1. PowerPoint Presentation Materials to accompany
Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 18 Part 1
RECOMBINANT
DNA TECHNOLOGY
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

2. 18-4
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3. Cloning Experiments Involve Chromosomal and Vector DNA
Cloning experiments usually involve two kinds of DNA molecules
Chromosomal DNA or cDNA
Serves as the source of the DNA segment of interest
Vector DNA
Serves as the carrier of the DNA segment that is to be cloned
18-5
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

4. The cell that harbors the vector is called the host cell
When a vector is replicated inside a host cell, the DNA that it carries is also replicated
The vectors commonly used in gene cloning were originally derived from two natural sources
1. Plasmids
2. Viruses
Commercially available plasmids have selectable markers
Typically, genes conferring antibiotic resistance to the host cell
Table 18.2 provides a general description of several vectors used to clone small segments of DNA
18-6
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

5. Cloning Experiments Involve Enzymes that Cut and Paste DNA
Insertion of chromosomal DNA into a vector requires the cutting and pasting of DNA fragments
The enzymes used to cut DNA are known as restriction endonucleases or restriction enzymes
These bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand
Figure 18.1 shows the action of a restriction endonuclease
18-8
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

6. To be continued 
Figure 18.1
18-9

7. Restriction enzymes are made naturally by many species of bacteria
They protect bacterial cells from invasion by foreign DNA, particularly that of bacteriophage
Currently, several hundred different restriction enzymes are available commercially
Table 18.3 gives a few examples
18-10
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

8. 18-11

9. Restriction enzymes bind to specific DNA sequences
These are typically palindromic
For example, the EcoRI recognition sequence is
5’ GAATTC 3’
3’ CTTAAG 5’
Some restriction enzymes digest DNA into fragments with “sticky ends”
Other restriction enzymes generate blunt ends
18-12
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

10. Figure 19-2 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure 19-2 Some common restriction enzymes, with their recognition sequences, cleavage patterns, and sources.
Figure Copyright © 2006 Pearson Prentice Hall, Inc.

11. A recombinant DNA molecule
This interaction is not stable because it involves only a few hydrogen bonds
To establish a permanent connection, the sugar-phosphate backbones of the two DNA fragments must be covalently linked
Add DNA ligase which covalently links the DNA backbones
A recombinant DNA molecule
Figure 18.1
18-13

12. The Steps in Gene Cloning
The general strategy followed in a typical cloning experiment is outlined in Figure 18.2
The procedure shown seeks to clone the human b-globin gene into a plasmid vector
The vector carries two important genes
ampR  Confers antibiotic resistance to the host cell
lacZ  Encodes b-galactosidase
Provides a means by which bacteria that have picked up the cloned gene can be identified
18-14
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

13. Figure 19-6 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure 19-6 A diagram of the plasmid pUC18 showing the polylinker region, located within a lacZ gene. DNA inserted into the polylinker region disrupts the lacZ gene, resulting in white colonies that allow direct identification of bacterial colonies carrying cloned DNA inserts.
Figure Copyright © 2006 Pearson Prentice Hall, Inc.

14. This is termed a hybrid vector
Note: In this case, the b-globin gene was inserted into the plasmid
It is also possible for any other DNA fragment to be inserted into the plasmid
And it is possible for the plasmid to circularize without an insert
This is called a recircularized vector
This is termed a hybrid vector
Figure 18.2
18-15

15. 18-16 Figure 18.2 This step of the procedure is termed transformation.
Cells that are able to take up DNA are called competent cells
Figure 18.2
18-16

16. Nonrecombinant: recircularized
Recombinant: vector plus inserted cloned gene
Selection for vector: ampicillin resistance
Selection for recombinant vs. nonrecombinant vector: b-galactosidase activity
Selection for for gene of interest?
18-17
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

17. The growth media contains two relevant compounds:
IPTG (isopropyl-b-D-thiogalactopyranoside)
A lactose analogue that can induce the lacZ gene
X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside)
A colorless compound that is cleaved by b-galactosidase into a blue dye
The color of bacterial colonies will therefore depend on whether or not the b-galactosidase is functional
If it is, the colonies will be blue
If not, the colonies will be white
In this experiment
Bacterial colonies with recircularized vectors form blue colonies
While those with hybrid vectors form white colonies
18-18
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

18. Recombinant DNA technology is not only used to clone genes
The net result of gene cloning is to produce an enormous amount of copies of a gene
During transformation, a single bacterial cell usually takes up a single copy of the hybrid vector
Amplification of the gene occurs in two ways:
1. The vector gets replicated by the host cell many times
2. The bacterial cell divides approximately every 30 minutes
Recombinant DNA technology is not only used to clone genes
Sequences such as telomeres, centromeres and highly repetitive sequences can be cloned as well
18-19
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

19. Experiment 18A: The First Gene Cloning Experiment
This was accomplished by Stanley Cohen, Annie Chang, Herbert Boyer, and Robert Helling in 1973
Several important discoveries led to their ability to clone a gene
DNA ligase covalently links DNA fragments together
EcoRI produces sticky ends when digesting DNA
Cohen et al realized that it is possible to create recombinant DNA molecules using
EcoRI then DNA ligase
18-20
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

20. Tetracycline resistance
They chose a plasmid for their vector
The plasmid was designated pSC101
The insertion of the gene will occur at the lone EcoRI site
As source of the gene, they obtained a second plasmid
They called it pSC102
One of the three EcoRI fragments is expected to carry the KanR gene
Kanamycin resistance
18-21
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

21. Testing the Hypothesis
A piece of DNA carrying a gene can be inserted into a plasmid vector using recombinant DNA techniques
If this recombinant plasmid is introduced into a bacterial host cell, it will be replicated and transmitted to daughter cells, producing many copies of the recombinant plasmid
Testing the Hypothesis
Refer to Figure 18.3
18-22
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

22. Figure 18.3
18-23

23. Figure 18.3
18-24

24. Figure 18.3
18-25

25. Figure 18.3
18-26

26. Figure 18.3
18-27

27. Density Gradient Centrifugation
Single peak
The Data
A single plasmid with intermediate density; NOT a mixture of two plasmids
Density Gradient Centrifugation
Control Experiment
pSC102
pSC101
18-28
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

28. This band corresponds to pSC101 This band is also found in pSC102
The Data
Gel Electrophoresis
This band corresponds to pSC101
This band is also found in pSC102
18-29

29. The recombinant plasmid is shown here
This experiment showed it is possible to create recombinant DNA molecules and to propagate them in bacterial cells
This hallmark achievement ushered in the era of gene cloning
18-30
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

30. cDNA To clone DNA, one can start with a sample of RNA
The enzyme reverse transcriptase is used
Uses RNA as a template to make a complementary strand of DNA
DNA that is made from RNA is called complementary DNA (cDNA)
It could be single- or double-stranded
Synthesis of cDNA is presented in Figure 18.4
18-31
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

31. polyA tail
Figure 18.4
18-32

32. This has two ramifications
From a research perspective, an important advantage of cDNA is that it lacks introns
This has two ramifications
1. It allows researchers to focus their attention on the coding sequence of a gene
2. It allows the expression of the encoded protein
Especially, in cells that would not splice out the introns properly
(e.g., a bacterial cell)
18-33
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

33. Gel electrophoresis
Nucleic acid electrophoresis separates DNA and RNA fragments by size
smaller fragments migrate at a faster rate through a gel than large fragments.

34. Figure 10-27 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure Electrophoretic separation of a mixture of DNA fragments that vary in length. The photograph shows an agarose gel with DNA bands corresponding to the diagram.
Figure Copyright © 2006 Pearson Prentice Hall, Inc.

35. Restriction Mapping
Sometimes, it is necessary to obtain smaller clones from a large chromosomal DNA insert
This process is termed subcloning
Cloning and subcloning require knowledge of the locations of restriction enzyme sites in vectors and hybrid vectors
A common approach to examine the locations of restriction sites is known as restriction mapping
Figure 18.5 outlines the restriction mapping of a bacterial plasmid, pBR322
18-34
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

36. Figure 18.5
18-35

37. Used for fragment size comparison
Figure 18.5
18-36

38. The restriction map can be deduced by comparing the sizes of DNA fragments obtained from the single, double and triple digestions
Figure 18.5
4,363 bp
18-37
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

39. Figure 19-22 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure An agarose gel containing separated DNA fragments stained with a dye (ethidium bromide) and visualized under ultraviolet light. Smaller fragments migrate faster and farther than do larger fragments, resulting in the distribution shown.
Figure Copyright © 2006 Pearson Prentice Hall, Inc.

40. Figure 19-23 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure Constructing a restriction map. Samples of the 7.0-kb DNA fragments are digested with restriction enzymes: One sample is digested with HindIII, one with SalI, and one with both HindIII and SalI. The resulting fragments are separated by gel electrophoresis. The separated fragments are measured by comparing them with molecular-weight standards in an adjacent lane. Cutting the DNA with HindIII generates two fragments: 0.8 kb and 6.2 kb. Cutting with SalI produces two fragments: 1.2 kb and 5.8 kb. Models are constructed to predict the fragment sizes generated by cutting with HindIII and with SalI. Model 1 predicts that 0.4-, 0.8-, and 5.8-kb fragments will result from cutting with both enzymes. Model 2 predicts that 0.8-, 1.2-, and 5.0-kb fragments will result. Comparing the predicted fragments with those observed on the gel indicates that model 1 is the correct restriction map.
Figure Copyright © 2006 Pearson Prentice Hall, Inc.