1. Chapter 20
Biotechnology

2. Overview: The DNA Toolbox
Sequencing of the human genome was completed by 2007
DNA sequencing has depended on advances in technology, starting with making recombinant DNA
In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule

3. Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes
DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products
An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

5. Insulin manufacturing

8. Why do we need insulin?
How does our body make insulin?
How can we make insulin as medication?

9. Learning objectives . Explain the role of restriction enzymes
Explain the process of DNA cloning

10. Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment
To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning

11. DNA Cloning and Its Applications: A Preview
Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids
Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
Cloned genes are useful for making copies of a particular gene and producing a protein product

12. Gene cloning involves using bacteria to make multiple copies of a gene
Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell
Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA
This results in the production of multiple copies of a single gene

13. Cell containing gene of interest Bacterium
Fig. 20-2a
Cell containing gene of interest
Bacterium 1
Gene inserted into plasmid
Bacterial chromosome
Plasmid
Gene of interest
Recombinant DNA (plasmid)
DNA of chromosome 2 2
Plasmid put into bacterial cell
Figure 20.2 A preview of gene cloning and some uses of cloned genes
Recombinant bacterium

14. Recombinant bacterium
Fig. 20-2b
Recombinant bacterium 3
Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
Gene of Interest
Protein expressed by gene of interest
Copies of gene
Protein harvested 4
Basic research and various applications
Basic research on gene
Basic research on protein
Figure 20.2 A preview of gene cloning and some uses of cloned genes
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hor- mone treats stunted growth

15. Using Restriction Enzymes to Make Recombinant DNA
Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
A restriction enzyme usually makes many cuts, yielding restriction fragments
The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments
Animation: Restriction Enzymes

16. DNA ligase is an enzyme that seals the bonds between restriction fragments

17. Restriction enzyme cuts sugar-phosphate backbones.
Fig
Restriction site
DNA 5 3 3 5 1
Restriction enzyme cuts sugar-phosphate backbones.
Sticky end
Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA

18. Restriction enzyme cuts sugar-phosphate backbones.
Fig
Restriction site
DNA 5 3 3 5 1
Restriction enzyme cuts sugar-phosphate backbones.
Sticky end 2
DNA fragment added from another molecule cut by same enzyme. Base pairing occurs.
Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA
One possible combination

19. Restriction enzyme cuts sugar-phosphate backbones.
Fig
Restriction site
DNA 5 3 3 5 1
Restriction enzyme cuts sugar-phosphate backbones.
Sticky end 2
DNA fragment added from another molecule cut by same enzyme. Base pairing occurs.
Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA
One possible combination 3
DNA ligase seals strands.
Recombinant DNA molecule

21. Cloning a Eukaryotic Gene in a Bacterial Plasmid
In gene cloning, the original plasmid is called a cloning vector
A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there

22. Expressing Cloned Eukaryotic Genes
After a gene has been cloned, its protein product can be produced in larger amounts for research
Cloned genes can be expressed as protein in either bacterial or eukaryotic cells

23. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)
The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA
A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules
Application of PCR:
Diagnose genetic diseases, do DNA fingerprinting, find bacteria and viruses, study human evolution, clone the DNA of an Egyptian mummy, establish paternity or biological relationships. PCR has become an essential tool for biologists, DNA forensics labs, and many other laboratories that study genetic material.

24. Taq ploymerase is used in PCR
Properties of Thermostable DNA Polymerase. The original report of this enzyme, purified from the hot springs bacterium Thermus aquaticus, was published in Roughly 10 years later, the polymerase chain reaction was developed and shortly thereafter "Taq" became a household word in molecular biology circles.

25. molecules; 2 molecules (in white boxes) match target sequence
Fig. 20-8
TECHNIQUE 5 3
Target sequence
Genomic DNA 3 5 1
Denaturation 5 3 3 5 2
Annealing
Cycle 1 yields 2
molecules
Primers 3
Extension
New nucleo- tides
Figure 20.8 The polymerase chain reaction (PCR)
Cycle 2 yields 4
molecules
Cycle 3 yields 8
molecules; 2 molecules (in white boxes) match target sequence

26. Gel Electrophoresis and Southern Blotting
One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis
This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size
A current is applied that causes charged molecules to move through the gel
Molecules are sorted into “bands” by their size
Video: Biotechnology Lab

27. Figure 20.9 Gel electrophoresis
TECHNIQUE
Mixture of DNA mol- ecules of different sizes
Power source –
Cathode
Anode +
Gel 1
Power source – +
Longer molecules 2
Shorter molecules
RESULTS
Figure 20.9 Gel electrophoresis

28. Mixture of DNA mol- ecules of different sizes
Fig. 20-9a
TECHNIQUE
Power source
Mixture of DNA mol- ecules of different sizes –
Cathode
Anode +
Gel 1
Power source
Figure 20.9 Gel electrophoresis – +
Longer molecules 2
Shorter molecules

29. Fig. 20-9b
RESULTS
Figure 20.9 Gel electrophoresis

30. RFLP( Restriction Fragment Length Polymorphism)
The DNA sample is broken into pieces (and digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths bygel electrophoresis
Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene.
The procedure is also used to prepare pure samples of individual fragments.

31. Fig
Normal -globin allele
Normal allele
Sickle-cell allele
175 bp
201 bp
Large fragment
DdeI
DdeI
DdeI
DdeI
Large fragment
Sickle-cell mutant -globin allele
376 bp
201 bp 175 bp
376 bp
Large fragment
DdeI
Figure Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the β-globin gene
DdeI
DdeI
(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

32. DNA Sequencing
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA.

33. Forensic Evidence and Genetic Profiles DNA fingerprinting
DNA profiling (also called DNA fingerprinting, DNA testing, or DNA typing) is a forensic technique used to identify individuals by characteristics of their DNA.

34. Murder investigation using DNA finger printing

35. Environmental Cleanup
Genetic engineering can be used to modify the metabolism of microorganisms
Algenol Biofuels
Rapeseed (i.e., canola)
Poplar trees remove groundwater contaminants
Able to digest and process phosphate
Enviropig i.e., “Frankenswine”

36. Agricultural Applications
DNA technology is being used to improve agricultural productivity and food quality
Golden rice

37. Animal Husbandry Banana vaccines
Genetic engineering of transgenic animals speeds up the selective breeding process
Beneficial genes can be transferred between varieties or species
Banana vaccines
Cancer fighting eggs

38. Genetic Engineering in Plants
Agricultural scientists have endowed a number of crop plants with genes for desirable traits
The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
Soybean
Roundup ready crops

39. Agrobacterium tumefaciens
Fig
TECHNIQUE
Agrobacterium tumefaciens
Ti plasmid
Site where restriction enzyme cuts
T DNA
RESULTS
DNA with the gene of interest
Figure Using the Ti plasmid to produce transgenic plants
For the Cell Biology Video Pronuclear Injection, go to Animation and Video Files.
For the Discovery Video Transgenics, go to Animation and Video Files.
Recombinant Ti plasmid
Plant with new trait