Biotechnology

1. Biotechnology Chapter 20

2. What is Cloning? • Cloning is the creation of an organism that is an exact genetic copy of another. This means that every single bit of DNA is the same between the two!

3. What animals have been cloned? • In 1952, the first animal, a tadpole, was cloned. • Before the creation of Dolly, the first mammal cloned from the cell of an adult animal, clones were created from embryonic cells. • Since Dolly, researchers have cloned: • Sheep, goats, cows, mice, pigs, cats, rabbits, and a gaur. • All these clones were created using nuclear transfer technology. • Some species may be more resistant to somatic cell nuclear transfer than others. • Traumatic experience , and improvements in cloning technologies may be needed before many species can be cloned successfully.

4. Types of Cloning • Somatic Cell Nuclear Transfer (Reproductive cloning) • Recombinant DNA Technology or DNA Cloning • Therapeutic cloning

5. Somatic Cell Nuclear Transfer (SCNT) • Produces an exact clone, or genetic copy, of an individual. • Method used to create Dolly the Sheep. • Somatic cell: Any cell in the body other than the gametes. In mammals, every somatic cell has two complete sets of chromosomes (diploid)

6. Somatic Cell Nuclear Transfer (SCNT) & Dolly (Reproductive Cloning ) • Researchers isolated a somatic cell from an adult female sheep. • Next, they transferred the nucleus from that cell to an egg cell from which the nucleus had been removed. • The egg cell, with its new nucleus, was behaving just like a fertilized zygote. • It developed into an embryo, which was implanted into a surrogate mother and carried to term.

7. Creating Dolly

8. Stage 1 Cell collected from a sheep’s udder.

9. Stage 2 Nucleus is removed from unfertilized egg of second sheep.

10. Stage 3 Udder cell is inserted into egg with no nucleus.

11. Stage 4 Insertion is successful.

12. Stage 5 Electrical charge is supplied.

13. Stage 6 Cells begin to divide.

14. Stage 7 Embryo implanted into a surrogate

15. Celebrity Sheep Died at Age 6 • Dolly was put down by lethal injection Feb. 14, 2003. • Prior to her death, Dolly had been suffering from lung cancer and crippling arthritis. • Although most Finn Dorset sheep live to be 11 to 12 years of age, postmortem examination of Dolly seemed to indicate that, other than her cancer and arthritis, she appeared to be quite normal. • The unnamed sheep from which Dolly was cloned had died several years prior to her creation. • Dolly was a mother to six lambs, bred the old-fashioned way.

16. Is cloning an organism the same as cloning a gene? • Cloning an animal, or any other organism, refers to making an exact genetic copy of that organism. • Cloning a gene means isolating an exact copy of a single gene from the entire genome of an organism. • Involves copying the DNA sequence of that gene into a smaller, more accessible piece of DNA, such as a plasmid. This makes it easier to study the function of the individual gene in the laboratory. Animation

17. Recombinant DNA Technology or DNA Cloning • Transfer of a DNA fragment from one organism to a self-replicating genetic element such as a bacterialplasmid. • The DNA is then be propagated in a foreign host cell. • Been around since the 1970s, and it has become a common practice in molecular biology labs today.

18. Plasmids • Self-replicating extra-chromosomal circular DNA molecules • Used by Human Genome Project (HGP) researchers to copy genes

19. To "clone a gene" • A DNA fragment containing the gene of interest is isolated from chromosomal DNA using restrictionenzymes and then united with a plasmid that has been cut with the same restriction enzymes. • When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA molecule." • Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA.

20. Restriction Enzymes • Aka: restriction endonucleases • DNA-cuttingenzymes • Found in bacteria & made exclusively by prokaryotes. • In order to sequence DNA, it is first necessary to cut it into smaller fragments. • A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. • Originally discovered through their ability to break down, or restrict, foreign DNA. • Can distinguish between the DNA normally present in the cell and foreign DNA • Ie. infecting bacteria virus DNA (bacteriophage). • Defend the cell from invasion by cutting the foreign DNA into pieces, thereby rendering the DNA nonfunctional.

22. Restriction Enzymes • Many restriction enzymes cut in an offset fashion. • The ends of the cut have an overhanging piece of single-stranded DNA. Known as "sticky ends" • They are able to form base pairs with any DNA molecule that contains the complementary sticky end. • The union can be made permanent by DNA ligase, that forms covalent bonds along the backbone of each strand. • The result is a molecule of recombinant DNA (rDNA). • Animation

23. Restriction Enzymes & Nucleases • Nuclease -any enzyme that cuts the phosphodiester bonds of the DNA backbone • bond between a two sugar groups and a phosphate group • Endonuclease -an enzyme that cuts some where within a DNA molecule. • Exonuclease- cuts phosphodiester bonds by starting from the free end of the a single DNA strand in the 3' to 5' direction

24. Restriction Enzymes • Animations

25. Palindrome DNA • A sequence of DNA which reads the same on both strands • Ie. a palindrome). • Such sequences often form the recognition sites for enzymes such as restriction endonucleases.

26. Restriction Enzyme EcoR1 • EcoRI, one of many restriction enzymes, is obtained from the bacteria Escherichia coli.

27. Benefits of Recombinant DNA • Revolutionized genetics & biotechnology industry • Examples: • Human insulin • Human factor VIII (for males with hemophilia A),

29. Other Cloning Vectors • Bacterial plasmids • Viruses • Bacteria artificial chromosomes (BACs) • Yeast artificial chromosomes (YACs) • Cosmids are artificially constructed cloning vectors for infection into E. coli cells. • Bacteria are most often used as the host cells for recombinant DNA molecules, but yeast and mammalian cells also are used.

30. Therapeutic Cloning • Aka: "embryo cloning“ • Production of human embryos for use in research. • Goal is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Animation

31. Can organs be cloned for use in transplants? • To do this, DNA would be extracted from the person in need of a transplant and inserted into an enucleated egg. • After the egg containing the patient's DNA starts to divide, embryonic stem cells that can be transformed into any type of tissue would be harvested. • The stem cells would be used to generate an organ or tissue that is a genetic match to the recipient. • In theory, the cloned organ could then be transplanted into the patient without the risk of tissue rejection.

32. Many challenges must be overcome before "cloned organ" transplants become reality. • More effective technologies for creating human embryos, harvesting stem cells, and producing organs from stem cells would have to be developed. • In 2001, scientists cloned the first human embryos; however, the only embryo to survive the cloning process stopped developing after dividing into sixcells. • In 2002, scientists successfully transplanted kidney-like organs into cows. The team of researchers created a cloned cow embryo by removing the DNA from an egg cell and then injecting the DNA from the skin cell of the donor cow's ear. Since little is known about manipulating embryonic stem cells from cows, the scientists let the cloned embryos develop into fetuses. The scientists then harvested fetal tissue from the clones and transplanted it into the donor cow. In the three months of observation following the transplant, no sign of immune rejection was observed in the transplant recipient.

33. What are the risks of cloning? • Reproductive cloning is expensive and highly inefficient. • More than 90% of cloning attempts fail to produce viable offspring. • More than 100 nuclear transfer procedures could be required to produce one viable clone. • Cloned animals tend to have more compromised immune function • Clones have been known to die mysteriously. • Example, Australia's first cloned sheep appeared healthy and energetic on the day she died, and the results from her autopsy failed to determine a cause of death.

34. More Risks • Programming errors in the genetic material from a donor cell. • When an embryo is created from the union of a sperm and an egg, the embryo receives copies of most genes from both parents. • A process called "imprinting" chemically marks the DNA from the mother and father so that only one copy of a gene (either the maternal or paternal gene) is turned on. • Defects in the genetic imprint of DNA from a single donor cell may lead to some of the developmental abnormalities of cloned embryos

35. PCR • Purpose: Used to identify and amplify (copy)DNA in a sample • Makes millions of copies of a gene • Necessary to have enough starting template for sequencing. Benefits • Takes only approximately 2 hours • Bypasses the need to use bacteria for amplifying DNA. • Relatively simple and inexpensive • Used every day to diagnose diseases, identify bacteria and viruses, match criminals to crime scenes, and in many other ways • Animation………….Animation

36. Steps of PCR • Pair of primers (short single-stranded DNA segments) are needed that attach to either side of the target DNA to be copied.  • The target DNA, the primers, a polymerase molecule (heat resistant)which duplicates DNA, and a supply of nucleotides are mixed together are go through a series of heating and cooling cycles. • After approximately 30cycles, the segment will have been copied 250 million times. • The results of PCR can then be analyzed in various ways, such as agarose gel electrophoresis or sequencing.

37. Enzymes & Primers • Primers: • About 20 nucleotides long • Complementary to the beginning and end of the DNA fragment of interest which one needs to amplify. • Select the boundaries of the region to be amplified by PCR. • During the PCR annealing cycle, PCR primers anneal to the complementary region of the DNA. • Anneal-to heat and then cool in order to separate strands and induce combination at lower temperatures especially with complementary strands

39. Heating & Cooling Process • 1st Heating Process • Samples heated to 95 degrees C • Purpose: Denature DNA- breaking bonds • 2nd Cooling Process (Annealing) • 60 degrees C • Allows primers to formHydrogen bonds or anneals with complementary DNA strand

40. Heating & Cooling Process • 3rd Heating Process • Temperature raised to 72 degrees C • Taq polymerase(optimal temperature) begins polymerization adding nucleotides to the 3’end of each primer attached to a DNA strand • Animation

41. The segment of the target DNA between the primers will be copied during each cycle, accumulating in the mixture in an exponential fashion. • After 1st complete cycle-----2 double stranded copies of the target DNA • After 2nd complete cycle----- 4 copies • After 3rd complete cycle----- 8 copies • After 25 cycles- 33 million copies

42. Steps of PCR

44. PCR

45. Steps of PCR

46. Enzymes & Primers • Taq polymerase • Obtained from hot spring bacteria • Able to withstand extreme PCR heat

47. Gel electrophoresis. • When restrictive endonuclease is used the DNA split to multiple mixed fragments, they are then separated by gel electrophoresis. • The gel is then blotted on nitrocellulose paper. • Turns to a solid form • Now we have separate solid DNA fragments. • A probe is then added to bind to its matching fragment which can then be visualized by autoradiography. • Animation

50. Types of Blotting • If the substance is DNA it is termed Southern Blotting. • If the substance is RNA it is termed Northern blotting. • If the substance is proteinit is termed Westernblotting.