1. Chapter 13 Biotechnology
13.1 What is biotechnology?
13.2 How does DNA recombine occur in nature?
13.3 How is biotechnology used in forensics science?
13.4 How is biotechnology used in agriculture?

2. Chapter 13 Biotechnology
13.5 How is biotechnology used to learn about the human genome?
13.6 How is biotechnology used for medical diagnosis and treatment?
13.7 What are the major ethical issues surround modern biotechnology?

3. 13.1 What Is Biotechnology?
Any industrial or commercial use or alternation of organisms, cells or biological molecules to achieve specific practical goals
Genetic engineering – modification of genetic material
GMOs (transgenic) – genetically modified organisms

4. Sexual Reproduction
Due to crossing over during meiosis, each chromosome in a gamete contains a mixture of alleles from the two parental chromosomes
Thus, eggs and sperm contain recombinant DNA

5. Transformation
Bacteria can naturally take up DNA from the environment (transformation) and integrate the new genes into the genome (recombination)
Transformation with DNA fragment
Transformation with plasmid

6. FIGURE 13-1a Recombination in bacteria
(a) In addition to their large circular chromosome, bacteria commonly possess small rings of DNA called plasmids, which often carry additional useful genes. Bacterial transformation occurs when living bacteria take up (b) fragments of chromosomes or (c) plasmids.

7. FIGURE 13-1bc Recombination in bacteria
(a) In addition to their large circular chromosome, bacteria commonly possess small rings of DNA called plasmids, which often carry additional useful genes. Bacterial transformation occurs when living bacteria take up (b) fragments of chromosomes or (c) plasmids.

8. Transformation
Small circular DNA molecules (plasmids) carry supplementary genes
Plasmid genes may allow bacteria to grow in novel environments
Plasmid genes may enhance virulence of bacteria in establishing an infection
Plasmid genes may confer resistance to antimicrobial drugs

9. Viral Transfer of DNA Viral life cycle
Viral particle invades host cell
Viral DNA is replicated
Viral protein molecules are synthesized
Offspring viruses are assembled and break out of the host cell

10. Viral Transfer of DNA Viral transfer of DNA
Viruses may package some genes from host cell into viral particles during assembly
Infection of new host cell injects genes from previous host, allowing for recombination
Viruses may transfer DNA between bacteria and between Eukaryotic species

11. FIGURE 13-2 Viruses may transfer genes between cells

12. Biotechnology and Forensics
Forensics is the science of criminal and victim identification
DNA technology has allowed forensic science to identify victims and criminals from trace biological samples
Genetic sequences of any human individual are unique
DNA analysis reveals patterns that identify people with a high degree of accuracy

13. 13.3 How Is Biotechnology Used in Forensics?
The Polymerase Chain Reaction Amplifies DNA
PCR copies a specific DNA sequence
Need high temperatures to separate DNA strands
But then a special DNA polymerase that wouldn’t fall apart at high temperatures is needed

14. Polymerase Chain Reaction
Four steps of a PCR cycle
Template strand separation
The test tube is heated to 90-95oC to cause the double stranded template DNA to separate into single strands…

15. Polymerase Chain Reaction
Four steps of a PCR cycle
Binding of the primers
The temperature is lowered to 50oC to allow the primer DNA segments to bind to the targeted gene sequences through hydrogen bonding…

16. Polymerase Chain Reaction
Four steps of a PCR cycle
New DNA synthesis at targeted sequences
The temperature is raised to 70-72oC where the heat-stable DNA polymerase synthesizes new DNA of the sequences targeted by the primers…

17. Polymerase Chain Reaction
Four steps of a PCR cycle
Repetition of the cycle
The cycle is repeated automatically (by a thermocycler machine) for cycles, producing up to 1 billion copies of the original targeted DNA sequence

18. FIGURE 13-3a PCR copies a specific DNA sequence
The polymerase chain reaction consists of a series of 20 to 30 cycles of heating and cooling. After each cycle, the amount of target DNA doubles. After just 20 cycles, a million copies of the target DNA have been synthesized.

19. FIGURE 13-3b PCR copies a specific DNA sequence
The polymerase chain reaction consists of a series of 20 to 30 cycles of heating and cooling. After each cycle, the amount of target DNA doubles. After just 20 cycles, a million copies of the target DNA have been synthesized.

20. Thomas Brock surveys Mushroom Spring. Thermus aquaticus
Figure: E13-1
Title:
Thomas Brock surveys Mushroom Spring
Caption:
The colors in hot springs arise from minerals dissolved in the water and from various types of microbes that live at different temperatures.
Thomas Brock surveys Mushroom Spring. Thermus aquaticus

21. Polymerase Chain Reaction
Choice of primers determines which sequences are amplified (copied)
Forensic scientists focus on short tandem repeats (STRs) found within the human genome

22. Polymerase Chain Reaction
STRs are repeated sequences of DNA within the chromosomes that do not code for proteins
STRs vary greatly between different human individuals
A match of 10 different STRs between suspect and crime scene DNA virtually proves the suspect was at the crime scene

23. 8 side-by-side (tandem) repeats of the same 4-nucleotide sequence,G A A G A T A G A T A G A T A G A T A G A T A G A T A G A T A G A T A G G T A T A T A A A A C T T C T A T C T A T C T A T C T A T C T A T C T A T C T A T C T A T C C A T A G A T
8 side-by-side (tandem) repeats
of the same 4-nucleotide sequence,
Figure: 13-4
Title:
Short tandem repeats are common in noncoding regions of DNA
Caption:
This STR, called D5, is not part of any known gene. The sequence AGAT may be repeated from 7 to 13 times in different individuals. T C T A
This STR, called D5, is not part of any known gene.

24. 13.3 How Is Biotechnology Used in Forensics?
Gel Electrophoresis Separates and Identifies DNA Segments
Gel electrophoresis is used to separate and identify segments of DNA
DNA fingerprinting
DNA probe

25. FIGURE 13-5a Gel electrophoresis is used to separate and identify segments of DNA

26. FIGURE 13-5b Gel electrophoresis is used to separate and identify segments of DNA

27. FIGURE 13-5c Gel electrophoresis is used to separate and identify segments of DNA

28. FIGURE 13-5d Gel electrophoresis is used to separate and identify segments of DNA

29. FIGURE 13-5e Gel electrophoresis is used to separate and identify segments of DNA

30. DNA Probes
DNA probes are short single-stranded DNA fragments used to identify DNA in a gel pattern
Probe sequence is complementary to a DNA fragment somewhere in the gel pattern

31. STR #1: probe base-pairs and binds
label
(colored
molecule)
STR #1: probe base-pairs and binds
Figure: 13-UN1
Title:
DNA probe
STR #2: probe cannot base-pair; does not bind

32. DNA Probes
DNA probes
Probes may have colored molecules attached to them to allow for visual identification of the bands to which they bind
Gel DNA pattern is usually transferred to piece of nylon paper before probing

33. DNA Fingerprinting
DNA from a crime scene sample can be amplified by PCR and run on a gel with suspect DNAs
Short tandem repeats (STRs) in the gel DNA can be identified by DNA probes

34. DNA Fingerprinting
Distinctive pattern of STR numbers and lengths are fairly unique to a specific individual (forming a DNA fingerprint)
DNA fingerprint from crime scene can be matched with DNA fingerprint of suspect

35. FIGURE 13-7 DNA profiling
The lengths of short tandem repeats of DNA form characteristic patterns on a gel; this gel displays six different STRs (Penta D, CSF, etc.). The evenly spaced yellow-green bands on the far left and far right sides of the gel show the number of repeats of the individual STRs. DNA samples from 13 different people were run between these standards, resulting in one or two bands per vertical lane. For example, in the enlargement of the D16 STR on the right, the first person's DNA has 12 repeats, the second person's has 13 and 12, the third has 11, and so on. Although some people have the same number of repeats of some STRs, no one has the same number of repeats of all the STRs. (Photo courtesy of Dr. Margaret Kline, National Institute of Standards and Technology.)

36. 13.4 How Is Biotechnology Used in Agriculture?
Many Crops Are Genetically Modified
Genetically Engineered Crops with USDA Approval
In 2002, 34% of corn, 71% of cotton & 75% of soybeans were GMOs
In 2005, 52% of corn, 79% of cotton & 87% of soybeans were GMOs
Not required to be labeled in US

37. Table 13-1 Genetically Engineered Crops with USDA Approval

38. Many Crops Are Genetically Modified
Crop plants are commonly modified to improve insect and herbicide resistance
Herbicide resistant crops withstand applications of weed-killing chemicals
Bt gene (from Bacillus thuringiensis bacterium) can be inserted into plants to produce insect-killing protein in crops

39. FIGURE 13-8 Bt plants resist insect attack
Transgenic cotton plants expressing the Bt gene (right) resist attack by bollworms, which eat cotton seeds. The transgenic plants therefore produce far more cotton than non-transgenic plants (left

40. Cloning of the Desired Gene
Modifying a plant genetically begins with gene cloning
Desired gene is first isolated from organism containing it
Desired gene may alternately be synthesized in the laboratory

41. Cloning of the Desired Gene
Modifying a plant genetically begins with gene cloning
Gene is next inserted into a small DNA circle called a plasmid which replicates itself autonomously in bacterial cells

42. Restriction Enzymes Cut DNA
A DNA sequence (e.g. a gene) can be removed from a chromosome using special enzymes
Restriction enzymes are nucleases that cut DNA at specific nucleotide sequences

43. FIGURE 13-9 Some restriction enzymes leave "sticky ends" when they cut DNA

44. 13.4 How Is Biotechnology Used in Agriculture?
The desired gene is cloned
Restriction enzymes cut DNA at specific nucleotide sequences
Cutting two pieces of DNA with the same restriction enzyme allows the pieces to be joined together
Using Agrobacterium tumefaciens to insert the Bt gene into plants

45. FIGURE 13-10a Using Agrobacterium tumefaciens to insert the Bt gene into plants

46. FIGURE 13-10b Using Agrobacterium tumefaciens to insert the Bt gene into plants

47. FIGURE 13-10c Using Agrobacterium tumefaciens to insert the Bt gene into plants

48. FIGURE 13-10d Using Agrobacterium tumefaciens to insert the Bt gene into plants

49. FIGURE 13-10e Using Agrobacterium tumefaciens to insert the Bt gene into plants

50. FIGURE 13-10 Using Agrobacterium tumefaciens to insert the Bt gene into plants

51. Figure: E13-2
Title:
Salmon
Caption:
Transgenic salmon (bottom) grow much faster than their wild relatives (top).
Transgenic salmon (bottom) grow much faster than their wild relatives (top)

52. 13.4 How Is Biotechnology Used in Agriculture?
Genetically modified plants may be used to produce medicines
Genetically modified animals may be useful in agriculture and medicine

53. 13.5/13.6 How Is Biotechnology Used for Medical Diagnosis and Treatment?
DNA technology can be used to diagnose inherited disorders
Restriction enzymes may cut different alleles of a homologous pair of chromosomes at different locations
RFLPs: Restriction length polymorphisms “riff-lips”

54. FIGURE 13-11a Diagnosing sickle-cell anemia with restriction enzymes
(a) The normal globin allele and the sickle-cell allele (both shown in red) are cut in half by the restriction enzyme MstII (far right arrow). The normal allele is also cut in another, unique location (middle arrow). Finally, regardless of which allele is present, the chromosome is cut somewhat ahead of the globin gene locus (far left arrow). A DNA probe (blue) is synthesized that is complementary to DNA on both sides of the unique cut site. Therefore, the probe will label two pieces of DNA from the normal allele but only a single piece of the sickle-cell allele.

55. FIGURE 13-11b Diagnosing sickle-cell anemia with restriction enzymes
(b) The cut DNA is run on a gel and made visible with the DNA probe. The large piece of DNA of the sickle-cell allele is close to the beginning of the gel, while the smaller pieces of the normal allele run further into the gel.

56. DNA Probes Defective alleles can also be identified using DNA probes
DNA probing is especially useful where there are many different alleles at a single gene locus
Cystic fibrosis is a disease caused by any of 32 alleles out of 1000 total possible alleles

57. DNA Probes
Arrays of single-stranded DNA complementary to each of the defective alleles can be bound to filter paper
A person’s DNA sample is cut up and separated into single-strands
The array is bathed in the DNA sample
Strands of DNA binding to complementary sequence on the paper indicate presence of a defective allele in person’s genome

58. FIGURE 13-12a DNA arrays in medicine and research
(a) DNA from a patient is cut into small pieces, separated into single strands, and labeled (blue, in this diagram). A cystic fibrosis screening array is bathed in this solution of labeled DNA. Each cystic fibrosis allele can bind to only one specific piece of complementary DNA on the array. In this simplified diagram, the patient has one normal allele (upper left) and one defective allele (middle bottom).

59. DNA Probes
An expanded version of this type of DNA analysis is known as a microarray
A microarray contains up to thousands of probes for a variety of disease-related alleles
Microarray analysis has the potential to comprehensively identify disease susceptibility

60. FIGURE 13-12b DNA arrays in medicine and research
(b) Each spot contains a DNA probe for a specific human gene. In most research applications, messenger RNA is isolated from a subject (for example, from a human cancer), and labeled with a fluorescent dye. The mRNA is then poured onto the array, and each base-pairs with its complementary template DNA probe. Genes that are particularly active in the cancer will "light up" the corresponding DNA probe.

61. Disease Treatment Treatments using DNA technology
Administration of proteins to treat but not cure a disorder
Human insulin produced inexpensively and rapidly in recombinant bacteria for diabetics
Growth hormone and blood clotting factors produced safely and inexpensively in recombinant bacteria

62. Disease Treatment Treatments using DNA technology
Replacing defective genes to possibly cure a disorder
Replacement of defective cystic fibrosis allele using a virus to carry in a functional gene sequence into patient lung cells
Defective bone marrow cell DNA replacement by functional gene in severe combined immune deficiency (SCID) patients

63. Table 13-2 Examples of Medical Products Produced by Recombinant DNA Method

64. Section 13.7 Outline 13.7 Biotechnology and Ethics
Issues Surrounding GM Organisms in Agriculture
Scientific Objections to Genetically Modified Organisms
Ethics of Using Biotechnology on the Human Genome

65. GM Organisms in Agriculture
The goal of breeding or genetically modifying plants or livestock is to make them more productive, efficient, or useful

66. GM Organisms in Agriculture
Genetic modification differs from selective breeding (“traditional biotechnology”)
Genetic engineering is much more rapid
Genetic engineering can transfer genes between species
Genetic engineering can produce new genes never seen before on Earth

67. GM Organisms in Agriculture
Benefits of genetically modified plants
Transgenic crops decrease applications of pesticides, saving fuel, labor, and money
GM plants can be sold at a lower price due to farm savings
Genetically engineered crops can deliver greater amounts of vitamins
e.g. “golden rice” which produces vitamin A

68. FIGURE E13-4 Golden Rice
Conventional milled rice is white or very pale tan (lower right). The original Golden Rice (upper right) was pale golden-yellow because of its increased beta-carotene content. Second-generation Golden Rice 2 (left) is much deeper yellow, because it contains about 20 times more beta-carotene than original Golden Rice does.

69. Scientific Objections to GMOs
Safety issues from eating GMOs
Could ingestion of Bt protein in insect-resistant plants be dangerous to humans?
Are transgenic fish producing extra growth hormone dangerous to eat?

70. Scientific Objections to GMOs
Safety issues from eating GMOs
Could GM crops cause allergic reactions?
USDA now monitors GM foods for allergic potential
Toxicology study of GM plants (2003) concluded that ingestion of current transgenic crops pose no significant health dangers

71. Scientific Objections to GMOs
Environmental hazards posed by GMOs
Pollen from modified plants can carry GM genes to the wild plant population
Could herbicide resistance genes be transferred to weed species, creating superweeds?

72. Scientific Objections to GMOs
Environmental hazards posed by GMOs
Could GM fish reduce biodiversity in the wild population if they escape?
Reduced diversity in wild fish makes them more susceptible to catastrophic disease outbreaks

73. Scientific Objections to GMOs
Environmental hazards posed by GMOs
US found to lack adequate system to monitor changes in ecosystem wrought by GMOs (National Academy of Science Study 2003)

74. The Human Genome
Should parents be given information about the genetic health of an unborn fetus?
Should parents be allowed to select the genomes of their offspring?
Embryos from in vitro fertilization are currently tested before implantation
Many unused embryos are discarded
Should parents be allowed to design or correct the genomes of their offspring?

75. FIGURE 13-13 Human cloning technology might allow permanent correction of genetic defects
In this process, human embryos are derived from eggs fertilized in culture dishes using sperm and eggs from a man and woman, one or both of whom have a genetic disorder. When an embryo containing a defective gene grows into a small cluster of cells, a single cell would be removed from the embryo and the defective gene replaced using an appropriate vector. The repaired nucleus could then be implanted into another egg (taken from the same woman) whose nucleus had been removed. The repaired egg cell would then be implanted in the woman's uterus for normal development.