Uses of DNA technology You will need to convince a grant committee to fund further research into your area of application of DNA technology Read your assigned section IN YOUR OWN WORDS, give an argument that includes 3 points that highlight that your field needs more money than any other Identify 2 counterarguments and explain why these may not be valid and you should still receive funding.

One of the key tools in DNA technology is the restriction enzyme

Where do these restriction enzymes come from???? What is their natural function??? How can we use them???

Recombinant DNA DNA from 2 sources combined Can be used to clone genes Used to produce a particular protein

Separate from main chromosome E. coli bacterium Plasmid Cell with DNA containing gene of interest 1 2 Isolate plasmid Isolate DNA Bacterial chromosome DNA Gene of interest A plasmid is a small circular piece of DNA found in some bacterial cells Separate from main chromosome May have genes that give the bacteria an advantage in certain circumstances Bacteria can take up plasmids from their environment Figure 12.1 An overview of gene cloning.

3 4 Cut cell’s DNA with same enzyme Gene of interest E. coli bacterium Plasmid Cell with DNA containing gene of interest 1 Isolate plasmid Bacterial chromosome 2 Isolate DNA DNA Gene of interest 3 4 Cut plasmid with enzyme Cut cell’s DNA with same enzyme Gene of interest Figure 12.1 An overview of gene cloning.

Combine targeted fragment and plasmid DNA E. coli bacterium Plasmid Cell with DNA containing gene of interest Isolate plasmid Bacterial chromosome 1 2 Isolate DNA 3 Cut plasmid with enzyme DNA Gene of interest 4 Cut cell’s DNA with same enzyme Gene of interest 5 Combine targeted fragment and plasmid DNA Figure 12.1 An overview of gene cloning.

Combine targeted fragment and plasmid DNA E. coli bacterium Plasmid Cell with DNA containing gene of interest 1 Isolate plasmid Bacterial chromosome 2 Isolate DNA 3 Cut plasmid with enzyme DNA Gene of interest 4 Cut cell’s DNA with same enzyme Gene of interest 5 Combine targeted fragment and plasmid DNA Figure 12.1 An overview of gene cloning. 6 Add DNA ligase, which closes the circle with covalent bonds Recombinant DNA plasmid Gene of interest

Put plasmid into bacterium Recombinant DNA plasmid Gene of interest Recombinant DNA plasmid Gene of interest Put plasmid into bacterium 7 Recombinant bacterium Figure 12.1 An overview of gene cloning.

Recombinant DNA plasmid Gene of interest Put plasmid into bacterium Recombinant DNA plasmid Gene of interest 7 Put plasmid into bacterium by transformation Recombinant bacterium Figure 12.1 An overview of gene cloning. 8 Allow bacterium to reproduce Clone of cells

Examples of gene use Genes may be inserted into other organisms Examples of gene use Genes may be inserted into other organisms Recombinant DNA plasmid Gene of interest 9 Genes or proteins are isolated from the cloned bacterium 7 Put plasmid into bacterium by transformation Recombinant bacterium Harvested proteins may be used directly Figure 12.1 An overview of gene cloning. 8 Allow bacterium to reproduce Clone of cells Examples of protein use

How is this recombinant DNA made? Important to use the same restriction enzyme to cut each source of DNA This allows complementary sticky ends to be created that can later base-pair to combine the DNA

Restriction enzyme recognition sequence DNA 1 Restriction enzyme cuts the DNA into fragments 2 Sticky end Figure 12.2 Creating recombinant DNA using a restriction enzyme and DNA ligase. This figure shows the production of recombinant DNA, using a restriction enzyme to produce complementary sticky ends and DNA ligase to seal the gaps when sticky ends associate with each other.

Restriction enzyme recognition sequence DNA 1 Restriction enzyme cuts the DNA into fragments 2 Sticky end Addition of a DNA fragment from another source 3 Figure 12.2 Creating recombinant DNA using a restriction enzyme and DNA ligase. This figure shows the production of recombinant DNA, using a restriction enzyme to produce complementary sticky ends and DNA ligase to seal the gaps when sticky ends associate with each other.

Restriction enzyme recognition sequence DNA 1 Restriction enzyme cuts the DNA into fragments 2 Sticky end Addition of a DNA fragment from another source 3 Two (or more) fragments stick together by base-pairing Figure 12.2 Creating recombinant DNA using a restriction enzyme and DNA ligase. This figure shows the production of recombinant DNA, using a restriction enzyme to produce complementary sticky ends and DNA ligase to seal the gaps when sticky ends associate with each other. 4

Restriction enzyme recognition sequence DNA 1 Restriction enzyme cuts the DNA into fragments 2 Sticky end Addition of a DNA fragment from another source 3 Two (or more) fragments stick together by base-pairing Figure 12.2 Creating recombinant DNA using a restriction enzyme and DNA ligase. This figure shows the production of recombinant DNA, using a restriction enzyme to produce complementary sticky ends and DNA ligase to seal the gaps when sticky ends associate with each other. 4 DNA ligase pastes the strands Recombinant DNA molecule 5

Steps in cloning a gene Plasmid DNA is isolated Plasmid DNA is isolated DNA containing the gene of interest is isolated Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle DNA with the target gene is treated with the same enzyme and many fragments are produced Plasmid and target DNA are mixed and associate with each other At step 6, there are actually three types of products that include plasmid DNA: (1) The ends of the plasmid can rejoin so that its original sequence is restored. (2) A recombinant DNA molecule can be formed containing part or all of the gene of interest. (3) Recombinant DNA molecules form that contain sequences unrelated to the gene of interest, representing the largest percentage of recombinant molecules. This mixture of products is typically used to transform bacteria under conditions where each cell is likely to take up only one plasmid. The cells are grown to form colonies, and properties of the plasmid and target DNA are used to detect the colony containing the recombinant plasmid carrying the gene of interest. Plasmids usually contain marker genes whose products indicate the presence of the plasmid within a bacterial host. A common approach is to use a plasmid with two markers, genes whose products indicate the presence of the plasmid within a bacterial cell. The site at which the plasmid is cut to add the target DNA is within one of the marker genes. Bacterial cells that show the action of both marker genes are not carrying target DNA and can be eliminated from the population. Bacterial cells expressing only the intact marker gene carry a recombinant plasmid. If the whole genome from the target organism is represented, this collection of clones is called a gene library (see Module 12.3). DNA from cells in this library can be tested for hybridization to a probe (see Module 12.5), to identify the cell carrying the gene of interest. Student Misconceptions and Concerns 1. Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11. 2. Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. Teaching Tips 1. Figure 12.1 is a synthesis of the techniques discussed in further detail in Modules 12.2–12.5. Figure 12.1 is therefore an important integrative piece that lays the foundation of most of the biotechnology discussion. Referring to this figure in class helps students relate the text to your lecture. 2. The general genetic engineering challenge discussed in Module 12.1 begins with the need to insert a gene of choice into a plasmid. This process is very similar to film or video editing. What do we need to do to insert a minute of one film into another? We will need techniques to cut and remove the minute of film and a way to cut the new film apart and insert the new minute. In general, this is also like removing one boxcar from one train, and transferring the boxcar to another train. Students can become confused by the details of gene cloning through misunderstanding this basic relationship. Copyright © 2009 Pearson Education, Inc.

Steps in cloning a gene Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together The recombinant DNA is taken up by a bacterial cell The bacterial cell reproduces to form a clone of cells At step 6, there are actually three types of products that include plasmid DNA: (1) The ends of the plasmid can rejoin so that its original sequence is restored. (2) A recombinant DNA molecule can be formed containing part or all of the gene of interest. (3) Recombinant DNA molecules form that contain sequences unrelated to the gene of interest, representing the largest percentage of recombinant molecules. This mixture of products is typically used to transform bacteria under conditions where each cell is likely to take up only one plasmid. The cells are grown to form colonies, and properties of the plasmid and target DNA are used to detect the colony containing the recombinant plasmid carrying the gene of interest. Plasmids usually contain marker genes whose products indicate the presence of the plasmid within a bacterial host. A common approach is to use a plasmid with two markers, genes whose products indicate the presence of the plasmid within a bacterial cell. The site at which the plasmid is cut to add the target DNA is within one of the marker genes. Bacterial cells that show the action of both marker genes are not carrying target DNA and can be eliminated from the population. Bacterial cells expressing only the intact marker gene carry a recombinant plasmid. If the whole genome from the target organism is represented, this collection of clones is called a gene library (see Module 12.3). DNA from cells in this library can be tested for hybridization to a probe (see Module 12.5), to identify the cell carrying the gene of interest. Student Misconceptions and Concerns 1. Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11. 2. Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. Teaching Tips 1. Figure 12.1 is a synthesis of the techniques discussed in further detail in Modules 12.2–12.5. Figure 12.1 is therefore an important integrative piece that lays the foundation of most of the biotechnology discussion. Referring to this figure in class helps students relate the text to your lecture. 2. The general genetic engineering challenge discussed in Module 12.1 begins with the need to insert a gene of choice into a plasmid. This process is very similar to film or video editing. What do we need to do to insert a minute of one film into another? We will need techniques to cut and remove the minute of film and a way to cut the new film apart and insert the new minute. In general, this is also like removing one boxcar from one train, and transferring the boxcar to another train. Students can become confused by the details of gene cloning through misunderstanding this basic relationship. Copyright © 2009 Pearson Education, Inc.

Use restriction enzymes to break DNA into manageable sized pieces that we can separate

What can we tell from this? It can be used to compare the DNA from different organisms Used to detect disease alleles Used to “match” DNA samples Determine parentage Crime scene forensics

Detecting disease alleles

Problem: if we’re trying to get a bacterium (prokaryote) to make our proteins, they don’t have introns… so, they can’t remove them Solution: Use reverse transcriptase (found in retroviruses) to make DNA from mature mRNA

PCR is used to amplify DNA sequences http://learn.genetics.utah.edu/content/labs/pcr/ Mix ingredients in a thermocycler What do you need to make lots of copies of DNA? From one target DNA sequence, 30 cycles of PCR will produce over 1 million copies. The use of primers is related to the native activity of DNA polymerase. To synthesize a DNA strand, DNA polymerase adds nucleotides to the 3′ end of a short nucleotide strand bound to the template. In the cell, primers are composed of RNA, synthesized by an enzyme called primase. These RNA segments are later removed from the DNA product. In PCR, synthetically produced DNA primers serve as the starting point for the polymerase. The heat-stable Taq polymerase was isolated from Thermus aquaticus, a bacterium found in hot springs. The enzyme can withstand heating to 94oC and synthesize DNA at 72oC during PCR. This is a helpful illustration of the effect of natural selection in favoring a form of the enzyme that would not denature with the high temperatures of the bacterium’s environment. Student Misconceptions and Concerns 1. Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete. 2. Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips 1. In PCR, the product becomes another master copy. Imagine that while you are photocopying, every copy is used as a master at another copy machine. This would require many copy machines. However, it would be very productive! Copyright © 2009 Pearson Education, Inc.

Figure 12.12 DNA amplification by PCR. Cycle 1 yields 2 molecules Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules Genomic DNA 3 5 3 5 3 5 5 1 Heat to separate DNA strands 2 Cool to allow primers to form hydrogen bonds with ends of target sequences 3 DNA polymerase adds nucleotides to the 3 end of each primer 3 5 5 3 Target sequence 5 5 3 5 3 5 3 Figure 12.12 DNA amplification by PCR. This figure shows several cycles of the PCR process, emphasizing that the number of templates doubles with each cycle. Primer New DNA

Cycle 1 yields 2 molecules Genomic DNA 3 5 3 5 3 5 5 DNA Cycle 1 yields 2 molecules Genomic DNA 3 5 3 5 3 5 5 DNA polymerase adds nucleotides to the 3 end of each primer 1 Heat to separate DNA strands 2 Cool to allow primers to form hydrogen bonds with ends of target sequences 3 3 5 5 3 Target sequence 5 Figure 12.12 DNA amplification by PCR. This figure shows several cycles of the PCR process, emphasizing that the number of templates doubles with each cycle. 5 3 5 3 5 3 Primer New DNA

Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules Figure 12.12 DNA amplification by PCR. This figure shows several cycles of the PCR process, emphasizing that the number of templates doubles with each cycle.