1. Genetic Engineering and Recombinant DNA
Microbiology: A Systems Approach
Genetic Engineering and Recombinant DNA
Chapter 10
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Chapter 10, pages 268 to 296

2. Learning Objectives:
Define restriction enzymes and outline how they are used to make recombinant DNA.
List the properties of vectors. Outline the process of genetic cloning.
List at least five applications of recombinant DNA technology
Explain the purpose and identify the steps in polymerase chain reaction (PCR).
Describe microarray technique and list its applications.
List and describe at least five applications of microbial and human genomics

3. Applications of Genetic Engineering
Artificial manipulation of genetic information
Characterize organisms
Indentify organisms
Modify organisms
More useful organisms
Produce useful products

4. DNA strands can be cut across
Enzymes that cut DNA
Each recognizes a known sequence of 4 to 10 base pairs
The sequences are usually palindromes (Madam, I’m Adam)

5. Some Common Restriction Enzymes
BamHI - Bacillus amyloliquefaciens G|GATCC CCTAG|G
EcoRI - Escherichia coli G|AATTC CTTAA|G
EcoRV - Escherichia coli GAT|ATC CTA|TAG

6. DNA fragments can be joined together
Restriction fragments.
Ligase rejoins the ends of restriction fragments by forming sugar-phosphate bonds

7. Genetic Cloning The required gene is isolated from a genetic donor.
Cloning vector (plasmid) is
commercially acquired.
Gene and plasmid are exposed to the same restriction endonuclease,
producing complementary
ends.
Complementary ends bind to form a single recombinant plasmid.

8. Cloning Human Insulin Gene
Isolate human insulin gene
Splice into a cloning vector
Insert recombinant plasmid in bacterial cells
Screen for recombinant plasmids
Identify clones carrying human insulin gene

9. Cloning Vectors Accept foreign DNA Able to “self-replicate”
Need a promoter
Selectable markers
Examples:
Plasmids
Bacteriophage
Phagemids
Cosmids
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EcoRI
SacI
lacZ
KpnI
MCS
SmaI
AmpR
BamHI
XbaI
pUC19
(2,686bp)
SalI
PstI
SphI
HindIII
ori

10. Transformation: Selecting Clones
Use selectable marker
Selective media
E.g. antibiotics
Differential media
E.g. X-gal
Indicates inserts
Culture of cloning
host after incubation
with recombinant
plasmid
Bacteria with
recombinant
plasmid
Bacteria lacking
plasmid
(1)
(2)
Bacteria carrying
plasmid
Ampicillin-
resistance gene
Regular nonselective
medium with two
types of colonies
Selective medium
with ampicillin
Pure culture
of bacteria
containing
cloned gene

11. Genetic Cloning Transformation. The recombinant plasmid
is introduced into a cloning host cell.
The transformed cell is grown in culture to increase numbers. Every cell will contain a replicated plasmid containing the desired gene.
Cells transcribe and translate the foreign gene.
This yields a protein product that is recovered from the culture and purified.

12. Recombinant DNA Strategies
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Human, other mammal,
microorganism, or plant cell gene identified.
DNA of interest is isolated.
Obtain target gene
Insert into vector
Introduce into hosts
Expression of desired gene product
DNA of interest is inserted
into a cloning vector.
Cloning host receives
vector, becomes a
recombinant
Cloning host provides abundant DNA for study.
Cloning host translates foreign
DNA into protein.
Pharmaceutical proteins
• insulin
• human growth hormone
Vaccines
• hepatitis B
Altered organisms with economically useful traits: transgenic plants
• pest resistance
• herbicide resistance
• improved nutritional value

13. Gene Therapy Some diseases due to the lack of a protein
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Gene Therapy 1
1. Normal gene is isolated from
healthy subject.
Some diseases due to the lack of a protein
Introduce good protein
Phenotype corrected 2
2. Gene is cloned.
3. Gene is inserted into retrovirus vector. 3
4. Bone marrow sample is taken from patient with genetic defect.
5. Marrow cells are infected with
retrovirus.
6. Transfected cells are reinfused
into patient. 7 5 6 4
7.Patient is observed for expression of normal gene.
Marrow cell

14. Genetic Probes Short single-stranded DNA sequences
Identity specific target sequences
Bound markers
Dye
Radioisotope
Enzyme

15. Polymerase Chain Reaction
Small amount of DNA or RNA limit some tests
Polymerase chain reaction (PCR) rapidly increases the amount of DNA in a sample
Can amplify a target DNA from a few copies to billions in a few hours
Can detect a single cell

16. Three Basic Steps that Cycle
Denaturation
Heat to 94°C to separate in to two strands
Cool to between 50°C and 65°C
Priming
Primers added that are complementary to the target
Extension
72°C
DNA polymerase and nucleotides are added
Polymerases extend the molecule
Repeat about 25 to 30 times

17. Polymerase Chain Reaction

18. Microarrays

19. Microarray Applications
Gene expression
Organism detection
Phylogenetic relationships

20. Microbial and Human Genomics
Many microbial genomes have been sequenced
Segments of the human genome have “microbial ancestors
Safer food production
Identification of uncultured microorganisms
Microbial forensics
New perspective to defining infectious diseases and studying evolution