Molecular Genetics: Implications and Applications Biology 3201 Unit III: Genetics p.613-618

Genetic Engineering: The manipulation of genetic material for practical purposes

The Sequence of Life In 1977, Frederick Sanger and his colleagues worked out the complete nucleotide sequence of DNA in a virus, which helped them understand how genetic material is organized.   They learned the following:  1. A way to break a DNA strand at specific sites along its nucleotide sequence. 2. The development of a process for copying or amplifying DNA samples. 3. Improvements in sorting/analyzing DNA molecules

What is Genetic Engineering? https://www.youtube.com/watch?v=3IsQ92KiBwM Making Insulin using Yeast https://www.youtube.com/watch?v=iMosKBs-v0E

Techniques Used in Genetic Engineering 1. Restriction Enzymes (Endonucleases) These enzymes are able to recognize a specific sequence of nucleotides on a strand of DNA and can then cut the strand at a particular point in that sequence. http://www.youtube.com/watch?v=lWXryzgRces&safety_mode=true&persist_safety_mode=1&safe=active The point at which the strand is cut is called the restriction site.

Two characteristics which have made restriction endonucleases useful to genetic researchers are: 1. Specificity The cuts made by these enzymes are specific and predictable. A certain enzyme will cut a particular strand of DNA the same way each time.

2. Staggered cuts These short, unpaired sequences are called sticky ends. The sticky ends can join with other short strands of DNA.

DNA from two different sources (species) 2.Recombinant DNA DNA from two different sources (species) DNA is cut with the same restriction enzyme to produce complementary sticky ends DNA ligase is then used to seal the strands together http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/plasmidcloning_fla.html http://www.youtube.com/watch?v=YdjvUv-1vCI&list=PL969072B9FEA0944E&safety_mode=true&persist_safety_mode=1&safe=active

Polymerase chain reaction (PCR) 3. DNA Amplification The process of producing a large sample of a target DNA sequence from a single gene or DNA fragment There are 2 methods: Cloning vectors Polymerase chain reaction (PCR)

a) Cloning Vectors Used to amplify large DNA sequences

Example: a DNA fragment of interest (e.g., a segment of human DNA) is inserted into a DNA molecule (called a vector) that is capable of independent replication in a host cell. The result is a recombinant molecule or molecular clone. Large quantities of the inserted DNA can be obtained if the recombinant molecule is allowed to replicate in an appropriate host.

b)Polymerase Chain Reaction (PCR) Makes copies of the target DNA in a shorter time than using cloning vectors Often used to amplify DNA samples that are very small, old or damaged

(v) Billions of copies of DNA can be generated in a few hours (i) The DNA sequence is placed in a solution with all four nucleotides as well as primers (needed for DNA polymerase to initiate DNA synthesis) (ii) The solution is heated to separate the 2 DNA strands (iii) The solution is cooled to allow the primers to bind to the ends of the targeted sequence (iv)  It takes about one minute to produce 2 copies and the cycle repeats itself (v)   Billions of copies of DNA can be generated in a few hours http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html

4. Gel Electrophoresis A method used to separate DNA fragments based on their mass and electrical charge (i) a solution of DNA restriction fragments is applied to one end of a gel. (ii) an electric current is applied to the gel and the DNA, which has a negative charge moves toward the opposite end of the gel which has a positive charge

(iii) shorter fragments move further along the gel (iv) the fragments eventually separate out into a pattern of bands = DNA fingerprint  The DNA fingerprint produced can then be analyzed E.g. To determine the paternity of a child or to solve a crime http://www.sumanasinc.com/webcontent/anisamples/majorsbiology/gelelectrophoresis.html

What can DNA fingerprinting be used for?

5. Sequencing DNA The process used to determine the order of DNA nucleotides in a DNA strand Uses chain termination sequencing, a modified form of PCR (i) A single-stranded DNA of unknown sequence is used as a template for the synthesis of complementary DNA. This replicated DNA is synthesized in a series of small fragments rather than in one strand

(ii) The nucleotide that ends each fragment is tagged with a fluorescent or radioactive marker (iii) When the fragments are separated using gel electrophoresis, the DNA fingerprint produced shows which fragments end with G nucleotides, which end with C nucleotides, etc. (iv) When all the fragments are analyzed together (read from the positive end to the negative end) the sequence of the synthesized strand is determined and can be used to deduce the sequence of the original single-stranded DNA of interest.

The Human Genome Project The HGP was a joint effort of thousands of laboratories worldwide that determined the sequence of the nearly three billion base pairs that make up the human genome. It was created to benefit mankind. Important Findings 1. DNA of all humans is more than 99.9% identical 2. The human genome contains only 35,000 genes (not 100,000)

Benefits of Knowledge Gained through HGP 1. Makes it easier to locate and study genes that are involved in human disease 2. Provide better ways to assess a person’s risk of developing a disease 3. Provide better ways to prevent a disease 4. The development of new drugs and treatments that are specifically tailored to an individual’s specific genetic make-up.  http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml

Risks of Applying Knowledge Gained through HGP  1. Privacy issues: who should have access to information about a person’s genetic make-up? 2. Emotional issues: stress of knowing you carry alleles for a serious illness 3. Ethical and Financial: who owns the information? The subjects or the drug company? 

Genetically Modified Organisms and Foods Also known as GMOs and GMFs These are transgenic = genetically engineered. Moving DNA from one organism to another creates a new combination =transgenic organism.

Examples and their Significance 1. Agriculture  a) Corn and Canola · Over 80% of the corn and Canola grown in North America are genetically-modified varieties · Herbicide-resistant strains of these crops will grow without being damaged when farmers apply herbicides to control weeds. Less weeds = higher crop production.

b) Milk Bovine Somatotropin (BST), a growth hormone, was the first transgenic product approved for use in North America When cows are given high doses they grow bigger and produce more milk c)  Rice “Golden Rice”, a strain modified to produce beta-carotene (precursor of vitamin A) and to have higher levels of iron was developed to treat malnutrition in many developing countries

d) Salmon Two different transgenic varieties have been developed in Atlantic Canada for the fish farming industry One type produces its own antifreeze hormone so it can tolerate the cold Atlantic ocean temperatures Another type grows at a rate much faster than normal so it can reach market size one year earlier than normal fish

2. Medicine Insulin-producing bacteria Transgenic bacteria containing the gene for human insulin provide a ready source for large-scale production of the hormone needed for treating diabetes Considered a better source than the traditional sources (from cows or pigs) Cheaper to produce and doesn’t cause allergic reactions

a) PCB-eating bacteria  3. Environment  a) PCB-eating bacteria Transgenic bacteria can break down PCBs (polychlorinated biphenyls), toxic byproducts of industry, which can accumulate in the environment b) Oil-eating bacteria Transgenic bacteria can clean up oil spills

Risks Associated with GMOs and GMFs: 1. Environmental Threats Most issues relate to the use of herbicide-resistant crops, including: a) Using these crops may encourage farmers to use higher levels of herbicides. This may lead to more leaching of herbicides into waterways b) The potential spread of genes from these crops to wild types may lead to decreased biodiversity c) Cross-breeding with related natural plants could produce “superweeds” d)  Using these crops could lead to the natural development of “superbugs” that would be immune to certain pesticides

2. Health effects Many consumer groups argue that not enough is known about the long-term effects of using transgenic products  3.Social and economic issues Does not solve the problem of world hunger because they do not address the real cause which is unequal food distribution Since large corporations develop and own the patent on these GMF’s that they will eventually control the world food supply

Cloning pgs. 627-630 What is a clone? A cell that develops into the exact copy of another organism. It is identical to the animal that donated its DNA.

Two types of human cloning: Therapeutic cloning: the culturing of human cells for use in treating medical disorders  Reproductive cloning: the development of a cloned embryo in order to create a cloned human being. Animals that have been successfully cloned include frog, mice and sheep.  Dolly, the sheep, was cloned from the udder cells of an adult. Dolly experienced premature aging through the loss of telomeres during DNA replication. See page 629 for steps.

Benefits and Risks: Benefits include the speed of reproduction, potential elimination of disease and manipulation of traits Risks include reduced genetic variability, loss of individuality and deliberate use and destruction of embryos http://www.youtube.com/watch?v=j-nBhl5wNOU&list=PL969072B9FEA0944E&safety_mode=true&persist_safety_mode=1&safe=active