Biotechnology 4.4 Syllabus points

4.4.1 PCR Outline use of PCR to copy and amplify minute quantities of DNA PCR = polymerase chain reaction Helps to get enough DNA to analyze when a sample is very small Thermocycler - machine automates the copying of the DNA Millions of copies in a few hours

PCR Uses DNA polymerase from bacteria that lives in hot springs Thermus aquaticus 3 steps Denature – heat up breaks double into single strand (about 98 degrees C) Anneal – cool and primers can attach to either a top or bottom strand Extension – Taq polymerase makes complementary strand

PCR Repeated cycles of heating and cooling stimulates the primers to attach to copies and copies of copies Result is exponential increase in number of copies of DNA

4.4.2 Gel electrophoresis separates DNA by size as move in electric field DNA negative charge move towards positive pole Agarose gel forms mesh of cross linked polymers DNA stained to visualize the separated bands

4.4.3 Gel electrophoresis of DNA used in DNA profiling Matching an unknown sample with a known to see if they match up is DNA profiling Identical band patterns means that is the individual in question – similar patterns usually mean the individuals are relatives

4.4.4 Applications of DNA profiling Paternity – legal identification of biological father Forensic investigations – compare samples from crime scene with suspect Cold cases reopen with new DNA technology such as PCR Release of wrongly imprisoned individuals

4.4.5 Analyze gels Use gel banding patterns to determine matches between individuals to solve crimes or determine paternity

4.4.6 Human Genome Project Outline three outcomes of sequencing the complete human genome international effort Government and private labs Aimed to identify the entire sequence of bases in human DNA Created in essence a map of all genes on all 23 chromosomes

4.4.6 Human genome Allows identification of location of genes causing diseases Production of new medications by identifying products made by healthy individuals determine the gene responsible copy the gene and use it to produce the desired molecule Evolutionary relationships and migration patterns of humans

4.4.7 Gene transfer Genes can be transferred between species because DNA is universal language and code for the same amino acids So gene will produce the same protein no matter what organism it is placed into

Examples gene transfer Cold resistant tomatoes Bt-corn (Bacillus thuringiensis) Spider silk goats

4.4.8 Technique Gene Transfer Cut and paste genes using restriction enzymes AKA endonucleases find and cut at target sequences Paste genes using DNA ligase enzyme which recognizes the sticky ends of the fragments and attaches them

Copy paste using plasmid DNA Use host cell often Escherichia coli or yeast Prokaryotes most DNA in single chromosome but also have extra DNA is small loops called plasmids To copy DNA it needs to be pasted into a plasmid

4.4.8 Pasting into plasmid Remove plasmid from cytoplasm of bacteria Cut open plasmid using restriction enzyme Paste new gene using DNA ligase into the open plasmid Modified plasmid called a recombinant plasmid Recombinant plasmid used to deliver new gene into genome of target organism

4.4.9 Genetically Modified organisms Golden rice – beta carotene into rice prevent blindness Salt resistant tomatoes Proponents – will help solve world hunger Critics – world hunger is distribution not production issue

Benefits/Risks GMO Benefits: Improve food production Reduce chemicals if plants have own pest control substances Reduce cost and pollution of making medicines Farmers have added control of crops and livestock Globally GM crops need less water and pesticides

Risks Risks- long term risks unknown Pollen escapes into wild integrated possibly into wild genome Genes could cross species – seen in labs could it happen in wild? Bt crops harmful to humans – pesticide throughout plant not just on surface Allergies Large corporations will own large part of food supply High tech not always better than simple solutions Decrease in biodiversity

Cloning Define clone Group of identically identical organisms or group of cells artificially derived from a single parent Farmers have cloned plants for years by regenerating plant material

Technique cloning Enucleate unfertilized egg and transfer nuclear material from donor cell Electrical current fuses egg and nucleus Zygote divides and forms embryo which is implanted in surrogate mother Donor cell may be adult differentiated cell (non gamete) rather than from an egg cell Offspring is clone of donor organism

Ethics therapeutic cloning Cloning using undifferentiated cells AKA embryonic stem cells Requires production of human embryos Is it ethical to generate new humans for medical research? However, stem cells allow repair of burns, new heart muscle, new kidney tissue

Types of Cloning Therapeutic cloning makes new tissues Reproductive cloning makes new organisms