IB BIOLOGYTOPIC 4.4: GENETIC ENGINEERING AND BIOTECHNOLOGY

I. What is Biotechnology?

A. Biotechnology: broadly defined as the use of living things and their products for the production of food, drink, medicine or for other benefits to humans or other animal species

1. Often involves the manipulation of DNA, often referred to as genetic engineering

2. Recombinant DNA: DNA from two different sources

3. Transgenic: refers to individuals that receive genes from other species

II. The Polymerase Chain Reaction (PCR)

Two animations of the polymerase chain reaction, they are very similar but the second one has some details that are not in the first one: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::640::480::/sites/dl/free/0073532223/811328/polymerase_chain_reaction.swf::Polymerase%20Chain%20Reaction

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::640::480::/sites/dl/free/0073532223/811328/pcr_reactions.swf::PCR%20Reactions

Figure 1. The steps in the polymerase chain reaction.

http://highered.mcgraw-hill.com/sites/0073532223/student_view0/chapter17/image_powerpoint_for_students.html

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A. Method used to make many copies of a small piece of DNA, even if the target DNA is only present in small quantities in a mixture of DNA

1. Invented by Kary Mullis for which he won a Nobel Prize in Chemistry in 1993, shared with Dr. Michael Smith (you know...from UBC)

http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/

B. Steps

1. First must know at least the beginning of the sequence of the target DNA

2. Synthesize primers that are about 20 nucleotides long (recall that primers are needed by DNA polymerase)

a. Need 2 primers for each strand of DNA, one for each of the 2 complementary strand

3. Make a mixture containing the target DNA, primers, all four deoxynucleotides (triphosphates), a DNA polymerase (common one is called the Taq polymerase (because it comes from a bacterium called Thermus aquaticus that grows in hot springs and is stable at high temperatures))

4. Put mixture in a thermocycler, a device that raises and lowers the temperature at set intervals

5. Set temperature high (98C for 5 minutes) to cause the DNA to denature and break into single strands

6. Lower the temperature (60C) so that the primers can anneal to the target strands

7. Heat mixture back to 70C to 72C, the optimal temperature for the Taq polymerase

8. Taq polymerase makes DNA strands that are complementary to the strands with the primer attached, other DNA strands in the mixture will not have primer and will not be replicated

9. A single strand of the target DNA has been made into 2 strands

10. Thermocycler automatically repeats the 3 temperature sequence and the number of target DNA strands increases exponentially 1, 2, 4, 8, etc.

11. Theoretically, after about 20 cycles, there will be about 1 million copies of the target DNA (220)

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a. in 0.1 microliters of saliva containing a small number of epithelial cells shed from the lining of the mouth, there is enough DNA to identify whether the DNA is human or not

b. by using different primers which represent regions of genes from different organisms or individuals for each PCR event, one can determine whether any DNA has been amplified

i. if no DNA was amplified, the target DNA most likely did not exist in the sample

*A little bit more detail can be found here: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml

To play a game using your knowledge of PCR: http://nobelprize.org/educational/chemistry/pcr/about.html

A virtual PCR is here: http://learn.genetics.utah.edu/

Animation and quiz about DNA microarrays: http://glencoe.mcgraw-hill.com/sites/0035456775/student_view0/chapter14/dna_printer.html

III. Gel Electrophoresis

A virtual gel electrophoresis is here: It is pretty slow but easy to understand.)http://learn.genetics.utah.edu/content/labs/gel/

Animation of DNA fingerprinting:http://glencoe.mcgraw-hill.com/sites/0035456775/student_view0/chapter14/dna_fingerprinting.html

A. A technique for separating DNA strands (or proteins) according to size

1. Requires a large amount of DNA which can be produced by PCR

B. Steps

1. cut DNA into fragments using enzymes called restriction enzymes/restriction endonucleases

An animation with a quiz about restriction enzymes: http://glencoe.mcgraw-hill.com/sites/0035456775/student_view0/chapter14/restriction_endonucleases.html

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An animation with a quiz about restriction fragment length polymorphisms: http://glencoe.mcgraw-hill.com/sites/0035456775/student_view0/chapter14/restriction_fragment_length_polymorphisms.html

a. each restriction enzyme recognizes a specific nucleotide sequence and “cuts” the DNA strand at that site

i. since everyone’s genome is different the number of times the restriction enzyme “cuts” and the sizes of the resulting fragments is unique to each individual

ii. by using different restriction enzymes, can isolate, sequence and manipulate individual genes

2. DNA sample with a dye to make the sample visible is put into a hole/well in a block of agarose gel

a. There can be a number of holes/wells in one block of gel so that different samples can be run at the same time

3. A reference DNA sample with DNA fragments of known size is placed in one of the wells

4. Gel is placed in an electrophoresis chamber that generates a current such that the side of the gel away from the wells is positively charged

a. since DNA fragments are negatively charged, they will move toward the positive electrode

i. larger fragments do not move through the gel as quickly as smaller fragments due to the size of the pores in the gel

5. DNA is stained with ethidium bromide which makes it visible under UV light

6. the sample DNA can be compared with the reference DNA to see what sizes of fragment are in the sample

Figure 2. Example of what a gel electrophoresis looks like over time. Note that Sample D does not match the other 3 samples.

http://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/Bio%20101/Bio%20101%20Lectures/Biotechnology/biotechn.htm

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C. DNA profiling

1. using gel electrophoresis to compare sample DNA with known DNA to see if they are the same or similar enough to draw conclusions about the identities of the people the samples came from or the relationships between the people the samples came from

a. some segments of the human genome are known to be more highly variable than others, so are chosen for DNA profiling

2. Used in paternity testing

3. Used in forensic investigations.

a. samples can come from any DNA-containing tissue of body such as cheek cells, hair, blood, skin, and semen

b. For example: suppose samples A and B in Figure 2 came from a crime scene and samples C and D came from 2 different suspects. What conclusion would you draw?

4. can be used to identify certain genetic diseases such as sickle cell disease, Huntington’s disease and Duchenne muscular dystrophy 5. can be used in taxonomy and evolutionary biology to determine the relative amount of similarity in the genomes of different organisms 6. in conservation biology, can be used to determine kinship among individuals so that inbreeding can be minimized in zoos or captive breeding programs

D. Practice

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1. http://www.life.illinois.edu/molbio/geldigest/assign1.html (question 2 is more than you need to know, has answers)

2. http://www.biology.arizona.edu/molecular_bio/problem_sets/ Recombinant_DNA_Technology/07Q.html

3. http://www.biology.arizona.edu/molecular_bio/problem_sets/ Recombinant_DNA_Technology/08q.html

IV. The Human Genome Project

http://sandwalk.blogspot.com/2007_02_25_archive.html

This prof’s blog is mostly about bio if that is what you are interested in ;)

http://www.nature.com/nature/journal/v404/n6774/full/404117a0.html

This one is kind of funny but did not copy over, http://questgarden.com/23/30/8/060422170530/process.htm

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http://dlibrary.acu.edu.au/research/theology/ejournal/aejt_2/curran.htm

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http://www.myspace.com/dna25

http://www.genome.gov/Images/press_photos/highres/38-300.jpg

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A. What was it?

Francis Collins, the director of NHGRI, noted that the genome could be thought of in terms of a book with multiple uses: "It's a history book - a narrative of the journey of our species through time. It's a shop manual, with an incredibly detailed blueprint for building every human cell. And it's a transformative textbook of medicine, with insights that will give health care providers immense new powers to treat, prevent and cure disease."

1. A project to completely sequence (write out all the nucleotides in order) the human genome

a. Began in 1990

b. In 1992 was expected to take 15 years to complete

c. Originally nearly 100 people donated their blood, but only a few of the samples were actually used

i. since no names were attached, this ensured anonymity for the donors

2. The first draft (about 90%) complete was published in February 2001 with the completed genome published in April 2003

B. The players

1. National Human Genome Research Institute represented by Dr. Francis Collins led an international consortium known as the International Human Genome Sequencing Consortium

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2. Celera Genomics representing J Craig Venter (recently in the news for the creation of “artificial life” (

An article about their testy relationship: http://www.nature.com/nature/journal/v404/n6774/full/404117a0.html

C. What did we find out?

1. There are about 20 500 human genes (many general estimates were about 30 000)

2. When the project began, fewer than 100 human disease genes had been identified but by then end, there were more than 1 400

3. In addition, DNA sequences from different populations was done to find out what kinds of variation there is by identifying large blocked of DNA sequences (called haplotypes) that tend to be inherited together

a. Called the HapMap

b. Haplotypes of people with a particular disease can be examined to look for associated genes

4. The Cancer Genome Atlas Project

a. Genetic factors involved in cancer

D. Ethical issues: potential benefits and harms

Extensive material can be found here: http://www.genome.gov/Issues/

1. Coverage and reimbursement in genetic testing

2. Intellectual property and genomics (gene patenting)

3. Health issues: genetic testing, personalized medicine

4. Genetic discrimination

5. Informed consent for research participants

E. How to Sequence a Genome

Animations for every step: http://www.genome.gov/25019885

1. Make “maps” of the human genome by identifying landmarks to help them know what sequences belonged to different chromosomes

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2. Then create “libraries” of clones that contain small fragments of human DNA

3. Store these “libraries” in bacteria (E. coli)

a. Can be stored in freezers indefinitely and brought to 37C when needed

b. The E. coli also copy the BAC

i. in one night, billions of E. coli cells can be made

4. Divide up the genome and distribute to labs around to world and coordinate the work

5. The clone libraries used bacterial artificial chromosomes or BAC’s containing 100 000 to 200 000 bases of DNA and were used to establish the order of the DNA sequences

6. To actually sequence the DNA, the BAC clones were cut into smaller fragments of about 2 000 bases

a. These fragments were stored in viruses called phage that infect E. coli cells

7. Sequencing requires the template DNA, free nucleotide bases (some of which have a fluorescent dye attached), primers, and DNA polymerase

a. Primer binds to the complementary base sequence on the DNA

b. Free nucleotides attach to the 3’ end of the primer and the strand grows

c. When a nucleotide with the dye attached is added, the growing DNA stops growing

8. The result is a whole lot of coloured DNA fragments: the shortest ones are the primer +1 nucleotide, the longest are 500 to 800 bases long

9. These are fed into an automatic sequencing machine that reads the samples

a. First the fragments are separated by gel electrophoresis

b. As the fragments reach the end of the gel, a laser activates the fluorescent dye and a camera detects the colour

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c. The machine reads the colours (remember each nitrogenous base has its own colour) and translates the colour into the letter corresponding to the nitrogenous base

10. each sequence reaction can find the order of several hundred bases

11. data from individual sequencing reactions are read and integrated by a computer in order to put them into the order in which they would appear on the original chromosome

a. many overlapping sequences are need to do this

b. on average, every base pair of DNA was sequenced 9 times

i. more than 50 million sequencing reactions were done by over 2000 scientists in more than 2 dozen labs around the world

V. Gene Transfer

A. the addition of a desired gene into the genome of a host cell so that the host cell will make a gene product that it could not have made before the alteration

B. Steps

Animation of steps of cloning a gene: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::640::480::/sites/dl/free/0073532223/811328/cloning_gene.swf::Steps%20in%20Cloning%20a%20Gene

Animation of gene transfer in plants using Ti-plasmid:http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::640::480::/sites/dl/free/0073532223/811328/Gene_Transfer_in_Plants_Using_the_Ti_plasmid.swf::Genes%20Transfer%20in%20Plants%20Using%20the%20Ti-plasmid

Figure 3. Steps in gene transfer to a plant.

http://highered.mcgraw-hill.com/sites/0073532223/student_view0/chapter17/image_powerpoint_for_students.html

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1. Gene of interest is cloned

a. Isolate the gene

b. Purify it and cut it with a restriction enzyme

i. Now it has 2 “sticky” ends

c. Remove a plasmid from Agrobacterium

d. Plasmid of Agrobacterium is cut open with same restriction enzyme/restriction endonuclease

i. Now it has 2 “sticky” ends, that match with the ends of the gene of interest

e. Insert the gene of interest into the plasmid

i. the gene will hybridize with the plasmid at the sticky ends

ii. ligase will join the ends together

f. Put the plasmid back into the Agrobacterium

g. Allow the Agrobacterium to divide in a medium

h. Isolate the clones of the Agrobacterium that have the gene of interest

i. usually this is done by attaching a second gene that codes for some ability that can easily be tested for such as antibiotic resistance or

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production of a fluorescing protein

2. Allow the Agrobacterium to infect host plant cells

3. Now the host plant cells will have the gene of interest and express the gene when it expresses its own genes

4. Any daughter cells will also have the gene of interest too

C. Current uses of genetically modified crops or animals

1. Salt tolerance in tomato plants

2. Synthesis of beta-carotene, the precursor of Vitamin A in rice

3. Herbicide resistant crop plants

a. Roundup resistance in plants designed by Monsanto

b. Government of Canada info about Roundup ready corn 2001: http://www.hc-sc.gc.ca/fn-an/gmf-agm/appro/roundup_ready_corn_603-mais_603_roundup_ready-eng.php

4. Factor IX (for human blood clotting) in sheep milk

5. Pest-resistant crops such as Bt cotton, Bt corn and Bt potatoes (more info: http://www.bt.ucsd.edu/index.html)

1. Produce a natural insecticide that is naturally produced by the bacterium Bacillus thuringiensis (Bt)

2. Sequences of genes from Bt to express the crystal protein made by the bacterium are inserted into plants so that the plants can make the crystal protein

3. Makes the crops resistant to: European corn borer, southwestern corn borer, tobacco budworm, cotton bollworm, pink bollworm, and the Colorado potato beetle

4. Other benefits: reduced use of pesticides, increases in beneficial insects (Bt proteins only work on specific insects)

5. Potential risks: potential for the transgenic organism to become invasive, potential for the pests to become resistant, potential cross-contamination of genes (genes transferred to native species)

A short animation and quiz about pest-resistant plants: http://glencoe.mcgraw-hill.com/sites/0035456775/student_view0/chapter14/pesticide_plants.html

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A short animation and quiz on GM Food Safety:http://glencoe.mcgraw-hill.com/sites/0035456775/student_view0/chapter14/gm_food_safety.html

D. Potential benefits and dangers

A summary of ideas is presented here, specific to Monsanto:http://web.mit.edu/demoscience/Monsanto/impact.html

VI. Cloning

A. Cloning: producing a genetically identical copy of a single gene, cell, group of cells or multicellular organism

1. Gene cloning: copying of a single gene (discussed earlier in these notes)

2. Reproductive cloning: creation of an individual using the genome of another currently or previously existing animal

3. Therapeutic cloning: the creation of an embryo to supply embryonic stem cells for medical use

B. Can be done on plants by cutting off stems or leaves and planting them (common technique for many houseplants)

a. A modification of what many plants do e.g. strawberries produce modified stems called runners, ferns produce specialized stem called rhizomes, onions, grass, and potatoes can also have this type of reproduction

C. First animal cloning done in the 1970’s by John Gurdon. The tadpoles did not survive and grow into adult frogs.

Figure 4. The steps in cloning from tadpoles.

http://science.howstuffworks.com/environmental/life/genetic/cloning2.htm

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D. 1997: Ian Wilmut et al produce a cloned sheep named Dolly.

a. Dolly lived until age 6 (most Finn Dorset sheet live 11 or 12 years)

b. Mother to 6 lambs

c. Dolly suffered from lung cancer and crippling arthritis

d. Dolly not identical to the nucleus donor because the mitochondria were from the egg donor

Figure 5. Steps in cloning Dolly: Somatic cell nuclear transfer (SCNT).

http://science.howstuffworks.com/environmental/life/genetic/cloning3.htm

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E. Steps in Somatic Cell Nuclear Transfer (using Dolly the sheep’s example)18

*first clone from adult cells

1. Remove the nucleus from and sheep egg cell

2. Isolate somatic cell from adult sheep

3. Fuse the somatic cell to the enucleated egg cell using electricity

a. Also stimulates cell division

4. New cell placed in surrogate mother and allowed to develop to term and delivered

5. It took 276 attempt before success!

F. Modifications of SCNT since Dolly

1. Can remove nucleus from somatic cell and insert it directly into the enucleated egg cell

G. Steps in Artificial Embryo Twinning

1. Manual separation of very early embryo into individual cells

2. Each cell then divides and separates on its own

3. Place resulting embryos into a surrogate mother

4. Embryos carried to term and delivered

H. Uses of reproductive cloning

1. Cloning of endangered animals:

a. First clone of an endangered animal: gaur (January 8, 2001), a large wild ox from India and SE Asia

b. Mouflon, endangered wild sheep also in 2001

c. Many animals that are potential candidates

d. For a short list of news about animals that have been cloned: http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml#animals

2. Cloning extinct animals

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a. Challenge because the egg donor and nucleus/genetic material/DNA donor would not be the same species

I. Therapeutic cloning

1. Uses

a. Produce whole organs from single cells

b. Replace damaged cells in degenerative diseases such as Alzheimer’s or Parkinson’s

2. Potential uses

a. Extract DNA from the person needing the transplant

b. Insert the DNA into an enucleated egg

c. Harvest the embryonic stem cells

d. Use the stem cells to produce any organ or tissue that is required

3. Challenges

a. Need more effective technologies for creating human embryos, harvesting stem cell, producing organs

b. In 2001, first cloned human embryos announced by Advanced Cell Technology but embryo stopped dividing after 6 cells

* Be sure to distinguish between reproductive cloning and therapeutic cloning.

Ethical issues associated with cloning:

Reproductive:

Therapeutic:

References:

1. http://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/Bio%20101/Bio %20101%20Lectures/Biotechnology/biotechn.htm

2. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/PCR.html 3. Teacher’s manual for Mader’s Inquiry into Life, 10th edition.4. http://people.ku.edu/~jbrown/pcr.html 5. http://biotech.about.com/od/whatisbiotechnology/a/whatisbiotech.htm 6. http://www.genome.gov/25019885

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7. http://genome.pfizer.com/tour.cfm This is the website for an exhibit that was at the Smithsonian in Washington DC.

8. http://www.accessexcellence.org/RC/AB/IE/Intro_The_Human_Genome.php 9. http://www2.needham.k12.ma.us/nhs/cur/Baker_00/2001_p2/

baker_dc_ad_p.2/human_genome.htm10. http://www.genome.gov/1801686311. http://science.howstuffworks.com/environmental/life/genetic/cloning.htm12. http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml13. http://learn.genetics.utah.edu/content/tech/cloning/whatiscloning/14. http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml#animalsQ

Extra:

Cloning and bacterial transformation: http://www.wiley.com/college/boyer/0470003790/animations/cloning/cloning.swf

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