1. Genetics Unit IV-Biotechnology
10/9/2017
Genetics Unit IV-Biotechnology
Dolly and surrogate Mom
Embryonic stem cells and gene therapy
Genetically modified rice.
G. Podgorksi, Biol. 1010

2. Recombinant DNA, Gene Cloning, and Pharmaceutical Production
Biotechnology
10/9/2017
Recombinant DNA, Gene Cloning, and Pharmaceutical Production
These are mature and widely utilized biotechnologies.
DNA can be cut at specific sequences using restriction enzymes.
This creates DNA fragments useful for gene cloning.
G. Podgorksi, Biol. 1010

3. Biotechnology
10/9/2017
Restriction Enzymes are Enzymes That Cut DNA Only at Particular Sequences
The enzyme EcoRI cutting DNA at its recognition sequence
Restriction enzyme animation
Different restriction enzymes have different recognition sequences.
This makes it possible to create a wide variety of different gene fragments.
G. Podgorksi, Biol. 1010

4. Restriction Enzymes
Discovered as a defense mechanism in bacteria (they cut invading DNA)
Used to cut valuable segments of DNA
Work in two ways:
a.) exonucleases – chop off bases at the ends (not useful)
b.) endonucleases – break DNA from the inside, allows you to stick in new molecules because of “sticky ends”
Both types recognize unique, specific sequences
About 600 exist, each recognizes a 5-7 base sequence in a palindrome

5. Sample Sequence Recognized by a Restriction Enzyme

6. How Restriction Enzymes Cut

7. DNAs Cut by a Restriction Enzyme Can be Joined Together in New Ways
Biotechnology
10/9/2017
DNAs Cut by a Restriction Enzyme Can be Joined Together in New Ways
These are recombinant DNAs and they often are made of DNAs from different organisms.
G. Podgorksi, Biol. 1010

8. Cloning Vectors
Organisms or pieces of DNA that will carry inserted DNA from another organism
Should be small, circular piece of DNA that will reproduce quickly and carry a selective advantage
Bacterial plasmids are choice #1
Viruses and yeast are also used

9. Plasmids are Used to Replicate a Recombinant DNA
Biotechnology
10/9/2017
Plasmids are Used to Replicate a Recombinant DNA
Plasmids are small circles of DNA found in bacteria.
Plasmids replicate independently of the bacterial chromosome.
Pieces of foreign DNA can be added within a plasmid to create a recombinant plasmid.
Replication often produces copies of a recombinant plasmid in each cell.
G. Podgorksi, Biol. 1010

10. Cloning a Gene Procedure
Use same restriction enzyme (RE) to cut both gene and cloning vector (plasmid)
Mix gene with vector in test tube
Add DNA ligase to bind gene and vector
Place plasmid in bacteria (now a recombinant molecule)
Place recombinant molecule in bacteria
Grow bacteria on plate with antibiotic
Only bacteria with plasmid will grow
Isolate multiple copies of gene or protein

11. Cloning a Gene

12. Producing Quantities of Protein
A.) Prokaryotes
Done easily, place gene in vector that contains a promoter (called an expression vector)
Gene will then be transcribed and translated
B.) Eukaryotes
Much more difficult, due to introns
Must start with mRNA
Use an enzyme, reverse transcriptase, to produce a DNA copy from the mRNA (called complementary DNA or cDNA)

13. Producing Quantities of Protein
Reverse transcriptase was discovered in retro-viruses (HIV, herpes)
Once cDNA is produced, clone as before, insert gene in vector with a promoter
Grow cloned bacteria or yeast in large vats, protein will be secreted, then purify and use
Hormones such as insulin and HGH are produced in this manner

14. Biotechnology
10/9/2017
Harnessing the Power of Recombinant DNA Technology – Human Insulin Production by Bacteria
G. Podgorksi, Biol. 1010

15. Human Insulin Production by Bacteria
Biotechnology
10/9/2017
Human Insulin Production by Bacteria
and cut with a restriction enzyme
6) join the plasmid and human fragment
G. Podgorksi, Biol. 1010

16. Human Insulin Production by Bacteria
Biotechnology
10/9/2017
Human Insulin Production by Bacteria
Mix the recombinant plasmid with bacteria.
Screening bacterial cells to learn which contain the human insulin gene is the hard part.
G. Podgorksi, Biol. 1010

17. Route to the Production by Bacteria of Human Insulin
Biotechnology
10/9/2017
Route to the Production by Bacteria of Human Insulin
One cell with the recombinant plasmid
A fermentor used to grow recombinant bacteria.
This is the step when gene cloning takes place.
Then the single cell with many recombinant plasmids produces trillions of like cells with recombinant plasmid – and the human insulin gene.
The single recombinant plasmid replicates within a cell.
G. Podgorksi, Biol. 1010

18. Route to the Production by Bacteria of Human Insulin
Biotechnology
10/9/2017
Route to the Production by Bacteria of Human Insulin
The final steps are to collect the bacteria, break open the cells, and purify the insulin protein expressed from the recombinant human insulin gene.
G. Podgorksi, Biol. 1010

19. Biotechnology
10/9/2017
Biotechnology
Biotechnology, defined broadly, is the engineering of organisms for useful purposes.
Often, biotechnology involves the creation of hybrid genes and their introduction into organisms in which some or all of the gene is not normally present.
Fourteen month-old genetically engineered (“biotech”) salmon (left) and standard salmon (right).
G. Podgorksi, Biol. 1010

20. What is Cloning?
Any manipulation of an organism’s genetic material to produce a new organism or organic product
Included in this definition are genetic engineering, cloning, biotechnology, and bioinformatics

21. Animal Cloning Dolly and her surrogate mother. Biotechnology 10/9/2017
G. Podgorksi, Biol. 1010

22. Why Clone Animals? To answer questions of basic biology
Biotechnology
10/9/2017
Why Clone Animals?
Five genetically identical cloned pigs.
To answer questions of basic biology
For pharmaceutical production.
For herd improvement.
To satisfy our desires (e.g. pet cloning).
G. Podgorksi, Biol. 1010

23. Is Animal Cloning Ethical?
Biotechnology
10/9/2017
Is Animal Cloning Ethical?
The first cloned horse and her surrogate mother/genetic twin.
As with many important questions, the answer is beyond the scope of science.
G. Podgorksi, Biol. 1010

24. A Cloned Mule and the First Cloned Equine
Biotechnology
10/9/2017
A Cloned Mule and the First Cloned Equine
G. Podgorksi, Biol. 1010

25. The Biotechnology of Reproductive Cloning
10/9/2017
The Biotechnology of Reproductive Cloning
Even under the best of circumstances, the current technology of cloning is very inefficient.
Cloning provides the most direct demonstration that all cells of an individual share a common genetic blueprint.
G. Podgorksi, Biol. 1010

26. Technique – The Nuclear Transfer Process
May be completed in a petri dish
Needed are eggs and mature skin cells (fibroblasts)
Each egg contains a nucleus (w/chromosomes), cytoplasm, cell membrane, and an extra protective layer called the zona pellucida
Inside the zona is an immature egg called a polar body

27. Technique – The Nuclear Transfer Process
Fibroblasts contain a nucleus + cytoplasm
1. How to carry out the process:
In petri dish, a group of eggs is stimulated to mature
Using a microscope and micropipette, the polar body and all chromosomes are removed from each egg
All that is left is the empty zona and cytoplasm
Fibroblasts are grown in a separate dish

28. Technique – The Nuclear Transfer Process
A fibroblast is then injected into each zona, not in the cytoplasm
The newly fused eggs are then given an electric shock (this mimics sperm entry)
The skin cell nucleus is now in cytoplasm
If lucky, cell division begins and a clone has been produced

29. The Nuclear Transfer Process
Biotechnology
10/9/2017
The Nuclear Transfer Process
G. Podgorksi, Biol. 1010

30. Cloning Tissues, Organs + Whole Organisms
A. Benefits
Skin grafts, tissue + organ replacements, without need for rejection drugs
Increased lifespan
Replacement of lost animals and people
B. Negatives
Expense
Reliability of technique
Population concerns
Ethics

31. Carbon Copy– the First Cloned Pet
Biotechnology
10/9/2017
Carbon Copy– the First Cloned Pet
(Science (2002) 295:1443)
Significantly, Carbon Copy is not a phenotypic carbon copy of the animal she was cloned from.
G. Podgorksi, Biol. 1010

32. Other Uses Production of Transgenic Organisms
Defined as an organism that lives with a foreign gene inserted into its genome
Procedure:
1. Clone multiple copies of the gene you wish to insert
2. Inject your gene into a fertilized egg cell
3. Implant your egg cell
4. Hope the gene “takes” and is expressed by the organism
5. Mate two organisms who have expressed the trait in hopes of starting a new line

33. The Next Step? Highly unlikely.
Biotechnology
10/9/2017
The Next Step?
Highly unlikely.
Attempts at human cloning are viewed very unfavorably in the scientific community.
G. Podgorksi, Biol. 1010

34. Biotechnology
Pharming
10/9/2017
Pharming is the production of pharmaceuticals in animals engineered to contain a foreign, drug-producing gene.
These goats contain the human gene for a clot-dissolving protein that is produced in their milk.
G. Podgorksi, Biol. 1010

35. The Promise and Possible Perils of Stem Cells
Biotechnology
10/9/2017
The Promise and Possible Perils of Stem Cells
G. Podgorksi, Biol. 1010

36. Biotechnology
10/9/2017
The Stem Cell Concept
A stem cell is an undifferentiated, dividing cell that gives rise to a daughter cell like itself and a daughter cell that becomes a specialized cell type.
G. Podgorksi, Biol. 1010

37. Biotechnology
10/9/2017
Stem Cells are Found in the Adult, but the Most Promising Types of Stem Cells for Therapy are Embryonic Stem Cells
G. Podgorksi, Biol. 1010

38. The Inner Cell Mass is the Source of Embryonic Stem Cells
Biotechnology
10/9/2017
The Inner Cell Mass is the Source of Embryonic Stem Cells
The embryo is destroyed by separating it into individual cells for the collection of ICM cells.
G. Podgorksi, Biol. 1010

39. Some Thorny Ethical Questions
Biotechnology
10/9/2017
Some Thorny Ethical Questions
Are these masses of cells a human?
Is it ethical to harvest embryonic stem cells from the “extra” embryos created during in vitro fertilization?
G. Podgorksi, Biol. 1010

40. Biotechnology
10/9/2017
Additional Potential Dilemmas – Therapeutic Cloning to Obtain Matched Embryonic Stem Cells
Cultured mouse embryonic stem cells.
Cells from any source other than you or an identical twin present the problem of rejection.
If so, how can matched embryonic stem cells be obtained?
A cloned embryo of a person can be made, and embryonic stem cells harvested from these clones.
G. Podgorksi, Biol. 1010

41. DNA, the Law, and Many Other Applications –
Biotechnology
10/9/2017
DNA, the Law, and Many Other Applications –
The Technology of DNA Fingerprinting
A DNA fingerprint used in a murder case.
The defendant stated that the blood on his clothing was his.
What are we looking at? How was it produced?
G. Podgorksi, Biol. 1010

42. DNA Fingerprinting Basics
Biotechnology
10/9/2017
DNA Fingerprinting Basics
Different individuals carry different alleles.
Most alleles useful for DNA fingerprinting differ on the basis of the number of repetitive DNA sequences they contain.
G. Podgorksi, Biol. 1010

43. DNA Fingerprinting Basics
Based on the fact that each individual has his/her own unique DNA sequence
Functional genes have same sequence
Introns are unique in each organism
Introns contain sequences that repeat over and over
Both length of sequence and # of times they repeat are unique to each person
Termed a Variable Number Tandem Repeat (VNTR)
Not even the same in identical twins
Your unique VNTR is termed your DNA Fingerprint

44. DNA Fingerprinting Basics
DNA fingerprints are used in a number of applications, including:
1. finding relatives and geneology
2. purebred pet disputes
3. paternity cases
Procedure
Obtain sample of DNA (blood, hair, cheek cells)
Amplify DNA
Done by using the Polymerase Chain Reaction (PCR)
PCR will make millions of copies of DNA from one strand

45. PCR Procedure Isolate the DNA (ds)
Heat the DNA to 90°C to separate the strands
Reduce temp to 55°C + add primers
Primers match the ends of your two strands
Add a DNA polymerase + bases, and copies are made
Reheat DNA to 90ºC to separate new strands
Repeat primer + synthesis steps
Now have 4 copies
Repeat over + over + over + over…….
Taq polymerase (from hot spring bacteria) is used
PCR machines are used to carry out the reaction

46. PCR Procedure After amplification, cut DNA with R.E.
Run DNA on gel to separate fragments by size, this is your fingerprint
Can compare to other samples in cases of dispute
Accurate to %
The length of fragments you produce is unique to you
This is termed a restriction fragment length polymorphism (a RFLP)

47. DNA Fingerprinting Basics
Biotechnology
10/9/2017
DNA Fingerprinting Basics
If DNA is cut with a restriction enzyme that recognizes sites on either side of the region that varies, DNA fragments of different sizes will be produced.
A DNA fingerprint is made by analyzing the sizes of DNA fragments produced from a number of different sites in the genome that vary in length.
The more common the length variation at a particular site and the greater the number the sites analyzed, the more informative the fingerprint.
G. Podgorksi, Biol. 1010

48. The DNA Fragments Are Separated on the Basis of Size
Biotechnology
10/9/2017
The DNA Fragments Are Separated on the Basis of Size
The technique is gel electrophoresis.
The pattern of DNA bands is compared between each sample loaded on the gel.
Gel electrophoresis animation
G. Podgorksi, Biol. 1010

49. Biotechnology
10/9/2017
A DNA Fingerprint
When many genes are analyzed, each with many different alleles, the chance that two patterns match by coincidence is vanishingly small.
DNA detective animation
HGP fingerprinting page
G. Podgorksi, Biol. 1010

50. Genetically Modified Foods
Biotechnology
10/9/2017
Genetically Modified Foods
Many of our crops in the US are genetically modified.
Should they be?
G. Podgorksi, Biol. 1010

51. Biotechnology
10/9/2017
GM Crops are Here Today
Source: Pew Initiative on Food and Biotechnology, August 2004.
G. Podgorksi, Biol. 1010

52. Biotechnology
10/9/2017
Methods for Plant Genetic Engineering are Well-Developed and Similar to Those for Animals
G. Podgorksi, Biol. 1010

53. Golden Rice is Modified to be Provide a Dietary Source of Vitamin A
Biotechnology
10/9/2017
Golden Rice is Modified to be Provide a Dietary Source of Vitamin A
Golden rice (yellow) with standard rice (white).
Worldwide, 7% of children suffer vitamin A deficiency, many of them living in regions in which rice is a staple of the diet.
G. Podgorksi, Biol. 1010

54. Genetically Modified Crops
Biotechnology
10/9/2017
Genetically Modified Crops
Genetically Modified Crops
Genetically Modified Cotton
(contains a bacterial gene for pest resistance)
Standard Cotton
G. Podgorksi, Biol. 1010

55. GMOs, Especially Outside the US, Are a Divisive Issue
Biotechnology
10/9/2017
GMOs, Especially Outside the US, Are a Divisive Issue
Protesters at the 2000 Montreal World Trade Summit
European sentiment
G. Podgorksi, Biol. 1010

56. Biotechnology
10/9/2017
Current Concerns by Scientists Focus on Environmental, Not Health, Effects of GM Crops
The jury’s still out on the magnitude of GM crop’s ecological impact, but the question is debated seriously.
G. Podgorksi, Biol. 1010

57. Biotechnology
10/9/2017
Current Concerns by Scientists Focus on Environmental, Not Health, Effects of GM Crops
G. Podgorksi, Biol. 1010

58. Other Techniques Autoradiography and Blotting
Used to detect DNA by making it radioactive
Isotopes of phosphorous (P32) are added to a growth medium, this is then incorporated into a DNA molecule
DNA separated by electrophoresis may be transferred to photographic film, which will show the radioactive DNA
In hybridization experiments, DNA or RNA is made single stranded, run on a gel, then a radioactive probe will be added
This probe will bind only to a complementary sequence, then it will show up on film

59. Other Techniques
Those hybridization experiments are the basis for 4 very important techniques called “blotting”
Southern blotting is a DNA/DNA hybridization technique
Northern blotting is a DNA/RNA technique
Western blotting is a protein/antibody technique
Southwestern blotting is a protein/DNA technique

60. Other Techniques Southern + Northern involve the following procedure:
1. cut nucleic acid with a restriction enzyme
2. denature to make single-stranded
3. run on gel
4. blot nucleic acid from gel onto filter paper
5. use radioactive probe to look for gene of choice
Northern blotting will allow you to find a gene’s matching mRNA sequence
Can then make cDNA from this mRNA