IB BIOLOGY
TOPIC 4.4 GENETIC ENGINEERING AND BIOTECHNOLOGY
I. WHAT IS BIOTECHNOLOGY?
A. biotechnology: the use of living things to create products that are useful to humans
1. these products can include new and/or improved food, medicines, vaccines, methods of
repairing damaged tissues/organs, tests to detect disease, treatment for human
infertility, bacteria that can produce substances that are useful to humans, bacteria
that are capable of cleaning the environment, biofuels and more!
B. biotechnology ranges from the application of selective breeding (something that humans
have done for centuries) which results in genetic modification to the more modern
methods of genetic engineering, cloning, genome sequencing, proteomics (the study of
the proteins that result from the genome) etc.
1. genetic modification refers to anything that can change the genetic material of an
organism
2. genetic engineering refers to genetic modifications that occur through direct
modification on the DNA (e.g. by the addition or removal of genes, also known as
recombinant DNA technology)
3. transgenic: refers to organisms that have a gene(s) from another organism inserted
into their chromosomes
4. mutagenesis: methods that cause mutations in the DNA without adding DNA from
another organism using radiation or mutagenic chemicals
a. site-directed mutagenesis can target specific genes
C. the IB Syllabus focuses on the following: the polymerase chain reaction (PCR), gel
electrophoresis, DNA profiling, the sequencing of the human genome, genetic modification
involving gene transfer between species using plasmids, and cloning.
II. VOCABULARY
A. plasmid: a small, circular DNA often found in bacteria
1. usually 2.5 to 20 kilobases
B. Vector: an agent used to carry genetic material into a cell
a. usually a virus or a plasmid

C. Recombinant DNA: DNA from two or more organisms joined into a single DNA molecule
D. Restriction enzyme: enzymes from bacteria that are able to cut the DNA at specific
nucleotide sequences to produce DNA fragments
1. the cut can produce either blunt or staggered/”sticky” ends on the DNA fragments
Figure 1 . Using restriction enzymes to make recombinant DNA. http://content.bfwpub.com/webroot_pubcontent/Content/BCS_3/Sadava_9e/Interactive%20Su mmaries/1810.html

2. used in making recombinant DNA and in DNA mapping
3. here is an interactive animation of how a restriction enzyme works: http://www.classzone.com/cz/books/bio_07/resources/htmls/animated_biology/unit3/bio_ch09_
0266_ab_enzyme.html
III. SELECTED TECHNIQUES USED IN MODERN BIOTECHNOLOGY AND THEIR

APPLICATIONS
A. The Polymerase Chain Reaction
Here are some animations of PCR:
1.
This is a 3D animation: http://www.dnalc.org/view/15475-The-cycles-of-the-polymerasechain-reaction-PCR-3D-animation-with-no-audio.html
2.
This is a step-through animation that you control yourself or a narrated animation: http://www.sumanasinc.com/webcontent/animations/content/pcr.html
3.
This is a step-through animation that you control yourself: http://www.dnalc.org/view/15924-Making-many-copies-of-DNA.html
4.
Another animation: http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html
1. invented by K. Mullis in 1985 (1983) and awarded the Nobel prize for Chemistry in
1993
a. he has his own, eponymous, website here: http://www.karymullis.com/pcr.shtml
b. Mullis, K. B., "The Unusual Origin of the Polymerase Chain Reaction", Scientific
American, April 1990. http://www.cs.brown.edu/courses/cs2952/papers/TheUnusualOriginofPCR.pdf
c. You can read his Nobel lecture here: http://books.google.ca/books?hl=en&lr=&id=XBM_VE6624MC&oi=fnd&pg=PA103&dq=the+poly merase+chain+reaction&ots=G52AiBeinT&sig=YdUXtGz11QjZo14LgboOGk9P4U8#v=onepage&q
=the%20polymerase%20chain%20reaction&f=false
2. is now automated and utilizes a machine called a thermocycler
3. used to make many (MANY!) copies of a segment of DNA (cloning a segment of DNA)
a. need to know what sequence of DNA you wish to copy
i. this is where sequencing comes in handy
4. synthesize (or purchase!) primers that are complementary to the 3’ end of the DNA
molecule that will be copied
5. make a mixture that includes:

a. the DNA section of interest
b. the required primer
c. the DNA deoxynucleotide triphosphates (all 4 of them)
d. DNA polymerase
i. the one most often used is called Taq polymerase because it comes from a
thermophilic bacterium called Thermus aquaticus
a . the bacterium normally lives in hot springs so the high temperatures used
for PCR will not cause it to denature b . the optimal temperature for this enzyme is between 75°C and 80°C.
ii. another enzyme used is called the Pfu polymerase from Pyrococcus furiosus a . also heat resistant
b . copies DNA more accurately than the Taq polymerase
e. DNA ligase
6. heat the DNA sample to be copied in order to separate the template strands
a. temperature is 90°C-95°C
7. now lower the temperature to about 50°C-65°C so that the primers will anneal/bind to
the complementary DNA
8. now raise the temperature to about 72°C so the DNA polymerases will add
DNA deoxyribonucleotides that are complementary to the parent strands
a. recall that polymerases must always add nucleotides to the 3’ end of the primer
or growing DNA strand
10. the ligase joins the sugar-phosphate backbone together
11. repeat the steps from 6-8 many times
a. each cycle doubles the number of DNA molecules and takes less than 5 minutes
b. after 30 cycles, there can be more than 1 billion copies of the original DNA strand

12. Applications of PCR
a. cloning
b. genetic engineering
c. sequencing
d. diagnosis of genetic disorders
13. Limitations of PCR
a. very sensitive to the levels of divalent cations and nucleotides
b. the primers must be designed/chosen very carefully
i. must not react with non-target DNA
ii. must not anneal to each other
c. contamination occurs easily
d. best results occur when the target/template DNA is 300 to 1000 bp long
14. A little trivia:
a. when Mullis invented PCR, he was working for Cetus, one of the first biotech
companies
b. before the discovery of the Taq polymerase, fresh enzyme had to be added at the
end of every cycle
c. Cetus developed the first thermocycling machine to address the issue above
d. purification of the Taq polymerase created the need for a machine that cycled
through the different temperatures more efficiently

d. Roche Molecular systems bought the PCR patent and associated technology from
Cetus for $ 300 000 000 (more info on this here: http://siarchives.si.edu/research/videohistory_catalog9577.html
)
Here is a virtual lab...you manipulate the lab equipment... (I personally have trouble...not enough gaming experience, I guess): http://learn.genetics.utah.edu/content/labs/pcr/
Here is an interactive explanation of this protocol: http://www.classzone.com/cz/books/bio_07/resources/htmls/animated_biology/unit3/bio_ch09_
0270_ab_pcr.html
B. Reverse Transcriptase PCR (RT-PCR)
1. the job is to create multiple copies of DNA that is complementary to a strand of mRNA
2. before PCR is done, RNA is copied into DNA (cDNA) by reverse transcriptase
a. the RNA can come from a virus
i. scientists at the Genome Sciences Centre in Vancouver were the first to
sequence the RNA genome of the SARS virus in 2003
( http://www.sciencemag.org/content/300/5624/1399.full
)
3. used to measure viral load (the amount of viral RNA in a sample)
C. Recombinant DNA Technology
1. Making recombinant DNA
a. See Figure 1 above.
b. treat both DNA samples (from different sources) with the same restriction enzyme
c. the cut ends are “sticky” so the DNA from the different sources can combine with
each other
d. DNA ligase will join the ends together
e. many copies of the recombinant DNA are needed
i. produced by DNA cloning using PCR or using living cells
2. Method of gene transfer

Figure 2. Recombinant DNA in the laboratory. http://content.bfwpub.com/webroot_pubcontent/Content/BCS_3/Sadava_9e/Interactive%20Su mmaries/1810.html

D. Cloning
1. clone: an individual with the identical DNA as another, the process of producing an
clone or identical DNA
2. DNA Cloning
a. in vitro by PCR
b. in vivo (in living cells) the recombinant DNA is taken up, replicated and
expressed by the cell
Here is a step-through tutorial that includes the transformation of bacteria with recombinant
DNA: http://biology.kenyon.edu/courses/biol09/plasmid/plasmidtut1.htmv
Here is another interactive tutorial: http://www.wiley.com/college/boyer/0470003790/animations/cloning/cloning.htm\
i. fragmentation: the DNA of interest is made into fragments of a suitable size
by restriction enzyme digestion so that it has “sticky ends”
ii. a bacterial plasmid is cut by the same restriction enzyme so that it also has
“sticky ends”
iii. recombinant DNA is produced when the fragments from the DNA of interest
and the bacterial plasmid fragment are joined by DNA ligase a . notice that there are a variety of different recombinant DNA molecules
that can result
Figure 3. Making a recombinant DNA plasmid. http://web.auckland.ac.nz/uoa/fms/default/science/about/departments/sbs/student_information
/schools/biotechnology/docs/genecloning.pdf

iv. the recombinant DNA plasmids are then mixed with bacteria that have
been treated to make them “competent” (capable of taking up plamids)
v. when the bacteria take up the recombinant DNA, they are said to be
“transformed”
vi. to check to see if the transformation has been successful, grow/culture
the bacteria a . the chosen vector usually has a marker than can be recognized as
some property of the bacteria such as antibiotic resistance b . the DNA of interest also makes the bacteria express a property/trait
that can be recognized
c . if the cultured bacteria express both of the traits, not just one, then
the production of the recombinant DNA and subsequent
transformation have been successful

c. Genetically modified crops/animals
i. salt-tolerant tomato plants
ii. golden rice
iii. Bt corn
iv. roundup ready crops
v. Factor IX in sheep milk
vi. spider silk in goat milk
d. recombinant DNA products being used in human therapy
i. insulin
ii. factor VIII for males with hemophilia A
iii. factor IX for haemophilia B
iv. erythropoietin for treating anemia
v. these and many more are from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html
e. Potential benefits and harms of genetic modification of organisms
3. Reproductive cloning
Cloning 101: http://www.dnalc.org/files/swfs/animationlib_swf/cloning_101.swf
a. reproductive: to produce individual organisms
i. could be used to bring back extinct species, increase the populations of
endangered species
ii. pro’s and con’s?
b. Method 1: Somatic Cell Nuclear Transfer
i. the method used to create Dolly the sheep
Figure 4. The process of somatic cell nuclear transfer.

http://www.biotechnologyonline.gov.au/popups/img_scnt.html
Note that the adult cell has to be “reprogrammed” so that it can behave as though it were unspecialized like a zygote. For more detail on this process: http://science.education.nih.gov/home2.nsf/Educational+ResourcesTopicsGenetics/BC5086E34E
4DBA0085256CCD006F01CB
Here is a paper about the progress of this process (as of 2002) written by one of the scientists that produced Dolly the sheep in 1996: http://cromatina.icb.ufmg.br/ciclo_celular/somatic.pdf
ii. Dolly the sheep: the first mammal to be cloned from an adult cell
a . born on July 5, 1996, died on February 14, 2003
b . cloned using a mammary gland cell from a healthy six-year old sheep
i. the c . had to be put down after developing a viral lung disease that usually
affects older animals d . she also developed arthritis earlier than is normal e . when she was one, her telomeres were shorter than would normally be
expected for a sheep of her age

i . speculation that animals cloned from adult cells would age
prematurely and die early was later shown to be untrue ii . telomere length is restored during the cloning process
f . she is on display at the National Museum of Scotland g . 4 other sheep have been cloned from the udder tissue that was used to
make Dolly ( http://www.dailymail.co.uk/sciencetech/article-
1334201/Dolly-reborn-Four-clones-created-sheep-changed-science.html
)
iii. mammals successfully cloned include sheep, goats, cows, mice, pigs, cats,
rabbits and a gaur
iv. the success rate of cloning is still very low and other cloned mammals have
tended to die early, have health issues, abnormal gene function, etc. http://www.newscientist.com/article/dn1903-cloned-animals-meet-early-deaths.html
http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml
c. Method 2: Twinning or Embryo Splitting
i. manual separation of the early embryo into individual cells
d. Method 3: the “Honolulu Technique”
i. similar to SCNT but used cells that are naturally usually in the G0 stage ofthe
cell cycle (Sertoli, brain and cumulus cells), the nuclei from the donor cells
were extracted and introduced into the enucleated ova immediately after the
donor cells were removed from the donor, and a chemical bath (not an
electrical stimulus) was used to stimulate cell growth and development
3. Therapeutic cloning
a. therapeutic: to replace cells, to cure genetic diseases, to generate organs for
transplant, to treat diseases such as Parkinson’s (mice with a disease similar to
Parkinson’s were made healthy again using neurons cloned using their own skin
cells)
b. Ethics
i. where do the donor cells come from?
ii. stem cells and cancer cells have similarities that are not well-understood

E. Gel Electrophoresis
Here are some animations: http://www.colorado.edu/Outreach/BSI/pdfs/gel_electrophoresis.pdf
http://www.dnalc.org/resources/animations/gelelectrophoresis.html
http://bcs.whfreeman.com/thelifewire/content/chp16/1602001.html
Here is an interactive tutorial: http://www.phschool.com/science/biology_place/biocoach/red/gel.html
Here is a virtual lab: http://learn.genetics.utah.edu/content/labs/gel/
This site walks you through the procedure and has photos of the actual equipment: http://www.life.illinois.edu/molbio/geldigest/electro.html
1. technique for separating DNA molecules by size
a. prepare a gel made of agarose
i. like a very firm jello that can be molded with wells for the initial placement
(loading) of the DNA sample
b. place the gel into a buffer solution (a salt solution that conducts electricity)
c. load the DNA samples into the wells
i. a tracking dye is mixed with the DNA so that the movement of the DNA can be
observed
ii. the DNA is stained with ethidium bromide so that it glows when exposed to UV
light
d. an electric current is applied to the gel by putting it into a
i. the samples are loaded at the negative end
ii. the DNA is negatively charged because of the phosphate groups in the backbone
so will migrate towards the positively charged end
iii. the agarose matrix provides resistance to the movement of the DNA samples
iv. smaller fragments of DNA are able to move more easily through the gel so they
move faster and travel farther towards the positive end of the gel

e. the gel is then exposed to UV light so that the locations of the DNA sample
fragments can be seen
ii. a photograph of the gel can be taken
2. uses include DNA fingerprinting, diagnosing genetic diseases, paternity testing,
forensics, mapping lineages in zoo populations, drawing conclusions about evolution of
species
3. samples can come from any body tissue or fluid that has cells with DNA
4. generally used after PCR in order to focus in on certain regions of DNA
a. if trying to identify humans, would focus in on more highly variable regions of DNA
and compare the gels resulting from the samples given by 2 or more individuals to
look for similarities
b. if trying to identify a virus, would look for similar patterns that resulted from cuts
made by restriction enzymes
5. can also be used to separate proteins by size but gel is made of polyacrylamide
a. SDS (sodium dodecyl sulphate) PAGE (SDS PolyAcrylamide Gel Electrophoresis) is
a technique where the proteins are coated with SDS to make them negatively
charged
6. scientific dyes are mostly negatively charged so can be separated by gel
electrophoresis
F. Sequencing DNA
Here are some animations: http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html
http://www.dnalc.org/resources/animations/sangerseq.html
http://www.wiley.com/college/pratt/0471393878/student/animations/dna_sequencing/index.ht
ml
1. essentially a PCR + gel electrophoresis
2. the difference is that the PCR reaction uses special deoxyribonucleotides called
dideoxyribonucleotides that lack an OH group at the 3’ end so cannot have another

deoxynucleotide added to it
a. will terminate the PCR reaction and cause the newly formed strands of DNA to be
shorter than the parent strand template
b. the dideoxyribonucleotides are also treated so that they fluoresce under UV light;
each nucleotide fluoresces a different colour
3. the PCR products are placed into different lanes based on the type of
dideoxyribonucleotide that was used
4. when the PCR products are separated by gel electrophoresis, the length of the
fragment and the end dideoxyribonucleotide can be identified so that the DNA
sequence can be known
5. now the process is automated so that we can now run one “test tube” containing all of
the different dideoxyribonucleotides
a. they use “capillary electrophoresis” where the fragments come out of a tiny tube in
size order
b. a UV laser “reads” the liquid as it comes out of the tube and the colours are
translated into the letters of the nucleotides
6. remember that this technique can only read about 900 bases at a time so how could
the human genome actually be sequenced?
a. using enough restriction enzymes and piecing together any overlapping segments
can result in the entire genome of an organism being sequenced
For more detail on how to sequence large segments or entire genomes for that matter: http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
G. Sequencing the Human Genome
NOVA movie (It’s almost 2 hours long but really good because it also illustrates the human side of the scientists, the conflicts between science for profit and science for benefitting humankind, some potential applications etc.): http://www.pbs.org/wgbh/nova/body/cracking-the-code-oflife.html
H. DNA profiling or DNA fingerprinting
1. makes use of inherited DNA variations (polymorphisms) such as single nucleotide
polymorphisms (SNPs), short tandem repeats (STRs) which have 2-8 nucleotide units
and variable number tandem repeats (VNTRs) which consist of 9-80 nucleotide units

a. most are found in non-coding regions of the DNA, especially near centromeres and
are always found in pairs
b. at a particular locus, there may be different numbers of repeated units creating
different alleles
i. the definition of allele now includes different structures or sequences of DNA
(compare/contrast with the definition we learned for classical/Mendelian
genetics)
ii. a person can be either homozygous or heterozygous for the number of repeats
at any particular locus
iii. since there are many different markers used and many different alleles for each
locus, the probability that any two people will have exactly the same profile is
extremely small
Go through the process of making a forensic profile here: http://www.dnai.org/d/index.html
1. for paternity testing
2. for forensic investigations
I. Individualized medicine
A. theoretically, knowing ones’s genetic makeup and personal proteome (all of one’s
proteins) can help us to know how to prevent and treat illness/disease in a way that
takes into account the unique characteristics of each individual
1. should be more effective and efficient: people should be able to be healthy at birth,
stay healthy for longer, get better results from any treatments, use the correct
medicines in the correct doses (pharmacogenomics) (e.g. some people metabolize
codeine into morphine much more efficiently than others leading to morphine
poisoning), and have shorter hospital stays thus reducing the cost of health care
Interested? You might want to read The End of Illness by Dr. David Agus. I’m sure that there are more resources out there but that is what I am reading now (May, 2012)
FYI: SOME (RANDOM) BIOTECH PROGRAMS IN CANADA
1.
http://www.calendar.ubc.ca/vancouver/index.cfm?tree=12,215,410,946
2.
This is the Michael Smith Laboratories at UBC: http://www.msl.ubc.ca/
3.
http://www.bcit.ca/study/programs/8910bsc
4.
This is the club for UBC/BCIT biotech current and prospective students: http://www.ubcbcitbiotech.com/
5.
http://www.biology.ualberta.ca/research/microbiology_biotechnology/

6.
http://www.enterprisesaskatchewan.ca/life-sciences
7.
http://explore.usask.ca/programs/bt/agriculturebiotechnology
8.
http://www.lauriercc.ca/career/students/planning/major/biochem.htm
9.
http://science.yorku.ca/Academic-Programs/Biotechnology/
10.
http://www.utm.utoronto.ca/mbiotech/
11.
http://biology.concordia.ca/graduate/graduate-diploma-in-biotechnology-and-genomics/
12. http://biology.uwaterloo.ca/
13.
http://www.uoguelph.ca/registrar/calendars/undergraduate/current/c10/c10bscbiot.shtml
14.
http://biotechnology.lakeheadu.ca/
15.
http://flemingcollege.ca/programs/by-campus
16. http://biology.mcgill.ca/undergrad/minorprog_biotech.html
17.This is the biotechnology centre at McGill University: http://www.mcgill.ca/sheldon/
18.
References:
1.
http://www.hc-sc.gc.ca/sr-sr/biotech/index-eng.php
2.
http://www.accessexcellence.org/RC/AB/BC/what_is_biotechnology.php
3.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Proteomics.html
4.
http://www.inspection.gc.ca/english/sci/biotech/reg/terexpe.shtml
5.
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
6.
http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml
7.
http://www.sciencemag.org/content/300/5624/1399.full
8.
http://www.webmd.com/hiv-aids/viral-load-measurement\
9.
http://www.medicinenet.com/pcr_polymerase_chain_reaction/page3.htm
10. http://siarchives.si.edu/research/videohistory_catalog9577.html
11.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html
12.
http://bcs.whfreeman.com/thelifewire9e/default.asp#542578__591344__
13. Way more detail about PCR than needed for the course but interesting because it points out some technical difficulties associated with PCR: http://www.escience.ws/b572/L3/L3.htm
14. http://faculty.plattsburgh.edu/donald.slish/pcr.html
15.There is a simple game here about PCR and it’s the Nobel Prize website: http://www.nobelprize.org/educational/chemistry/pcr/
16. http://www.faseb.org/portals/0/pdfs/opa/The%20Polymerase%20Chain%20Reaction.pdf
17.
http://web.auckland.ac.nz/uoa/fms/default/science/about/departments/sbs/student_infor mation/schools/biotechnology/docs/genecloning.pdf
18. http://www.colorado.edu/Outreach/BSI/pdfs/gel_electrophoresis.pdf
19. http://biotech.about.com/od/pcr/a/sequencing.htm
20. http://www.dnai.org/
21. http://www.nature.com/clpt/journal/v82/n5/full/6100390a.html
22. http://www.newscientist.com/article/dn13523-therapeutic-cloning-used-to-treat-braindisease.html
23. http://www.newscientist.com/article/dn3393-dolly-the-sheep-dies-young.html
24. http://www.roslin.ed.ac.uk/public-interest/dolly-the-sheep/a-life-of-dolly/

25.
http://science.education.nih.gov/home2.nsf/Educational+ResourcesTopicsGenetics/BC508
6E34E4DBA0085256CCD006F01CB
26.
http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml
27.
http://www.genome.gov/25020028
28.
For further reading:
1.
http://www.ncbi.nlm.nih.gov/pubmed/15164072
2.
http://www.mayoclinic.org/development/individualized-medicine.html
3.
http://www.sciencedaily.com/releases/2005/01/050123213425.htm
4.
http://health.usnews.com/health-conditions/cancer/personalized-medicine
5.