR-Cas9 technology are a guide RNA and an endonuclease enzyme, Cas9.
• Earlier gene therapy techniques added DNA segments to genomes but at unpredictable locations.
• CRISPR-Cas9 technology enables precise genomic editing edits, modifies, disables, and deletes DNA from the genome.

How CRISPR Repairs DNA

• The cut DNA can be repaired using one of the following methods:
  • Gene knock in (“gene editing") inserts a new DNA sequence into the DNA break. For example allows a faulty gene sequence to be replaced.
  • Gene knock out “gene silencing" normal processes mend broken DNA, errors result and resulting frame-shift mutation changes the way nucleotide sequence is read, either disabling gene function or producing a STOP signal.

Technique: Amplifying DNA – Polymerase Chain Reaction (PCR)

• PCR amplifies the amount of DNA by using DNA replication with Taq polymerase to make exact copies of the original DNA.
• Through repeating the cycle, sufficient DNA can be generated for testing from only trace amounts.

Polymerase Chain Reaction

• PCR was developed in 1984.
• The Taq DNA polymerase enzyme comes from Thermus aquaticus, the bacterial species that inhabits hot springs.
• Thermus aquaticus lives at high temperatures, so its enzymes tolerate high temperatures and aren't denatured, which then makes PCR possible.

Steps in the PCR Process

• PCR involves the following steps 1-3 each cycle:
  • Separate strands by heating the target DNA at 98°C for 5 minutes.
  • Add primers that are short nucleotide strands (A, T, G, C) to provide a starting sequence for DNA replication.
  • Incubate by cooling to 60°C for few minutes which allow primers attach to single-stranded DNA and for DNA polymerase to synthesize.

Review: The Process of PCR

• To calculate the number of DNA fragments of PCR use 2n where n is the cycle number. Steps are:
  • Obtain a DNA sample called target DNA.
  • Denature DNA strands (separate DNA) sample by heating for 5 minutes at 98°C.
• Anneal primers to the single strands of DNA.
• Cool the sample to 60°C.
• A thermally stable DNA polymerase enzyme binds to exposed DNA strand and free nucleotides which synthesises a complementary strand. After one cycle, there are two copies of the original sample.

Technique: Sorting DNA Fragments using Gel Electrophoresis

• When DNA is cut the fragments are sorted using gel electrophoresis.
• The DNA mixture is run through an agarose gel which is bathed in an electrolyte and exposed to an electric field, causing it to be pulled through the gel to the positive terminal because DNA has a negative charge.
• Smaller fragments travel faster and move further in time because they encounter less resistance from the gel.
• Gel electrophoresis separates molecules by size, electrical charge, physical properties. DNA is prepared by cutting into smaller pieces with restriction digest.
• To carry out electrophoresis, the DNA samples are placed in wells and covered with a buffer solution that gradually dissolves them into solution

Running the Gel

• An electric field is applied to the solution which causes molecules to move depending on the charge on the molecule.
• DNA is negatively charged because the phosphates have a negative charge.
• Molecules of different sizes become separated because of their molecular weights.
• Bands can be visualised by dyes or radio-labelled probes.

Visualizing and Interpreting Gel results

• Fragments in the gel are visualized using DNA binding dye, such as ethidium bromide which under UV light appears orange.
• To identify the bands of DNA a dye that can be seen under UV light is often added to the DNA.
• To estimate the sizes of unknown DNA fragments, markers or standards of known lengths of DNA are often run in the same gel as the sample.

GMOs: Genetically Modified vs Transgenic

• A genetically modified organisms has its own genome altered or contains DNA from a donor in same species
• A transgenically modified organism contains DNA from a donor of a different species

What is The Difference Between Genetically Modified and Transgenic Organisms

• GMOs are artificially manipulated through genetic engineering to express new traits.
• Agricultural crops are commonly modified to improve yield, growth rates, and nutritional value.
• Genetically modified animals may be used to grow human transplant tissues and organ through xenotransplantation.
• Several microorganisms fuel and biodegraders, and pharmaceutical companies are exploring the possibilities of introducing vaccines through foods.

What Is Transgenesis

• Transgenesis inserts a gene from one species into a different species, so its protein product is expressed in the second species.
• Direct modification of a genome can introduce a novel trait into an organism.
• Organisms that have undergone transgenesis are called transgenic organisms.

Genetically Modified Organisms

• Genetically modified organisms are widely used in agriculture.
• Transgenesis can be used for extending the shelf life of fresh produce, engineering plants in the to have pest and herbicide resitance.
• Transgenesis improves crops through drought tolerance and higher protein levels, as well as livestock through desirable qualities such as meat or wool production.

Agricultural Applications

• Genetic engineering is used to produce modified cotton, corn, and potato varieties that produce the Bt toxin.
• The gall forming bacterium Agrobacterium tumefaciens commonly transfers the Bt gene into plants through recombinant plasmid.
• Bacillus thuringiensis is used to make cotton plants that are genetically engineered to include cry1Ac.
• cry1AC gene codes for Bt protein that acts as an insecticide for Cottonboll worm working by rupturing the gut and killing the worm.
• The PSY gene for more beta-carotene was isolated from daffodil, and the CRT1 gene isolated from the bacteria Erwinia uredovora helped produce golden rice

Benefits of Genetic Modification

• Potential benefits include: increase in crop yields, decrease in pesticides, production of drought or salt tolerant crops, manufacturing and health industries, etc.

Risks of Genetic Modification

• Potential risks linked to:
  • Possible (uncontrollable) spread of transgenes into other plants or animals.
  • Concerns that the release of GMOS into the environment may be irreversible.
  • Poor animal welfare and ethical issues as well as GM animal reduced life span.
  • GMOs may cause the growth of pest, insect, or microbial resistance to traditional control methods.
  • The possibility of creating a monopoly and dependency of developing countries on companies who control the world's seed supply.