Genetic Modification & Biotechnology (3.5)IB Diploma Biology

Essential Idea: Modern understandings of genetics and

biochemistry allow biologists to modify and manipulate the

traits of organisms

3.5.1 Gel electrophoresis is used to separate proteins or fragments of DNA according to size and charge.

Gel Electrophoresis:• Samples of either DNA or protein are

inserted into wells in a gel

• Gel is placed in a conducting fluid and a current is passed through it

• Molecules move through gel based on their charge (ex. DNA molecules move to positive electrode since they’re negatively-charged)

• Smaller fragments / molecules move further through the pores in the gel, while larger pieces don’t travel as far

3.5.2 PCR can be used to amplify small amounts of DNA.

The Polymerase Chain Reaction (PCR)

Synthetic method of amplifying specific sequences of DNA. Useful when only a small amount of DNA is available for testing e.g. crime scene samples of blood, semen, hair, etc.

http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

• Processes artificially recreates DNA replication

• Taq DNA Polymerase is used for PCR

• Comes from heat-resistant bacterium, Thermus aquaticus, that lives in hot springs…

• Can resist denaturation at high temperatures required to separate DNA strands in PCR

• Copies up to 1000 nucleotides / minute

3.5.2 PCR can be used to amplify small amounts of DNA.

The PCR Process:PCR occurs in a thermal cycler and involves 3 steps:

1. Denaturation: DNA sample is heated to 95⁰C to break hydrogen bonds and separate it into two strands

2. Annealing: DNA sample is cooled to 54 ⁰C, allowing primers attach to opposite ends of the target sequence

3. Elongation: A heat-tolerant DNA polymerase (Taq) copies the strands

• One cycle of PCR yields two identical copies of the DNA sequence

• A standard reaction of 30 cycles would yield 1,073,741,826 copies of DNA (230)

3.5.3 DNA profiling involves comparison of DNA.

http://learn.genetics.utah.edu/content/labs/gel/

http://www.dnalc.org/resources/animations/gelelectrophoresis.html

3.5.3 DNA profiling involves comparison of DNA.

AHL: 7.1.8 Explain how tandem repeats are used in DNA profiling.

• Variable Number Tandem Repeats (VNTRs) are short base sequences that show variation between individuals in terms of the number of repeats

• These are examples of Highly Repetitive Sequences – useful for DNA profiling due to uniqueness

3.5.9 Use of DNA profiling in paternity and forensic investigations.

Forensic Investigations:

• Straightforward – just look for a full match between the DNA sample and the potential suspects

Paternity Investigations:

• More complicated – since offspring inherits a mix of DNA from parents, the child will show bands unique to each parent

• Each band in the child’s profile must match to a either a band in the mother’s profile OR a band in the father’s profile (usually ~50-50 split)

3.5.14 Analysis of examples of DNA profiles.

3.5.14 Analysis of examples of DNA profiles.

3.5.14 Analysis of examples of DNA profiles.

3.5.14 Analysis of examples of DNA profiles.

3.5.14 Analysis of examples of DNA profiles.

3.5.14 Analysis of examples of DNA profiles.

3.5.14 Analysis of examples of DNA profiles.

3.5.14 Analysis of examples of DNA profiles.

3.5.14 Analysis of examples of DNA profiles.

3.5.14 Analysis of examples of DNA profiles.

Since the genetic code is universal, genes can be transferred between species and still allow for the same polypeptide to be translated.

Gene transfer can be used to give organisms new characteristics usually only found in other species.

Left, a tobacco plant that has been modified by the addition of a glowing gene (for the enzyme Luciferase) naturally found in fireflies.

These organisms are known as GMOs or Transgenic organisms

3.5.4 Genetic modification is carried out by gene transfer between species.

3.5.4 Genetic modification is carried out by gene transfer between species.

3.5.4 Genetic modification is carried out by gene transfer between species.

3.5.10 Gene transfer to bacteria with plasmids using restriction endonucleases (enzymes) and DNA ligase

Transferring genes into bacteria, such as E. coli requires plasmids, restriction endonucleases, and DNA ligase enzymes

• An mRNA transcript encoding the desired protein is obtained from the eukaryotic cell and then made into cDNA (complimentary DNA using reverse transcriptase)• Not only is mRNA easier to extract (since

eukaryotic DNA is bound up with histones in the nucleus), but this also ensures the inserted gene will already be spliced and have no introns (which bacteria do not have)

• A bacterial plasmid and the eukaryotic cDNA are both cut with the same restriction enzymes

• DNA Ligase is used to seal the eukaryotic sequence into the bacterial plasmid

3.5.11 Assessment of the potential risks and benefits associated with genetic modification of crops.

GMO Description

Golden Rice Rice modified with daffodil genes to have more beta-carotene, which body converts to Vitamin A

Salt-resistant Tomatoes

Tomatoes modified to grow well in saline soils

Bt Corn Corn modified with a bacterial insecticide gene so that it produces insect toxins within its cells

Factor IX Sheep

Sheep modified with human clotting factor IX gene so that they produce clotting factor in their milk for hemophiliacs

Round Up Ready Soy

Soybeans modified with a herbicide resistance gene so farmers can spray fields and kill weeds, not soybean plants

Rainbow Papaya

Papaya modified with viral genes that make it immune to the Papaya Ringspot Virus

3.5.11 Assessment of the potential risks and benefits associated with genetic modification of crops.

Benefits of GMOsEnvironmental Health Agricultural

Pest-resistant crops mean less chemical insecticides are used

Less need to plow and spray crops also save fuel, reduced carbon footprint

Improved shelf-life means less wasted / spoiled food in stores

Nutritional value of foods can be improved by enhancing vitamins

Crops can be produced that lack natural allergens or toxins

GM crops can be engineered to produce cheap, edible vaccines

GM bacteria produce cheap medical compounds such as insulin and clotting factor

Crops can be made to be drought, cold, and salinity-resistant, expanding range for farming and increasing crop yields

Herbicide resistant GM crops allow for easy killing of weeds that sap nutrients from crop plants

Crop varieties can be produced that are resistant to viruses

3.5.11 Assessment of the potential risks and benefits associated with genetic modification of crops.

Risks of GMOsEnvironmental Health AgriculturalToxins in pest-resistant GMOs could negatively impact non-target organisms and harm ecosystems

Cross-species pollination could spread herbicide resistance genes and create ‘super-weeds’

Biodiversity could be negatively affected by destruction of pests, weeds, and even competing plants

Proteins transcribed and translated from transferred genes could cause allergic reactions in humans or other animals – currently GM foods are not necessarily labeled

Antibiotic resistance genes used as markers during gene transfer could spread to pathogenic bacteria

Transferred genes could mutate and cause unexpected risks

GMOs with pest toxins could increase evolution of resistance in certain pest populations

Big biotech companies hold monopolistic legal rights (patents) over GM seeds and farmers must pay large sums for seeds each year. They are not permitted to save and re-sow seeds, so strains are not able to adapt to local conditions.

3.5.15 Analysis of data on risks to monarch butterflies of Bt crops

Previously, farmers would protect crops from pests by spraying with chemical pesticides. Today, may crops are genetically-modified with a gene from the bacterium Bacillus thuringiensis (Bt) that produces a protein toxic to insects

The Bt toxin kills targeted pest like the corn-borer worm, but also kills non-target insects

Monarch butterflies feed on milkweed which often grow near Bt crops. When pollen from the Bt crops ends up dusting milkweed plants, butterflies consume the toxin and die

In the graph above, blue represents plants not

dusted with any pollen, yellow represents non-GM pollen dusting, and

red represents Bt pollen dusted milkweed plants

3.5.5 Clones are groups of genetically identical organisms, derived from a single original parent cell

Clone:A group of genetically-identical organisms derived from a single original parent cell

Organisms that reproduce asexually always produce genetically-identical offspring (clones)

Clones are rarer in sexually-reproducing organisms (i.e. monozygotic twins)

A clone can be very large, such as in the case of commercially grown potatoes, but it can always be traced back to an original parent cell.

3.5.6 Many plant species and some animal species have natural methods of cloning

Many plants can naturally produce clones (term derives from Greek word for twig)

Examples:• A single garlic bulb will clone itself to

form many identical bulbs in a growing season

• Strawberry plants grow stems with plantlets that can become independent parent plants

• Hydra create clones by budding

3.5.13 Design an experiment to assess one factor affecting the rooting of stem cuttings

A stem cutting is a short length of plant stem that can be used to clone a plant. If roots develop from the cut stem, it can become a new, independent parent plant

Nodes are parts of stem where leaves attach. Cuttings are made below nodes. Normally takes several weeks for ‘rooting’ (new root growth)

Possible factors to study:• Cutting above or below node• Length of cutting• Whether end is left in air or

compost / water• How many leaves are left on• Use of hormone root powder• Type of compost• Temperature

3.5.7 Animals can be cloned at the embryo stage by breaking up the embryo into more than one group of cells

In early stages of embryo development when cell are still pluripotent, an embryo can be fragmented or split to create animal clones. This is how monozygotic twins can occur in humans.

Animal embryos created through in vitro fertilization can be artificially fragmented and then transplanted into surrogate mothers. This is most effectively done when the embryo is at the 8-cell stage

Less interest in this cloning process since the 8-cell embryo stage is still too early to assess if a clone will have desired traits or not…

3.5.8 Methods have been developed for cloning adult animals using differentiated cells

In the 1950s, John Gurdon, then a student at Oxford, removed the nucleus from the body cell of a

tadpole and transplanted the nucleus into a tadpole egg cell.

The egg cell with the transplanted nucleus developed as a normal zygote

and created a tadpole with the same genome as the body cell nucleus

In 1996, Dolly the sheep became the first mammal cloned in this way

In 2012, Gurdon received the Nobel Prize in Medicine for his research

3.5.12 Production of cloned embryos by somatic cell nuclear transfer

https://www.hhmi.org/biointeractive/somatic-cell-nuclear-transfer-animation

Cloned embryos, such as Dolly, are produced by a process called Somatic Cell Nuclear Transfer (SCNT)

1. Somatic cells are taken from adult organism to be cloned and grown in a low-nutrient medium. This inactivates genes to wipe out any previous pattern of differentiation

2. Unfertilized egg cells are taken from a female of same species and their nuclei are removed

3. Cultured somatic cells and enucleated egg cells placed side-by-side and zapped with a small electric pulse to fuse them together (about 10% of cells fuse)

4. Fused egg cells containing somatic cell nucleus develop into embryos for seven days and are then implanted into surrogate mother (in the case of Dolly, only 1 of 29 successfully implanted and completely developed)

3.5.12 Production of cloned embryos by somatic cell nuclear transfer

3.5.12 Production of cloned embryos by somatic cell nuclear transfer

Bibliography / Acknowledgments

Bob Smullen