1. 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.

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

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

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

7. 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.

8. 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.

9. 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.

10. 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

11. 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.

12. 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

13. 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

15. 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

16. 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.

17. 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.

18. 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

19. 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

20. 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.

21. 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.

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

24. 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

25. Detecting disease alleles

27. 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

28. PCR is used to amplify DNA sequences
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.

29. 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 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

30. 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 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

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