1. Recombinant DNA Technology
Chapter 15
Recombinant DNA Technology

2. Some terminology genetic engineering recombinant DNA technology
deliberate modification of organism’s genetic information by directly changing the sequence of nucleic acids in its genome
recombinant DNA technology
procedures used to carry out genetic engineering
biotechnology
use of organisms to form useful products

4. Restriction enzymes
bind to DNA at specific sequence called the recognition site
cleave DNA at this site or a defined distance from it
hundreds are commercially available
Figure 14.2

5. Restriction Endonucleases and Their Recognition Sequences
Table 14.2

6. Tools used in genetic engineering Restriction enzymes
Bacteria – restriction enzymes
To breakdown phage DNA
Extract the restriction enzyme from bacteria and use it genetic engineering
EcoRI, BamHI – recognize specific sequence
Staggered cuts on DNA
Ends of the fragment are single stranded

7. Staggered cuts
Sticky ends
Cohesive ends
ligase

8. Reverse transcriptase
synthesizes double stranded DNA from RNA template
used to construct complementary DNA (cDNA)
independently discovered by Temin and Baltimore in 1970

9. cDNA Complementary DNA Eukaryotic genes – introns and exons
Introns – noncoding regions
Exons - coding regions
cDNA – synthetic gene – has only exons
c stands for complementary DNA

11. If we want to introduce eukaryotic gene into a prokaryotic cell, we should use cDNA
If we place natural eukaryotic gene into a
Bacterial cell, it cannot remove the introns.
Functional protein will not be produced by the prokaryotic cell.

12. Southern blotting technique
developed by Edwin M. Southern (1975)
used to detect specific DNA fragments
often uses radioactive DNA hybridization probes
autoradiography
method for detecting radioactively labeled molecules

13. Genetic screening. If a person is a carrier for cystic fibrosis (genetic disorder)
Mutation in a gene that codes for a membrane protein
Thick mucus

14. Person has the disease – symptoms
Some people are carriers
One cystic fibrosis gene (father) one normal gene (mother) – heterozygous for cystic fibrosis
Two carriers – have a baby – the baby can have the disease
Genetic screening is used – procedure known as southern blotting.

15. Gel electrophoresis
hybridize
Single stranded
DNA complementary

16. Synthetic DNA oligonucleotides (can be DNA or RNA)
synthesized in stepwise process in which single nucleotides are added to end of a growing DNA chain
chain length can vary from short (2-30 nucleotides) to long ( nucleotides)
can be synthesized to have known nucleotide sequences
DNA synthesizer – products are used in PCR

17. The Polymerase Chain Reaction (PCR)
Amplify
enables the rapid synthesis of many copies of a specific DNA fragment from a complex mixture of DNA and other cellular components
reaction mix contains
primers
target DNA
thermostable DNA polymerase such as Taq polymerase
each of the four deoxyribonucleotide triphosphates
thermocycler is the instrument used

18. Polymerase
Chain
Reaction
Amplify DNA
Target DNA
Nucleotides
DNA polymerase

19. Gel Electrophoresis
used to separate molecules based on their charge and size
agarose or acrylamide gels can be used to separate DNA fragments
DNA is acidic; it migrates from the negative to the positive end of the gel
Smaller fragments migrates faster

20. Gel Electrophoresis of DNA
Figure 14.11

21. Cloning Vectors and Creating Recombinant DNA
there are four types of cloning vectors
plasmids (most commonly used)

22. Cloning Vectors and Creating Recombinant DNA
each type of cloning vector generally has
an origin of replication
a selectable marker
a multicloning site or polylinker

24. Vectors – carry the gene of interest into a bacterial cell.
Plasmids
Small enough – they can enter into the cell
Selection markers – antibiotic resistance genes.
Help in selecting the cells that have the gene of interest.

25. Inserting Recombinant DNA into Host Cells
most common hosts
E. coli - procaryotic host
S.cerevisiae – eucaryotic host
DNA introduction into microbes
transformation

26. The Numerous Applications of Genetic Engineering
in medicine
many useful proteins
gene therapy - mutant gene replaced by normal gene.
in agriculture

27. vaccine Hepatitis B vaccine Genetically engineered Saccharomyces cells
Vaccine has only the protein part of the virus.
Does not have the genetic material of the virus
No chance of getting the disease due to vaccination

28. Table 14.4

29. Agriculture

30. Forensic medicine
Each person has a unique set of introns.
restriction enzyme fragment pattern of DNA

31. Social Impact of Recombinant DNA technology
there is scientific and philosophical concern about
the use of embryonic stem cells in gene therapy. This is currently a major controversial issue in the U.S.
the production of genetically engineered food
the possibility of the production of genetically engineered organisms by bioterrorists
and other issues