2. Types of cloning DNA Cloning (Recombinant DNA Technology) Reproductive Cloning (in animals) Cloning in agriculture

3. DNA Cloning review Bacteria can be transformed with recombinant plasmids; plasmids (carry- ing a specific gene can then be cloned to make many copies)

4. Reproductive Cloning Two types: 1. Somatic nuclear cell transfer Animals can be cloned by removing the nucleus from a somatic cell and injecting it into an egg cell that has had its nucleus removed (somatic cell nuclear transfer). After some chemical treatment, the cell starts dividing and develops into an embryo, is implanted into a surrogate mother who carries it to term. Can be fusion of somatic cell and egg cell or microinjection of only somatic cell nucleus.

6. Some notes about animal reproductive cloning Dolly the Sheep – this was done to determine if a specialized somatic cell could be re-programmed to produce a new organism Not a total clone since there is always some mitochondrial DNA that contributes to the genetic make-up of an organism

7. Reproductive cloning 2. Artificial Embryo Twinning Mimics the natural process of creating identical twins in a Petri dish. Embryos are separated into individual cells early on; those cells develop into embryos which are carried to terms by surrogate mothers

8. Therapeutic cloning Also called ‘embryo cloning’ – the production of human embryos for use in research (illegal in Canada) Generating stem cells to grow new tissues and organs

9. Cloning in agriculture Tissue culture – small piece of plant grown in test tube, produces shoots that are identical OR Cloning vector – use of a bacterial plasmid, infects plants (Ti plasmid can incorporate into host genome)

10. Foreign genes can be inserted by using a bacteria, Agrobacterium tumefaciens, that can carry a recombinant plasmid into a plant.

11. Limitations A. tumefaciens only infects dicots (e.g. soybeans, potatoes, tobacco, etc.) Now, DNA molecules can be injected directly into plant cells (microinjection, electroporation and particle bombardment)

12. Applications in agriculture Designing transgenic (also known as genetically modified) plants. Why? Resist herbicides (round-up ready corn) Resist insect pests and diseases (Bt corn) Less need for artificial fertilizers Resist environmental stresses Produce plants with new characteristics (e.g. longer shelf life)

13. Examples of transgenic plants FlavrSavr tomato Golden rice Triple stack corn

14. Applications in agriculture cont’d Increasing desirable characteristics in livestock (e.g. cows, sheep, goats, chickens, etc.). Such as? Increased production of meat, milk, eggs and other animal products Better disease resistance Changing the fat:lean ratio of meat

15. Techniques used in biotechnology research and applications

16. RFLP analysis Restriction fragment length polymorphism (RFLP – pronounced rif-lip) is a difference in homologous DNA sequences RFLP analysis is the original method used for profiling DNA and was used in genetic fingerprinting and genome mapping before cheaper methods such as PCR (polymerase chain reaction) and DNA sequencing came along

17. How does it work? DNA samples are digested by restriction enzymes Then the DNA fragments are run on an electrophoresis gel Then an RFLP probe (radioactive DNA probe – sequence known) is added and will bind to certain matching bases – this is how they started mapping the human genome

18. RFLP Analysis Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals or to detect disease

19. A pioneering method but….. It takes about one month for the process It requires a large amount of DNA Still used to detect genetic variation between individuals, for inherited diseases and more generally for disease detection

21. A quick and easy way to make lots of copies of the DNA we want Polymerase Chain Reaction (PCR)

22. Why PCR? Often a gene of interest is not in large enough quantities to analyze Much faster than cloning a certain gene or piece of DNA in bacteria

25. All happens in a test tube: 1st step: Denaturation – the break down of double-stranded DNA using heat 2 nd step: Annealing – primers attach to either end; lower temperature 3rd step: Extension - DNA polymerase extends the strand using nucleotides that have been added, medium temperature

26. And the test tubes are in this……

27. Application DNA fingerprinting for forensics and paternity Genetic testing for hereditary and infectious diseases

28. Move aside PCR Constant T PCR has been developed Recombinase polymerase amplification No need for sophisticated PCR equipment

29. Agarose Electrophoresis of DNA used to separate fragments of DNA DNA is negatively charged, charge is proportional to size agarose can be used as a molecular strainer (sieve) to separate the pieces of DNA by size

30. Gel electrophoresis A current is run through the buffer surrounding the gel and it pushes the DNA away from the negative anode, towards the positive cathode

31. Gel Electrophoresis

33. DNA sequencing Used to determine the bases along a particular stretch of DNA Was developed during the time of the Human Genome Project – allowed it to proceed much faster

34. What do you need? First we need: DNA of interest, with known sequences at the ends DNA polymerase ATCG primers, modified A, T, C and G in each test tube.

35. How does it work? 1st. Double-stranded DNA is denatured 2 nd. Primers attach to known ends 3rd. DNA polymerase adds nucleotides 4th. All four nucleotides are mixed into test tubes with the DNA of interest

36. continued 5th. One of the four nucleotides mixtures has an ‘A’ that has been modified – the ‘terminator’. Then separate the pieces by running an them on an agarose gel (electrophoresis) – the place where an A (adenine) is will show up The same happens for the other bases…..