Topic 4: Genetics 4.4: Genetic engineering and biotechnology 8 class periods
Objectives Outline the use of PCR to copy and amplify minute quantities of DNA. State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size. State that gel electrophoresis of DNA is used in DNA profiling. Describe the application of DNA profiling in paternity tests and forensic investigations Analyze DNA profiles to draw conclusions about paternity tests and forensic investigations. Outline three outcomes of the sequencing of the complete human genome.
Objectives (part II) Outline a basic technique used for gene transfer involving plasmids, a host cell, restriction enzymes (endonucleases) and DNA ligase. State two examples of the current uses of genentically modified crops or animals. Discuss the potential benefits and possible harmful effects of one example of genetic modification Define clone Outline a technique for cloning using differentiated animal cells Discuss the ethical issues of therapeutic cloning in humans.
Biotechnology and genetic engineering Biotechnology: The modern forms of industrial production that use living organisms, especially microorganisms. Some biotechnology processes involve naturally occurring organisms, but others involve organisms that have been produced by genetic engineering. Genetic engineering is a group of techniques, which allow genes to be transferred between species (genetic modification). This is possible because the genetic code is universal. Recombinant DNA technology is the set of laboratory techniques that combines genes from different sources into a single DNA molecule
Manipulating DNA Plasmid: is a small, circular DNA molecule separate from the much larger bacterial chromosome. A plasmid may carry a number of genes, and can make copies of itself. A method often used for gene transfer in bacteria involves plasmids, restriction enzymes and DNA ligase. Bacteria naturally absorb plasmids and incorporate them to their main DNA molecule
Gene transfer Plasmid: A small extra circle of DNA used to exchange genes. Restriction enzymes: Endonucleases, are enzymes that cut DNA molecules at specific base sequences. Some restriction enzymes can cut the two strands of DNA at different points, leaving single-stranded sections called “sticky ends”. Sticky ends can be used to link together pieces of DNA by hydrogen bonding between the bases. DNA ligase: Enzyme that joins DNA molecules together firmly by making sugar-phosphate bonds between nucleotides.
Gene transfer A copy of the gene being transferred is needed, and can be obtained from messenger RNA transcripts using reverse transcriptase that can make DNA copies of RNA molecules (cDNA)
The specific sequence lets the restriction enzyme know where to cut Sticky ends
Examples of genetic modification Animals: Goats: Modified to secrete an anti-clotting agent (anti-thrombin) in their milk. Sheep: Modified to produce alpha-1-antitrypsin, a protein used in the treatment of emphysema. Plants: Many crop plants have been engineered to produce a protein that makes them resistant to the herbicide glyphosate.
Examples of genetic modification “Golden rice”: The introduction of 3 genes, two from daffodil plants and one from a bacterium, so that β-carotene, a precursor of vitamin A, is produced in the rice grains. The development of golden rice was intended as a solution to the problem of vitamin A deficiency. The economic benefits of genetic modification to biotechnology companies is considerable, but there is also the possibility of harmful changes to local economies and inequalities in wealth may become greater.
Recombinant DNA: UsingE. colifortheproduction of human insulin
Libraries of cloned genes By cloning recombinant DNA, as in the example of E. coli bacteria, the procedure produces many different clones, each containing a different portion of the source DNA . The procedure captures a large number of additional genes because the restriction enzyme makes cuts all over the source DNA. The result is that many genes are cloned in addition to the target gene. The complete collection of cloned DNA fragments from an organism is called a genomic library.
A typical plasmid contains a DNA fragment big enough to carry only one or a few genes. Together, the different recombinant plasmids in a genomic library contain the entire genome of the organism from which the DNA was derived.
Once a genomic library is created for an organism, how does a biologist find a specific gene in that library? One method requires knowing at least part of the gene's nucleotide sequence. For example, suppose that the gene for protein V contains the base sequence TAGGCT. Knowing this, a biologist can use nucleotides labeled with a radioactive isotope to build a complementary single strand of DNA with the sequence ATCCGA . This complementary, radioactively labeled nucleic acid molecule is called a nucleic acid probe. Video: A green light for biology
Mini research Prepare a short presentation (5 minutes) about a usefulproductusinggeneticallymodifiedmicroorganisms. Examples: Escherichiacolithat produces human insulin. Work in pairs Importantissuestoaddress: name and source of theorganism, name and source of the gene, techniqueusedforthemassproduction of theproduct.
GMO’s Genetic engineering is replacing traditional methods of plant breeding in many situations. It is used most often when a plant's useful traits are determined by one or only a few genes. A genetically modified organism (GMO) is any organism that has acquired one or more genes by artificial means. Biologists often use a plasmid from the soil bacterium Agrobacterium tumefaciensto introduce new genes into plant cells.
Figure 13-11To genetically modify a plant, researchers insert a plasmid containing the desired gene into a plant cell. There, the gene is incorporated into the plant cell's DNA. The engineered plant cell then grows into a genetically modified plant.
Example of GM animal Factor IX: The protein (factor IX) is expressed in milk from which it must be isolated before use by hemophiliacs. A ewe is treated with fertility drugs to create super-ovulation. Eggs are inseminated. Each fertilized egg has the transgene injected. A surrogate ewe has the egg implanted for gestation. Lambs are born which are transgenic, GMO for this factor IX gene. Each Lamb when mature can produce milk. The factor IX protein is in the milk and so must be isolated and purified before use in human.
GM animals Genetically modifying animals is more difficult than producing GM plants. It usually takes many attempts before an egg actually incorporates the DNA. If the embryo develops successfully, the result is a GM animal. The offspring contains a gene or genes from a third "parent" that may even be of another species. http://learn.genetics.utah.edu StemCells Somatic nuclear transfer video
Potential benefits and disadvantages of GMOs Class discussion: In groups find arguments in favor of and against the relative importance of various factors concerning the modification of genes The advantages and disadvantages of GMOs is a controversial topic with wide political, environmental, health and social effects.
Benefits of GMOs Increased yields particularly in regions of food shortage. Yields of crops with specific dietary requirement such as vitamins and minerals. Crops that do not spoil so easily during storage. GM animals produce similar effect including higher meat yields.
Disadvantages The foods (animal and plant) are considered un-natural and unsafe for human consumption. There is a risk of the escape of 'genes' into the environment where they may be passed to other organisms with unknown effects. Disruption of local economies
Cloning Clone: a group of genetically identical organisms or a group of cells derived from a single parent Somatic cell cloning: Dolly the sheep was cloned with this technique. http://learn.genetics.utah.edu/content/tech/cloning/whatiscloning/ Let’s clone a mouse, mouse, mouse http://learn.genetics.utah.edu/content/tech/cloning/clickandclone/
The risks of cloning 1. High failure rate Cloning animals through somatic cell nuclear transfer is simply inefficient. The success rate ranges from 0.1 percent to 3 percent, which means that for every 1000 tries, only one to 30 clones are made. Why is this? The enucleated egg and the transferred nucleus may not be compatible An egg with a newly transferred nucleus may not begin to divide or develop properly Implantation of the embryo into the surrogate mother might fail The pregnancy itself might fail
2. Problems during later development Cloned animals that survive tend to be much bigger at birth than their natural counterparts. Scientists call this "Large Offspring Syndrome" (LOS). Clones with LOS have abnormally large organs. This can lead to breathing, blood flow and other problems = Dolly Because LOS doesn't always occur, scientists cannot reliably predict whether it will happen in any given clone. Also, some clones without LOS have developed kidney or brain malformations and impaired immune systems, which can cause problems later in life.
3. Abnormal gene expression patterns Are the surviving clones really clones? The clones look like the originals, and their DNA sequences are identical. But will the clone express the right genes at the right time? One challenge is to re-program the transferred nucleus to behave as though it belongs in a very early embryonic cell. In a naturally-created embryo, the DNA is programmed to express a certain set of genes. Later on, as the embryonic cells begin to differentiate, the program changes. For every type of differentiated cell - skin, blood, bone or nerve, for example - this program is different. In cloning, the transferred nucleus doesn't have the same program as a natural embryo. It is up to the scientist to reprogram the nucleus, like teaching an old dog new tricks. Complete reprogramming is needed for normal or near-normal development. Incomplete programming will cause the embryo to develop abnormally or fail.
4. Telomeric differences As cells divide, their chromosomes get shorter. This is because the DNA sequences at both ends of a chromosome, called telomeres, shrink in length every time the DNA is copied. The older the animal is, the shorter its telomeres will be, because the cells have divided many, many times. This is a natural part of aging. So, what happens to the clone if its transferred nucleus is already pretty old? Will the shortened telomeres affect its development or lifespan? When scientists looked at the telomere lengths of cloned animals, they found no clear answers. Chromosomes from cloned cattle or mice had longer telomeres than normal. These cells showed other signs of youth and seemed to have an extended lifespan compared with cells from a naturally conceived cow. On the other hand, Dolly the sheep's chromosomes had shorter telomere lengths than normal. This means that Dolly's cells were aging faster than the cells from a normal sheep. To date, scientists aren't sure why cloned animals show differences in telomere length.
Therapeutic cloning in humans HOMEWORK The discussion is about the creation of an embryo to supply stem cells for medical use. Research what is meant by therapeutic cloning. Decide what the ethical issues are in therapeutic cloning. What is an embryo? Where would they be obtained from? Alternatives? Try to make yourself aware of the position of interest groups on the issues.
PCR Polymerase Chain Reaction PCR is the cloning of DNA (amplification). Copies are made and the amount of DNA can be rapidly increased. Useful if the source of DNA is small. Temperature is used instead of enzymes like helicases (95oC ). DNA polymerase is thermostable to protect it against the reaction temperatures. This is an automated process and can produce sufficient DNA in 20 cycles. http://learn.genetics.utah.edu/content/labs/pcr/
Gel electrophoresis Sample of fragmented DNA is placed in one of the wells on the gel. An electrical current is passed across the gel. Fragment separation is based on charge and size. Large fragments move slowly. Negative fragments are moved to the right. http://learn.genetics.utah.edu/content/labs/gel/
Gel after staining This diagram shows the separation of 6 separate mixtures of DNA. The dark bands to the left are those with a large molecular mass or a positive charge (a) contains 5 fragments of DNA. Each bands corresponds to a group of DNA molecules of the same size and charge. (b) and (e) have the same bands. They are identical Direction of movement
DNA profiling and forensics PCR and gel electrophoresis are techniques commonly used for finding the identity of a person, determining the source of a sample of DNA, determining paternity, forensic investigations, animal breeding, disease detection. Satellite (Tandem repeating) DNA are highly repetitive sequences of DNA from the non coding region of DNA. Different individuals have a unique length to their satellite regions. These can be used to differentiate between one individual and another. There are different types of 'DNA fingerprinting' for different circumstances
(a) The mothers chromosome provides a DNA STR cutting the chromosome with particular restriction enzymes. (b)The fathers chromosome provides the same fragment using the same restriction enzymes. (c) The mother DNA fragment placed in the well of the gel. (d) The mother DNA fragment placed in the well of the gel. (e) Mothers fragment produces 5 STR and moves a short distance along the electrophoresis gel. (f) fathers fragment produces 2 STR and moves a longer distance along the electrophoresis gel. (g) The child is heterozygous for the fragment having on homologous chromosome form the father and one form the mother. Both 5 STR and 2 STR are shown in the electrophoresis.
Animal Cloning In cloning an entire animal, the nucleus from a single cell of that adult animal replaces the nucleus of an unfertilized egg cell from another animal of the same species. In livestock, an egg can be fertilized in vitro and allowed to develop into a multicellular embryo. Cells can be separated while they still are pluripotent (capable of developing into all types of tissue) and transplanted into surrogate mothers. Somatic cell nuclear transfer video
Cloning Anothermethodinvolvesthe use of non-reproductivecells: Dolly thesheep. Celltakenfromudder and culturedfor 6 daysisfusedusing a spark of electricitywithaneggcellthat has no nucleus. Resultingembryoistransferredtosurrogatemother.
Cloning Let’s clone a mouse, mouse, mouse activity.
The polymerase chain reaction (PCR) It’s a technique that can be used to amplify small quantities of DNA. This is especially useful when samples are limited as are fossil samples or small samples used for forensic investigations. 1. The first stage involves denaturing the DNA sample using heat (separating the two strands) 2. The second stage involves annealing with a primer that matches a particular target within the DNA. The final stage involves the extension of the primer using a DNA polymerase from bacteria such as Thermophilusaquaticus.
PCR PCR involves repeated cycles as shown in the figure:
Gel electrophoresis A technique that involves separating charged molecules in an electric field.