This document outlines techniques in genetic engineering and biotechnology such as polymerase chain reaction (PCR), gel electrophoresis, and DNA profiling. It discusses how PCR is used to copy and amplify small amounts of DNA, how gel electrophoresis separates DNA fragments by size, and how DNA profiling determines paternity and is used in forensics. Additional topics covered include human genome sequencing, gene transfer between species using plasmids and restriction enzymes, examples of genetically modified crops, cloning techniques using differentiated cells, ethical issues around human therapeutic cloning, and definitions of terms like clone.

1. Topic 4.4 - Genetic Engineering and 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.
• DNA profiling can be used in criminal investigation, including murders and rape.
• DNA can be isolated from blood, semen or any other tissue available.
• DNA profiling is then carried out on these specimens and on the suspect.
• The results using this technique are reliable, however contamination of the samples with bacteria
or other DNA sources can interfere with the results to a great extent.
• DNA profiling can also be used in paternity suits.
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.
• Lead to an understanding of many genetic diseases
• The development of genome libraries
• The production of gene probes to detect sufferers and carriers of genetic diseases (e.g. Duchenne
muscular dystrophy).
• It may also lead to production of pharmaceuticals based on DNA sequences.
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.
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2. 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.
Plasmids are smaller circles of DNA found in prokaryotes (e.g. E.coli). They are used as a vector
(medium by which genes of interest or “TARGET DNA” are transferred to host)
A host cell (bacterium) receives the target DNA via a plasmid vector (= gene transfer). This cell replicates
repeatedly, passing on the target DNA to its offspring (= cloning)
Restriction enzymes (endonucleases) are produced naturally by bacteria as a defense against viruses. In
genetic engineering they are used to cut (called cleaving) the desired section of the DNA. A restriction
enzyme recognises unique sequences of DNA in the plasmid and in the target DNA. It will cut DNA,
producing “sticky ends”. Complementary sticky ends in target DNA and the plasmid allow incorporation
of the target DNA into the plasmid, producing recombinant DNA.
DNA ligase creates covalent bonds joining together the target DNA within the plasmid (called splicing),
producing recombinant DNA
Host cells often also serve to test if the DNA recombination has been successfully conducted by adding
onto the recombinant strand some gene sequence that will cause the host to display an easily observable
characteristic. Such a sequence that is often used codes for phosphorescence, causing the host cell to glow
if the transfer has been completed successfully.
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3. 4.4.9. State two examples of the current uses of genetically modified crops or animals.
• Salt tolerance in tomato plants, which allow them to grow in overly irrigated farmlands
• Factor IX (human blood clotting) in sheep milk.
4.4.10. Discuss the potential benefits and possible harmful effects of one example of genetic
modification.
• Some gene transfers are regarded as potentially harmful. A possible problem exists with the
release of genetically engineered organisms in the environment. These can spread and compete
with the naturally occurring varieties. Some of the engineered genes could also cross species
barriers, and many genetically modified organisms display surprising and unforeseen side effects
due to their modification. An excellent example of this is a corn variety modified to be more
resistant to several types of disease. While the plant did indeed become more resistant, in the
process the modification had affected the chemical composition of their pollen coat. The pollen
was now toxic to the Monarch butterfly, and thousands of them died during their migration
through the Midwest, where the corn was planted. The result of all this could be massive
disruption of the ecosystem. Benefits include more specific (less random) breeding than with
traditional methods.
4.4.11. Define clone.
• Clone - a group of genetically identical organisms or a group of cells derived from a single parent
cell.
4.4.12. Outline a technique for cloning using differentiated cells.
Cloning farm animals, eg. Dolly the sheep.
• Differentiated mammary cells extracted from parent sheep
• Grown in nutrient-deficient solution to stop the cell cycle
• Undifferentiated egg cells extracted from egg donor
• Nucleus removed and discarded
• Mammary cell placed next to enucleated egg cell
• Electric shock causes two cell membranes to fuse, and mitosis to trigger
• Mitotic division continues, producing embryo
• Embryo implanted into surrogate mother
• After 5-month gestation, Dolly the lamb born with identical genotype to parent donating nucleus
from mammary cell
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4. 4.3.12. Discuss the ethical issues of therapeutic cloning in humans.
Therapeutic cloning is the creation of an embryo to supply embryonic stem cells for medical use.
• Opposition to human cloning is very strong, based on a variety of arguments most of which
invoke a violation of “the sanctity of life”
• Cloning happens naturally, for example monozygotic twins.
• Some may regard the invitro production of two embryos from one to be acceptable.
• Others would see this as leading to the selection of those "fit to be cloned" and visions of
"eugenics and a super-race".
• Perhaps the most pressing question, however, is that of the status and rights of a theoretical human
clone.
• What is being debated and discussed right now by lawmakers, ethicists and religious leaders is
exactly this.
• Is a clone its own unique human being?
• Is cloning strictly for the purpose of stem cell production or organ harvesting legal or right?
• What about reproductive cloning?
These are only a very few of the issues that must be decided in the human cloning debate.
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