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4.4 Genetic Engineering & Biotechnology 3/30/11 3:03 PM
Topic 4: Genetics4.4: Genetic Engineering & Biotechnology
4.4.1 Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA.
4.4.2 State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size.
4.4.3 State that gel electrophoresis of DNA is used in DNA profiling.
4.4.4 Describe the application of DNA profiling to determine paternity and also in forensic investigations.
4.4.5 Analyse DNA profiles to draw conclusions about paternity or forensic investigations.
4.4.6 Outline three outcomes of the sequencing of the complete human genome.
4.4.7 State that, when genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal.
4.4.8 Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase.
4.4.9 State two examples of the current uses of genetically modified crops or animals.
4.4.10 Discuss the potential benefits and possible harmful effects of one example of genetic modification.
4.4.11 Define clone.
4.4.12 Outline a technique for cloning using differentiated animal cells.
4.4.13 Discuss the ethical issues of therapeutic cloning in humans.
4.4.1 PCR 3/30/11 3:03 PM
4.4.1 Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA. (Details of method are NOT required).
Orange book pg. 163Green book pg. 64WS1: Polymerase Chain Reaction
To do:Remember - as you view the following animations – you do NOT need to know the process, the idea is to get you to understand that the purpose if to copy and amplify small quantities of DNA.
http://www.dnalc.org/resources/animations/pcr.html
http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
http://www.maxanim.com/genetics/PCR/PCR.htm
http://www.sumanasinc.com/webcontent/animations/content/pcr.html
http://learn.genetics.utah.edu/content/labs/pcr/
Complete the following questions to act as a summary in your books.
1. What is the purpose of the Polymerase Chain Reaction?
2. Briefly describe how the Polymerase Chain Reaction works (temperatures, names etc are NOT needed).
3. How has the development of the Polymerase Chain Reaction improved the technique of DNA fingerprinting?
The Polymerase Chain ReactionPCR can clone (or amplify) DNA samples as small as a single molecule. The polymerase chain reaction is simply DNA replication in a test tube. If a length of DNA is mixed with the four nucleotides (A, T, C and G) and the enzyme DNA polymerase in a test tube, then the DNA will be replicated many times. The details are shown in this diagram:
1. Start with a sample of the DNA to be amplified, and add the four nucleotides (sugar, phosphate and base) and the enzyme DNA polymerase (sticks the new nucleotides to the old nucleotides).
2. The two strands of the DNA double helix are separated by heating to 95°C for two minutes. This breaks the hydrogen bonds.
3. The DNA must be cooled to 40°C to allow new nucleotides to be added to the original DNA strand..
4. Each original DNA molecule has now been replicated to form two molecules.
5. The cycle is repeated from step 2 and each time the number of DNA molecules doubles. This is why it is called a chain reaction, since the number of molecules increases exponentially, like an explosive chain reaction. Typically PCR is run for 20-30 cycles.
PCR can be completely automated, so in a few hours a tiny sample of DNA can be amplified millions of times with little effort. The product can be used for further studies, such as cloning and electrophoresis. Because PCR can use such small samples it can be used in forensic medicine (with DNA taken from samples of blood, hair or semen), and can even be used to copy DNA from mummified human bodies, extinct woolly mammoths, or from an insect that's been encased in amber since the Jurassic period. One problem of PCR is having a pure enough sample of DNA to start with. Any contaminant DNA will also be amplified, and this can cause problems, for example in court cases.
The following silly links are only if you have time “The PCR Song”http://www.youtube.com/watch?v=7uafUVNkuzg
“Enzyme – DNA – PCR song”http://www.youtube.com/watch?v=CQEaX3MiDow
4.4.2 Electrophoresis 3/30/11 3:03 PM
4.4.2 State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size.
Orange book pg. 164Green book pg. 65WS2: Gel Electrophoresis
To do:View the animations – details of the process are NOT needed:
http://www.dnalc.org/resources/animations/gelelectrophoresis.html
http://learn.genetics.utah.edu/content/labs/gel/
http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html
Read the information provided below.
In your green books, explain how the DNA fragments are separated.
This is a form of chromatography used to separate different pieces of DNA on the basis of their length. It might typically be used to separate restriction fragments (produced by the use of restriction enzymes on DNA). The DNA samples are placed into wells at one end of a thin slab of gel and covered in a buffer solution (allows the electricity to flow). An electric current is passed through the gel. Each nucleotide in a molecule of DNA contains a negatively-charged phosphate group, so DNA is attracted to the anode (the positive electrode). The molecules have to diffuse through the gel, and smaller lengths of DNA move faster than larger lengths, which are retarded by the gel. So the smaller the length of the DNA molecule, the further down the gel it will move in a given time. At the end of the run the current is turned off.
Unfortunately the DNA on the gel cannot be seen, so it must be visualised. There are three common methods for doing this:
The gel can be stained with a chemical that specifically stains DNA. The DNA shows up as blue bands. This method is simple but not very sensitive.
The DNA samples at the beginning can be radiolabelled. Photographic film is placed on top of the finished gel in the dark, and the DNA shows up as dark bands on the film. This method is extremely sensitive.
The DNA fragments at the beginning can be labelled with a fluorescent molecule. The DNA fragments show up as coloured lights when the finished gel is illuminated with invisible ultraviolet light.
4.4.3 DNA Profiling 3/30/11 3:03 PM
4.4.3 State that gel electrophoresis of DNA is used in DNA profiling.
Orange book pg. 164Green book pg. 65WS3: DNA Profiling using PCR
To do:Go through the following website in the order stated below:“DNA Profiling”: http://www.dnai.org/d/index.html Human Identification (bottom of screen) Profiling (top of screen) 1st Circle – DNA Variations and Fingerprints
A – DNA Variations (watch animation)V – Tandem Repeats (listen to audio explanation of Tandem
Repeats) Return Circle – The First DNA fingerprints
A – The First DNA “fingerprints”No need to look at any other sections just yet.
Read the section below “Which sections of DNA are used for profiling?”1. Explain in your own words why exons are no use for profiling.
2. Describe the sections that are used for profiling and why they can be used.
Read the section below “Profiling – the technique”Explain the role of Electrophoresis in profiling.
Which sections of DNA are used for Profiling?About 90% of human genome has no known function. The non-coding regions of DNA – introns are removed before translation.
Exons that code for a protein vary little, since any change in the structure of an essential protein is likely to be harmful.
Introns vary greatly in a population and are the basis for DNA profiling.
Intron DNA consists of regions called minisatellites – where there is great variability from one individual to another.
Minisatellite – short sequences of 20-100 nucleotides called variable number tandem repeats (VNTRs).
Each chromosome has many VNTRs and the number of repeats differs from person to person and so does the length of the VNTR region.
Each individual has a unique profile.
Variation in fragment length depends on: the number of repeats number of bases in each repeated sequence.
E.g. CGCGCGCGCGCG (6 repeats of 2 bases) this will have the same fragment length as 3 repeats of 4 bases.
Profiling - The Technique** You do NOT need to learn the technique.You just need to understand where gel electrophoresis fits in.
Extraction of the DNA from a sample.Using chemicals and enzymes the DNA can be removed from a sample of tissue and then purified.
DNA cut up.Using restriction enzymes the DNA is digested. The restriction enzymes do not attack any site with a repeated sequence and so the repeats are left intact.
Fragments separated by gel electrophoresis.
The DNA would have been labeled in some way and will show up as dark bands on the gel.The spacing of these and is unique to the individual.
4.4.4 Uses of Profiling 3/30/11 3:03 PM
4.4.4 Describe the application of DNA profiling to determine paternity and also in forensic investigations.
Orange book pg. 164Green book pg. 65WS4: DNA ProfilingWS5 : Manual DNA Sequencing
To do:Read the information below.
In your green books make a list of application of DNA profiling.
Explain how DNA profiling can be used to determine paternity and also in forensic investigations.
Applications/ UsesDNA profiling can be used in paternity suits when the identity of someone’s biological father must be known for legal reasons.
Cases of incest where a child has been born can be used to prove the accused is genetically more similar to a child than if the father were a non-relative.
At a crime scene, forensic specialists can collect samples such as blood or semen which contain DNA. Gel electrophoresis is used to compare the collected DNA with that of suspects. If they match, the suspect has a lot of explaining to do. A match can confirm the guilt of a suspect. If the samples do not match it can also be used to clear someone who is claiming to be innocent.Criminal cases are sometimes reopened many years after a judgement in order to consider new DNA profiling results. In the United States, this has lead to the liberation of many individuals who had been wrongly sent to jail for crimes they did not commit.
In Europe it can be used to establish if a person has relatives in the country they are trying to enter (for immigration purposes).
Captive breeding of endangered species. When numbers are low used to prevent inbreeding by making it possible to select non-related animals.
In studies of ecosystems, DNA samples taken from animals can be used to establish better understanding of social relationships, migrating patterns and nesting habits. In addition, the study of DNA in the biosphere has given new credibility to the ideas of evolution: DNA evidence can often reinforce previous evidence of common ancestry based on anatomical similarities between species.
Advantages over blood testing.Blood groups are not unique; they can be used to eliminate suspects not to convict. DNA profiling can give a positive identification – it is unique.Any tissue containing cells can be used e.g. skin, hair, bone can all serve as a DNA source
4.4.5 Analysing Profiles 3/30/11 3:03 PM
4.4.5 Analyse DNA profiles to draw conclusions about paternity or forensic investigations.
Orange book pg. 164Green book pg. 66-67
To do:The following website gives an example of a paternity test and also examples of forensic investigations.http://www.dnai.org/d/index.htmlGo through the following website in the order stated below: Human Identification (bottom of screen)
Family (top of screen) Murder Innocence
For each of the three cases:1. Read through all the information.2. Try the comparison – match the DNA fragments yourself.3. In your own words summarise how DNA profiling was used each of the three cases.
Exam Questions:1. The following is a DNA gel. The results are from a single probe showing a DNA profile for a man, a woman and their four children. Which fragment of DNA is the smallest?
2. The following is a DNA gel. The results are from a single probe showing a DNA profile for a man, a woman and their four children.
Which child is least likely to be the biological offspring of the father?
4.4.6 Human Genome Project 3/30/11 3:03 PM
4.4.6 Outline three outcomes of the sequencing of the complete human genome.
Orange book pg. 158-159Green book pg. 67WS6: The Human Genome Project
The website below is the official Human Genome Project website.http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml
The following all require the same login:Username: BiologyhelpPassword: Ilovebiology
“The Human Genome Project”http://www.yteach.ie/page.php/resources/view_all?id=restriction_endonuclease_nitrogenous_base_nucleotide_base_chromosomal_aberration_mitotic_division_amnion_denaturation_sequencing_cytosine_thymine_uracil_denaturation_gene_t_page_21&from=search
“Sequencing the Human Genome”http://www.yteach.ie/page.php/resources/view_all?id=restriction_endonuclease_nitrogenous_base_nucleotide_base_chromosomal_aberration_mitotic_division_amnion_denaturation_sequencing_cytosine_thymine_uracil_denaturation_gene_t_page_22&from=search
4
To do:Have a browse through the official Human Genome Project website.
View the clip: “Introduction”http://www.genome.gov/25019885
Read the corresponding page in the green text book and the information below.
Complete the objective: ”Outline three outcomes of the sequencing of the complete human genome”.
General InformationThe Human Genome Project was an international co-operative venture which set out to sequence the complete human genome. Because the genome of an organism is a catalogue of all the bases it possesses, the HGP hoped to determine the order of all the bases A, T, C and G in human DNA. In 2003, the HGP announced that it had succeeded in achieving its goal. Now, scientists are working on deciphering which sequences represent genes and which genes do what. The human genome can be thought of as a map which can be used to show the locus of any gene on any one of the 23 pairs of chromosomes.
As you have seen, some diseases are sex linked, so it is relatively easy to determine which chromosome the gene responsible for the disease is found on; often the locus is on the X-chromosome. In traits which show no sex linkage, it is difficult to know which of the 22 other chromosomes carries the gene. With genome libraries of genetic diseases, doctors can find out exactly where to look if they think one of their patients might posses a disease-carrying allele.
Another advantageous use of the HGP is the production of new medications. This idea involves several steps: Find beneficial molecules which are produced naturally in healthy
people
Find out which gene controls the synthesis of a desirable molecule Copy that gene and use it as instructions to synthesize the molecule in
a laboratory. Distribute the beneficial molecule as a new medical treatment.
This is not science fiction; genetic engineering firms are finding such genes regularly.
In addition, by comparing the genetic make-up of populations around the world, countless details could be revealed about ancestries and how humans have migrated and mixed their genes with other populations over time.
4.4.7 Genetic Code 3/30/11 3:03 PM
4.4.7 State that, when genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal.
Orange book pg. 161Green book pg. 67
To do:First a recap on the genetic code below.
Username: BiologyhelpPassword: Ilovebiology“Characteristics of the genetic code”http://www.yteach.ie/page.php/resources/view_all?id=termination_site_gene_codon_operator_operon_promoter_polymerase_binds_polymerase_dna_rna_transcription_translation_gene_gene_expression_polymerase_nucleosome_uracil_mrna_triplet_code_anticodon_t_page_7&from=search
In your own words, in your green books, explain the significant to the genetic code being universal.
Recap on the Genetic CodeAll living organisms use the same bases:
This means that base sequences can be transferred from one organism to another without changing their function.
So …We could take the gene for healthy insulin production from a human and insert it into a bacterial plasmid – and the bacteria will then be able to produce human insulin.
4.4.8 Transgenesis 3/30/11 3:03 PM
4.4.8 Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase.
Orange book pg. 161Green book pg. 68-69WS7 : Restriction EnzymesWS8 : LigationWS9: Gene Cloning Using PlasmidsWS10 : Transgenic Organisms
Introduction - **Do NOT learn – this is simply to set the scene:Since the discovery of the hormone insulin in 1921, diabetic patients, whose elevated sugar levels are due to impaired insulin production, have been treated with insulin derived from the pancreas glands of slaughtered animals. The hormone, produced and secreted by the beta cells of the pancreas' islets of Langerhans, regulates the use and storage of food, particularly carbohydrates
Although bovine (cow) and porcine (pig) insulin are similar to human insulin, their composition is slightly different. Consequently, a number of patients' immune systems produce antibodies against it, neutralising its actions and resulting in inflammatory responses at injection sites. Added to these adverse effects of bovine and porcine insulin, were fears of long term complications ensuing from the regular injection of a foreign substance, as well as a projected decline in the production of animal derived insulin. These factors led researchers to consider synthesising Human insulin by inserting the insulin gene into a suitable vector, the E. coli bacterial cell, to produce insulin that is chemically identical to its naturally produced counterpart. This has been achieved using Recombinant DNA technology. This method is a more reliable and sustainable method than extracting and purifying the abattoir by-product.
To do:In no more than a couple of sentences (in your books) explain the benefits of producing insulin from genetically modified bacteria compared to how it was previously obtained from animals.
The Process of TransgenesisThe following website give you an overview of the entire process before we break it down. Go through the webpage to get an overview before moving on.http://www.abpischools.org.uk/page/modules/diabetes_16plus/diabetes8.cfm?cositenavigation_alltopic=1
1. Identifying the human insulin gene The human insulin gene was isolated, cloned and sequenced in the 1970s, and so it became possible to insert this gene into bacteria, who could then produce human insulin in large amounts.We know which gene codes for insulin.
2. Inserting the DNA into a plasmid vector using restriction enzymes and DNA ligase
Restriction EnzymesView the animations: “Restriction Endonucleases”http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html
“DNA Restriction”http://www.dnalc.org/resources/animations/restriction.html
“Restriction Enzymes”Username: BiologyhelpPassword: Ilovebiologyhttp://www.yteach.ie/page.php/resources/view_all?id=hydrogen_bonds_conjugation_transformation_ligase_clone_replication_transcription_vector_plasmids_retrovirus_nucleotides_dna_pcr_hybridization_polymerase_exons_introns_t_page_3&from=search
Read the section below on restriction enzymes:
These are enzymes that cut DNA at specific sites. They are properly called restriction endonucleases because they cut phosphodiester (Between sugar and phosphate) bonds in the middle of the polynucleotide chain. Some restriction enzymes cut straight across both chains, forming blunt ends, but most enzymes make a staggered cut in the two strands, forming ‘sticky ends’.
The cut ends are “sticky” because they have short stretches of single-stranded DNA with complementary sequences. These sticky ends will stick (or anneal) to another piece of DNA by complementary base pairing, but only if they have both been cut with the same restriction enzyme. Restriction enzymes are highly specific, and will only cut DNA at specific base sequences, 4-8 base pairs long, called recognition sequences.
In your books summarise the role of restriction enzymes to include: specificity bonds broken sticky ends
DNA LigaseWatch the animations:“Steps in cloning a gene”http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.html
Username: BiologyhelpPassword: Ilovebiology“Basic Tools in Genetic Engineering”
http://www.yteach.ie/page.php/resources/view_all?id=hydrogen_bonds_conjugation_transformation_ligase_clone_replication_transcription_vector_plasmids_retrovirus_nucleotides_dna_pcr_hybridization_polymerase_exons_introns_t&from=search
Read through section below:
This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It is commonly used in genetic engineering to do the reverse of a restriction enzyme, i.e. to join together complementary restriction fragments.
The sticky ends allow two complementary restriction fragments to anneal, but only by weak hydrogen bonds, which can quite easily be broken, say by gentle heating. The backbone is still incomplete.
DNA ligase completes the DNA backbone by forming covalent phosphodiester bonds. Restriction enzymes and DNA ligase can therefore be used together to join lengths of DNA from different sources.
In your books – explain, in a couple of sentences, the role of DNA ligase in transgenesis.
VectorsIn biology a vector is something that carries things between species. For example the mosquito is a disease vector because it carries the malaria parasite into humans. In genetic engineering a vector is a length of DNA that carries the gene we want into a host cell. A vector is needed because a length of DNA containing a gene on its own won’t actually do anything inside a host cell. Since it is not part of the cell’s normal genome it won’t be replicated when the cell divides, it won’t be expressed, and in fact it will probably be broken down pretty quickly. A vector gets round these problems by having these properties:
It is big enough to hold the gene we want. It is circular (or more accurately a closed loop), so that it is less likely
to be broken down (particularly in prokaryotic cells where DNA is always circular).
It contains control sequences, such as a replication origin and a transcription promoter, so that the gene will be replicated, expressed, or incorporated into the cell’s normal genome.
It contains marker genes, so that cells containing the vector can be identified.
PlasmidsView the animation:“Construction of a plasmid vector”http://highered.mcgraw-hill.com/sites/0072552980/student_view0/chapter10/animation_quiz_1.html
Username: BiologyhelpPassword: IlovebiologyView the animation: “Features of a good plasmid vector”http://www.yteach.ie/page.php/resources/view_all?id=amplification_cloning_insulin_phospholipid_plasmid_polymerase_primer_restriction_endonuclease_plasmid_single_stranded_electrophoresis_denaturation_primers_nucleotides_polymerase_matrix_bioreactors_gene_vectors_sticky_blunt_ends_dna_purification_t_page_7&from=search1. Why do plasmid vectors need to be small?
2. Which “genetic tool” will be used to cut the plasmid?
3. Explain the purpose of choosing plasmids which contain antibiotic resistant genes.
View the animation: “Use of plasmids in gene cloning”http://www.yteach.ie/page.php/resources/view_all?id=amplification_gene_cloning_insulin_phospholipid_plasmid_polymerase_primer_restriction_endonuclease_plasmid_dna_single_stranded_dna_electrophoresis_denaturation_primers_nucleotides_page_2&from=search1. What is a gene vector?
2. What is the first stage of gene cloning?
3. Why is the gene inserted into a plasmid?
4. Into which microorganism are the plasmids introduced?
5. Due to the addition of the plasmid containing the gene, what is the bacterial cell capable of producing?
Read the section below on plasmids.
Plasmids are by far the most common kind of vector, so we shall look at how they are used in some detail. Plasmids are short circular bits of DNA found naturally in bacterial cells. A typical plasmid contains 3-5 genes and there are usually around 10 copies of a plasmid in a bacterial cell. Plasmids are copied separately from the main bacterial DNA when the cell divides, so the plasmid genes are passed on to all daughter cells. They are also used naturally for exchange of genes between bacterial cells (the nearest they get to sex), so bacterial cells will readily take up a plasmid. Because they are so small, they are easy to handle in a test tube, and foreign genes can quite easily be incorporated into them using restriction enzymes and DNA ligase.
The diagram below shows how DNA fragments can be incorporated into a plasmid using restriction and ligase enzymes.
The foreign DNA anneals with the plasmid and is joined covalently by DNA ligase to form a hybrid vector (in other words a mixture or hybrid of bacterial and foreign DNA).
Make sure you understand this diagram
Use the diagram above to help you draw and annotate your own diagram to show the process of transgenesis that has been covered so far, so you should include: finding and cutting out the desired gene Cutting the plasmid Sticking the desired gene into the plasmid Formation of the recombinant plasmid.
3. Inserting the plasmid vector into the host bacteriumThe plasmid must now be introduced into a bacterial cell, which will allow the vector to multiply (clone itself and the foreign donor DNA it contains). The bacterium commonly used is Escherichia coli (E. coli) a normal inhabitant of the human gut and was chosen for this task because a great deal is known about its genetics and because it grows rapidly with a doubling time of 30 minutes. A mutant form of E. coli was specially developed for genetic engineering. This form can only survive in special laboratory conditions. Therefore if it escapes with foreign genes inserted, it cannot infect humans.
The process of adding new DNA to a bacterial cell is called transformation.
4. Cloning the bacteria and harvesting the human insulinThe bacterial cells need nutrients in order to grow, divide, and live. While they live, the bacterial cell processes turn on the gene for human insulin and the insulin is produced in the cell. When the bacterial cells reproduce by dividing, the human insulin gene is also reproduced in the newly created cells.
Human insulin protein molecules produced by bacteria are gathered and purified. Millions of people with diabetes now take human insulin produced by bacteria or yeast (biosynthetic insulin) that is genetically compatible with their bodies, just like the perfect insulin produced naturally in your body.
Username: BiologyhelpPassword: IlovebiologyView the animation: “Biotechnology Past and Present - Biofermenters”http://www.yteach.ie/page.php/resources/view_all?id=asepsis_dna_recombination_dna_insulin_somatostatin_transgenic_organism_somatotrophin_fermentation_pasteurization_malting_milling_whirling_brewing_maturation_filtration&from=search1. What containers are needed for the large scale production of Biological molecules such as the protein insulin?
2. What conditions must be maintained?
View the animation: “Insulin producing bacteria”http://www.bbc.co.uk/schools/gcsebitesize/science/edexcel/genes/genesrev3.shtml
View the animation: “Use of bioreactors in industrial scale drug production”http://www.yteach.ie/page.php/resources/view_all?id=amplification_gene_cloning_insulin_phospholipid_plasmid_polymerase_primer_restriction_endonuclease_plasmid_dna_single_stranded_dna_electrophoresis_denaturation_primers_nucleotides&from=search1. What is the role of the biotechnologists.
2. What role do the engineers play?
3. List the necessary conditions for optimum bacterial growth.
4. Explain the role of the propellers/ paddles in the bioreactor/ fermenter.
QuestionsQuestion One (8)Recombinant DNA technique (transgenesis) has enabled researchers to insert DNA from one organism into the genome of bacteria for later multiplication.
The numbered diagrams on the following page show the sequence of events in the process of gene cloning.
(a) Match the numbers of the diagram with the descriptions below. (The first answer has been done for you). (4) A selected restriction enzyme cuts the donor DNA and the bacterial
DNA do that they that the same sticky ends. = 3
The plasmids with the recombinant DNA are reincorporated into bacteria.
The donor DNA is joined by a ligase to produce recombinant DNA molecules.
Recombinant bacteria are selected out to produce large numbers of recombinant plasmids.
Donor cell DNA The restriction enzyme is used again to cut the segments of donor DNA
from the plasmids. Bacterial cell DNA. Donor DNA is incorporated into the bacterial plasmid. Recombinant DNA plasmids multiply inside the bacteria.
(b) Refer to the following and give the numbers of the two ‘sticky ends’ resulting from a cut by the same restriction enzyme. (2)
1. GAA 2. GCCTGGC 3. AAGCCT CTTAAG ACCG TTC
4. CTGCAG 5. GAA 6. AAGCTT GAC GGACTT
GAA
(c) State TWO reasons why an experimenter might want to insert some donor DNA into a bacterial plasmid. (2)
Question Two (14)Read the following extract carefully, then answer the questions which follow.
Volunteers are being recruited to eat raw potatoes in the first human trials of a vaccine grown in genetically engineered potatoes. Researchers hope that people who eat the potatoes will be protected from common gut infections.
The team first tried out the technique in tobacco plants. They took a strain of Escherichia coli bacteria that causes food poisoning and identified that toxin as a protein molecule. The toxin binds to receptor molecules on the cell surface membrane of the gut cells of its victim. The researchers then used a modified bacterium called Agrobacterium tumefaciens. Under normal circumstances, these bacteria transfer plasmids into plant cells causing the plant to manufacture the nutrients the bacteria need. In the modified bacteria, however, the gene for producing the E. coli toxin had been inserted into the plasmid DNA.
Once inside the tobacco cells, the foreign DNA becomes incorporated into the tobacco chromosomes. These genetically engineered tobacco plants were grown and propagated asexually by taking cuttings. These cuttings all contained the gene for, and produced, the E. coli toxin.
Proof of success came when the tobacco leaves were mashed up and fed to mice. Within days the mice started producing antibodies specific to the E. coli toxin. The team then produced genetically engineered potatoes and fed these to mice with similar results.
One problem with growing potatoes to produce vaccines is that the potatoes are usually cooked before being eaten. Bananas, which are usually eaten raw, might prove to be a better option.
(a) Use the information in this passage to help explain what is meant by:(i) recombinant DNA (2)
(ii) a vector (2)
(b) Explain why:(i) the tobacco cuttings all contained the gene for producing the toxin. (2)
(ii) Only some of the plants grown from the seeds of the genetically engineered tobacco plants would be expected to contain the gene. (2)
(c) Explain why it is thought that bananas might be a better option than potatoes for producing the vaccine. (2)
(d) What is a plasmid? (1)
(e) The gene for the toxin may be isolated and put into a plasmid from Agrobacterium tumefaciens. Describe in detail the part played by restriction enzymes in these processes. (3)
4.4.9 Uses of GMOs 3/30/11 3:03 PM
4.4.9 State two examples of the current uses of genetically modified crops or animals.
Orange book pg. 161Green book pg. 69WS11 :What is Genetic Modification”WS12 : Edible VaccinesWS13 : Using Recombinant BacteriaWS14 : Genetically Modified Plants
To do:Read the two sections below: “Transgenic Plants” and “Transgenic Animals”.
Read pg. 69 in the Green Text book and make SHORT notes on the examples given
In you books, state two example from the plant list (pick ones you think you will remember) and the one example from the animal list.
Username: BiologyhelpPassword: Ilovebiologywww.yteach.ieIf you search the term “genetic engineering” you will find numerous animations. They are all really short 1-2 mins and worth going through.The following are relevant:
Traditional farming techniques and genetic engineeringGenetic engineering methods in agricultureLegislation and the use of genetic engineering
Genetically Modified FoodsThe simplest kinds of GM food is one in which an undesirable gene is removed. In some cases, another more desirable gene is put in its place but in other instances, only the introduction of a new gene is needed, no DNA has been removed.Whichever technique is applied, the end result is either that the organism no longer shows the undesired trait or that it shows one which genetic engineers want.
Transgenic PlantsThe first commercial example of a GM food was the ‘Flavr Savr’ tomato. It was first sold in the US in 1994 and has been genetically modified to delay the ripening and rotting process so that is would stay fresher longer.
Another species of tomato was modified by a bioengineering company to make it more tolerant to higher levels of salt in the soil. This makes it easier to grow in certain regions of high salinity. One of the claims of the biotech industry is that GM foods will help solve the problem of world hunger by allowing farmers to grow foods in various otherwise unsuitable conditions. Critics point out that the problem of hunger in the world is one of food distribution, not food production.
Another plant of potential interest to the developing world is a genetically modified rice plant which has been engineered to produce beta carotene in the rice grains. The aim is that the people who eat this rice will not have deficiencies in vitamin A (the body used beta carotene to form vitamin A). the presence of beta carotene give a yellow colour “Golden Rice”
Other crop examples include:Bt Gene in Crops
Bt gene within maize makes a protein, which is toxic to insects feeding on the maize.
Potatoes Modified to Increase Starch ContentThey contain less water and absorb less oil when fried - healthier.
Antifreeze Gene in CropsAn antifreeze gene from Arctic flounder has been introduced to fruit and veg to prevent frost damage.
Transgenic AnimalsOne way of genetically engineering an animal is to get to produce a substance which can be used in medical treatment. Consider the problem faced by some people with haemophilia a blood condition in which their blood does not clot because they lack a protein called factor IX. If such people can be supplied with factor IX, their problem will be solved. The least expensive way of producing large amounts of factor IX is to use transgenic sheep. If a gene which codes for the production of factor IX is associated with the genetic information for milk production in a female sheep, she will produce that protein in her milk.
4.4.10 Pros and Cons of GMOs 3/30/11 3:03 PM
4.4.10 Discuss the potential benefits and possible harmful effects of one example of genetic modification.
Orange book pg. 161Green book pg. 70WS15 : Ethics of GMO Technology
As an introduction, watch the Youtube clip: “GM foods and You”http://www.youtube.com/watch?v=B8p7M0WF_7A
The information in the green book is good for this section so make sure you read through it.
Is genetic engineering a good or bad thing?Genetic engineering raises many profound social and ethical questions. As you read through the ideas below, think about which ones you agree with and can you justify your opinions?
Benefits, promises and hopes for the future GM crops will help farmers by improving food production GM crops which produce their own pest-control substances will be
beneficial to the environment because fewer chemical pesticides will be needed. Reduction in use of pesticides leads to less contamination of ground water around farmland, less harm to non-target species. Financial benefit not having to pay for pesticides and a ‘greener’ image.
Using GMOs to produce rare proteins for medications or vaccines could be, in the long run, less costly and produce less pollution than synthesizing such proteins in laboratories.
Farmers can be more in control of what crops or livestock they produce. There is always some randomness in breeding; genetic modification makes the process less of a gamble. It is also much quicker than selective breeding.
The traits agricultural scientists are incorporating into our crops through genetic engineering are the same as those traits that have been incorporated by selective breeding e.g. improved nutritional content, delayed ripening, resistance to disease caused by bacteria, fungi and viruses, better taste, ability to withstand harsh environmental conditions, resistance to pests such as insects and weeds.
The multinational companies who make GM plants claim that they will enable farmers in developing nations to help reduce hunger by using pest-resistant crops of GM plant which require less water.
As of January 2000 in over 30 countries, 10000 field tests of over 100 species have been carried out. Over 100 million acres of genetically modified foods have been grown – there have yet to be any unanticipated adverse environmental consequences.
Harmful effects, dangers and fears No-one knows the long-term effects of GMOs in the wild. Efforts to keep
GM plants under control in well-defined areas have failed and pollen from GM crops has escaped to neighbouring field. Genes from GM plants could be integrated into wild species giving them an unnatural advantage over other species and an ability to take over the habitat.
There is a danger that the genes could cross species. It has been proves possible in laboratories, so there is a possibility in nature too. Again, no one knows the consequences of genes crossing species.
BT-crops which produce toxins to kill insects could be harmful to humans because, unlike chemical pesticides which are only applied to the outer surface, the toxins are found throughout the plant.
There are risks for allergies: if someone is not allergic to natural tomatoes, but is allergic to GM tomatoes, they will need to know which ones they are eating. But there is no difference in the outward appearance of the fruit and food labelling is not always clear.
Critics are worried that large portions of the human food supply will be the property of a small number of corporations
High-tech solutions are not necessarily better than simpler solutions. Crop production could be increase by teaching farmers how to use water and natural pest-control systems more efficiently
A proliferation of genetically modified organisms may lead to a decrease in biodiversity.
4.4.11 Clone 3/30/11 3:03 PM
4.4.11 Define clone.
Orange book pg. 165Green book pg. 70
To do:Do exactly as it says in the objective - but write it in your green books.
Clones and CloningThe definition of a clone is a group of genetically identical organisms or a group of cells artificially derived from a single parent. In either case, the resulting cells or organisms were made using laboratory techniques. In farming, clones have been made for decades by regenerating plant material or by allowing an in-vitro fertilized egg to divide to make copies of itself.
4.4.12 Cloning 3/30/11 3:03 PM
4.4.12 Outline a technique for cloning using differentiated animal cells.
Orange book pg. 165Green book pg. 71-72WS16: Cloning by Nuclear Transfer
To do:“Click and Clone”http://learn.genetics.utah.edu/content/tech/cloning/clickandclone/
“Try cloning a dog using the same method to create Dolly”http://www.vtaide.com/png/cloning.htm
Draw and annotate a diagram to show the process in nuclear transfer cloning.
CloningCloning is of great commercial importance, as brewers, pharmaceutical companies, farmers and plant growers all want to be able to reproduce “good” organisms exactly.
Natural methods of asexual reproduction are often quite suitable for some organisms (such as yeast, potatoes and strawberries), but many important plants and animals do not reproduce asexually, so artificial methods have to be used.
Until recently, cloning was only possible using genetic information from an egg cell. Fertilised eggs are not differentiated (specialized) yet. After dividing many times, some cells will specialize into muscle cells, others into nerve cells, others into skin cells and so on until a foetus forms. For a long time, it was thought that once a cell has gone through differentiation, it cannot be used to make a clone. But then along came Dolly …
Cloning using a differentiated animal cellSomatic Cell Cloning (or Nuclear Transfer)Until recently it was thought impossible to grow a new animal from the somatic cells of an existing animal (in contrast to plants). However, techniques have gradually been developed to do this, first with frogs in the 1970s, and most recently with sheep (the famous “Dolly”) in 1996.
1. From the original donor sheep to be cloned, a somatic cell (non-gametic cell) from the udder was collected and cultured. The nucleus was removed from a cultured cell.
2. An unfertilized egg was collected from another sheep and its nucleus was removed.
3. The somatic udder cell was fused with the unfertilised egg cell which had had its nucleus removed.
4. This combination of a diploid nucleus in an unfertilised egg cell was a bit like a zygote, and sure enough it developed into an embryo in vitro.
5. The embryo was implanted into the uterus of a surrogate mother, and developed into an apparently normal sheep, Dolly.
It took hundreds of attempts to achieve success with Dolly, but once the technique is improved it will be possible to combine this technique with embryo cloning to make many clones of an adult animal. Dolly’s “mother” was just an ordinary sheep, but in the future prize animals (or genetically engineered ones) could be cloned in this way.
4.4.13 Ethics 3/30/11 3:03 PM
4.4.13 Discuss the ethical issues of therapeutic cloning in humans.
Orange book pg. 165Green book pg. 72
Read the information in the green book and below.
In your books, write a paragraph summarizing some of the ethical issues surrounding therapeutic cloning in humans.
Ethical issues surrounding therapeutic cloningSince therapeutic cloning starts with the production of human embryos, it raises fundamental issues of right and wrong. Is it ethically acceptable to generate a new human embryo for the sole purpose of medical research? In nature, embryos are created only for reproduction and many people believe that using them for experiments is unnatural and wrong.
However, the use of embryonic stem cells has lead to major breakthroughs in the understanding of human biology. What was once pure fiction is coming closer and closer to becoming an everyday reality thanks to stem cell research: Growing skin to repair a serious burn Growing new heart muscle to repair an ailing heart Growing new kidney tissue to rebuild a failing kidney.
With very rare exceptions, the vast majority of researchers and medical professionals are against the idea of reproductive cloning in humans. However, there is a growing popularity for the pursuit of therapeutic cloning since the promises of stem cell research are so enticing.
Reproductive cloning is performed with the express intent of creating another organism. This organism is the exact duplicate of one that already exists or has existed in the past. Cloning of plants, animals, and humans falls into the class of reproductive cloning.
Therapeutic cloning is performed, not to produce another organism, but to harvest embryonic stem cells for use in medical treatments. Embryonic stem cells are those cells found inside of developing embryos. They can be used to produce a number of different cells including tissue, muscle, and organ cells
