1. Copyright © 2009 Pearson Education, Inc.. Lectures by Gregory Ahearn University of North Florida Chapter 12 Biotechnology

2. Copyright © 2009 Pearson Education Inc. 12.1 What Is Biotechnology?  Biotechnology is any use or alteration of organisms, cells, or biological molecules to achieve specific practical goals. Today, selective breeding is an important part of biotechnology. Modern biotechnology frequently uses genetic engineering. This term refers to direct methods for modifying genetic material.

3. Copyright © 2009 Pearson Education Inc. 12.1 What Is Biotechnology?  A key tool in genetic engineering is recombinant DNA, which is DNA that has been altered to contain genes or parts of genes from different organisms. Large amounts of recombinant DNA can be grown in bacteria, viruses, or yeasts, and then transferred into other species. Plants or animals that express DNA that has been modified or derived from other species are called transgenic, or genetically modified, organisms (GMOs).

4. Copyright © 2009 Pearson Education Inc. 12.1 What Is Biotechnology?  Modern biotechnology also includes many methods of manipulating DNA, whether or not the DNA is subsequently put into a cell or an organism.  For example, determining the nucleotide sequence of specific pieces of DNA is crucial to forensic science and the diagnosis of inherited disorders.

5. Copyright © 2009 Pearson Education Inc. 12.1 What Is Biotechnology?  This chapter is organized around five themes. 1.Recombinant DNA mechanisms found in nature 2.Biotechnology in criminal forensics (DNA matching) 3.Biotechnology in agriculture, and production of transgenic plants and animals 4.The Human Genome Project 5.Biotechnology in medicine; the treatment of inherited disorders

6. Copyright © 2009 Pearson Education Inc. 12.2 How Does DNA Recombine In Nature?  Many natural processes can transfer DNA from one organism to another, sometimes even to organisms of different species. Sexual reproduction recombines DNA from two different organisms. Every egg and sperm contain recombinant DNA, derived from the organism’s two parents. When a sperm fertilizes an egg, the resulting offspring also contains recombinant DNA.

7. Copyright © 2009 Pearson Education Inc. 12.2 How Does DNA Recombine In Nature?  Transformation may combine DNA from different bacterial species. Bacteria can undergo several types of recombination that allow gene transfer between species. In a process called transformation, bacteria pick up pieces of DNA from the environment. The DNA may be part of the chromosome of another bacterium or from another species, or may be in the form of tiny circular DNA molecules called plasmids.

8. Copyright © 2009 Pearson Education Inc. 12.2 How Does DNA Recombine In Nature?  Transformation in bacteria Fig. 12-1 plasmid bacterial chromosome DNA fragments bacterial chromosome A DNA fragment is incorporated into the chromosome bacterial chromosome The plasmid replicates in the cytoplasm (a) Bacterium (b) Transformation with a DNA fragment (c) Transformation with a plasmid 1 micrometer

9. Copyright © 2009 Pearson Education Inc. 12.2 How Does DNA Recombine In Nature?  Viruses may transfer DNA between species. Viruses are genetic material encased in a protein coat, and they transfer their genetic material to the host cells that they infect. Once inside a host cell, the viral genes replicate and use the infected cell’s enzymes and ribosomes to synthesize viral proteins. These proteins form new viruses that are released to infect other cells.

10. Copyright © 2009 Pearson Education Inc. 12.2 How Does DNA Recombine In Nature?  Viruses may transfer genes between cells. Fig. 12-2 The virus releases its DNA into the host cell; some viral DNA (red) may be incorporated into the host cell’s DNA (blue) Viral genes encode the synthesis of viral proteins and viral gene replication; some host cell DNA may attach to the replicated viral DNA (red/blue combination) New viruses assemble; some host cell DNA is carried by “hybrid viruses” The host cell bursts open, releasing newly assembled viruses; if “hybrid viruses” infect a second cell, they may transfer genes from the first cell to the second cell viral proteins “hybrid virus” virus viral DNA A virus attaches to a susceptible host cell host cell host cell DNA The virus enters the host cell

11. Copyright © 2009 Pearson Education Inc. 12.2 How Does DNA Recombine In Nature? Animation—Genetic Recombination in Bacteria PLAY

12. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  The polymerase chain reaction (PCR) amplifies DNA. PCR produces virtually unlimited amounts of selected pieces of DNA. Primers (small pieces of complementary RNA) tell the DNA polymerase where on the DNA molecule to start copying.

13. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  The polymerase chain reaction (PCR) amplifies DNA (continued). One primer is complementary to the beginning of the DNA strand to be copied. The other primer is complementary to the other end, so DNA replication occurs in both directions. PCR consists of the following steps repeated as often as needed to make enough copies of DNA.

14. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  In a small test tube, DNA is mixed with primers, free nucleotides, and a special heat-resistant DNA polymerase. 1.The test tube is heated to 90°C, which breaks the hydrogen bonds between complementary bases, separating the DNA into single strands. 2.The temperature is lowered to about 50°C to allow the primers to form complementary base pairs with the original DNA strands.

15. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science? 3.The temperature is raised to 70–72°C so DNA polymerase can use the primers to make copies of the DNA segment bounded by the primers. 4.The cycle is repeated as many times as desired.

16. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  PCR copies a specific DNA sequence. Fig. 12-3a original DNA 194°F (90°C)122°F (50°C)161°F (72°C) DNA polymerase new DNA strands primers Heating separates DNA strands Cooling allows primers and DNA polymerase to bind New DNA strands a synthesized (a) One PCR cycle

17. Copyright © 2009 Pearson Education Inc. Fig. 12-3b 123 1248 PCR cycles DNA copies 4 etc. 16 etc. DNA fragment to be amplified (b) Each PCR cycle doubles the number of copies of the DNA 12.3 How Is Biotechnology Used In Forensic Science?

18. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Differences in short tandem repeats can identify individuals. In many criminal investigations, PCR is used to amplify the DNA so that there is enough to compare the DNA left at the crime scene with the suspect’s DNA. Forensic experts have found that small segments of DNA, called short tandem repeats (STRs), can be used to identify people with astonishing accuracy.

19. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Short tandem repeats Fig. 12-4 Eight side-by-side (tandem) repeats of the same four-nucleotide sequence A T G C A T T A A T G C A T T A T A A T A T T A T A T A T A G C A T A T G C A T T A A T G C A T T A A T G C A T T A A T G C A T T A A T G C A T T A A T G C A T T A A T G C G C T A A T A T G C A T T A

20. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Differences in short tandem repeats can identify individuals (continued). People have different numbers of repeated nucleotides in their STRs. A perfect match of 10 STRs in a suspect’s DNA and the DNA found at a crime scene means that there is less than one chance in a trillion that the two DNA samples did not come from the same person.

21. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Gel electrophoresis separates DNA segments. A mixture of DNA pieces is separated by a technique called gel electrophoresis. The mixture of DNA is loaded onto a slab of agarose. The gel is put in a chamber with electrodes connected to each end; one is positive, the other negative. Current is allowed to flow between the electrodes through the gel. The flowing current separates the DNA fragments, forming distinct bands on the gel.

22. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Gel electrophoresis Fig. 12-5(1) gel power supply wells pipetter DNA samples are pipetted into wells (shallow slots) in the gel. Electrical current is sent through the gel (negative at the end with the wells, positive at the opposite end).

23. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Gel electrophoresis (continued) Fig. 12-5(2) DNA “bands” (not yet visible) Electrical current moves the DNA segments through the gel. Smaller pieces of DNA move farther toward the positive electrode.

24. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  DNA probes are used to label specific nucleotide sequences. Unfortunately, the DNA bands are invisible, so how can anyone identify which band contains a specific STR? Usually, the two strands of the DNA double helix are separated during gel electrophoresis. This allows pieces of synthetic DNA, called DNA probes, to base-pair with specific DNA fragments in the sample.

25. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  The DNA probes are labeled, either by radioactivity or by attaching colored molecules to them.  A given DNA probe will only label certain DNA sequences and not others.

26. Copyright © 2009 Pearson Education Inc. STR #1: The probe base-pairs and binds to the DNA STR #2: The probe cannot base-pair with the DNA, so it does not bind probe label (colored molecule) TTTGA T A C G T A A T T A C G T A A T T A C G T A A T GAATACTG T A C A T T A G T A C A T T A G T A C A T T A G 12.3 How Is Biotechnology Used In Forensic Science?  DNA probes identify specific DNA sequences. Fig. 12-6

27. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  DNA probes are used to label specific nucleotide sequences (continued). The single-stranded DNA segments are transferred out of the gel and onto a piece of nylon paper. The paper is next bathed in a solution containing a specific DNA probe that will base- pair with, and bind to, only a specific STR, making this STR visible.

28. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Gel electrophoresis (continued) Fig. 12-5(3) gel nylon paper The gel is placed on special nylon “paper.” Electrical current drives the DNA out of the gel onto the nylon.

29. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Gel electrophoresis (continued) Fig. 12-5(4) nylon paper solution of DNA probes (red) The nylon paper with the DNA bound to it is bathed in a solution of labeled DNA probes (red) that are complementary to specific DNA segments in the original DNA sample.

30. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Gel electrophoresis (continued) Fig. 12-5(5) Complementary DNA segments are labeled by the probes (red bands).

31. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Every person has a unique DNA profile. In the early 1990s, forensic scientists ran DNA samples from a crime scene and from the suspects side-by-side on a gel, to see which suspect’s DNA matched that found at the scene. In modern STR analysis, a suspect and crime scene DNA samples can be run on different gels in different locations. The reason is that people have different numbers of repeats of their STRs, so that every person on Earth—except for identical twins—has a unique set of STRs.

32. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  When DNA samples are run on STR gels, they produce a pattern, called a DNA profile Fig. 12-7 STR name Penta D CSF D16 D7 D16: An STR on chromosome 16 DNA samples from 13 different people Number of repeats 15 14 13 12 11 10 9 8

33. Copyright © 2009 Pearson Education Inc. 12.3 How Is Biotechnology Used In Forensic Science?  Every person has a unique DNA profile. Anyone convicted of certain crimes must give a blood sample. Forensic technicians then determine the criminal’s DNA profile. This DNA profile is coded and stored in computer files. Because all forensic labs use the same STRs, computers can easily determine if DNA left at a crime scene matches one of the millions of profiles stored in a profile database.

34. Copyright © 2009 Pearson Education Inc. 12.4 How Is Biotechnology Used In Agriculture?  Many crops are genetically modified. Many herbicides kill plants by inhibiting an enzyme that is used by plants, but not animals, to synthesize essential amino acids. Many herbicide-resistant transgenic crops have been given a bacterial gene encoding an enyzme that functions even in the presence of these herbicides. These plants continue to synthesize normal amounts of amino acid and proteins. Less competition from weeds—which are killed by the herbicide, but not the crops—provides larger harvests.

35. Copyright © 2009 Pearson Education Inc. 12.4 How Is Biotechnology Used In Agriculture?  Many crops are genetically modified (continued). Insect resistance of many crops is enhanced by giving them a gene, called Bt, from the bacterium, Bacillus thuringiensis. The protein encoded by the Bt gene damages the digestive tract of insects, but not mammals. Bt crops therefore suffer less damage from insects, and farmers have to apply less pesticide to their fields.

36. Copyright © 2009 Pearson Education Inc. 12.4 How Is Biotechnology Used In Agriculture?  How would a seed company make insect- resistant Bt plants? 1.The desired gene is cloned. 2.First, one must obtain the gene; then, it must be inserted into a plasmid so that huge numbers of copies can be made. 3.Restriction enzymes cut the DNA at specific nucleotide sequences. 4.Genes are inserted into plasmids through the action of restriction enzymes isolated from bacteria.

37. Copyright © 2009 Pearson Education Inc. 12.4 How Is Biotechnology Used In Agriculture? 5.Each restriction enzyme cuts DNA at a specific nucleotide sequence. Fig. 12-8 A specific restriction enzyme (EcoRI) binds to the GAATTC sequence and cuts the DNA, creating DNA fragments with “sticky ends.” single-stranded “sticky ends” double- stranded DNA EcoRI restriction enzyme A TT G C T T A GAATT CT CGAT T T G TTA C GAATCTT AT AAGCTAA A C... A TT G C T T AG TTAACG AATCTTA AA A A A T TCG A T T T G GCTAA A C...

38. Copyright © 2009 Pearson Education Inc. 12.4 How Is Biotechnology Used In Agriculture? 6.Cutting two pieces of DNA with the same restriction enzyme allows the pieces to be joined together. 7.The Bt gene is inserted into a plasmid by cutting the DNA on either side of the Bt gene and splitting open the circle of the plasmid with the same restriction enzyme.

39. Copyright © 2009 Pearson Education Inc. 12.4 How Is Biotechnology Used In Agriculture? Fig. 12-9 DNA including Bt gene (blue) plasmid The DNA containing the Bt gene and the plasmid are cut with the same restriction enzyme.

40. Copyright © 2009 Pearson Education Inc. 12.4 How Is Biotechnology Used In Agriculture? 8.As a result, the ends of the Bt gene and the opened-up plasmid both have complementary bases in their ends, and can base-pair with each other. 9.When the cut Bt genes and plasmids are mixed together, some of the Bt genes will be temporarily inserted between the ends of the plasmid. 10.Adding DNA ligase permanently bonds the Bt genes into the plasmid.