1. Recombinant DNA Technology
Chapter 14
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. Staggered cuts
Sticky ends
Cohesive ends
ligase

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

8. 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

10. 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.

11. 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

12. 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

13. 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.

14. Gel electrophoresis
hybridize
Single stranded
DNA complementary

15. 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

16. 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

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

18. More About PCR… in the reaction DNA is denatured
primers anneal to target DNA
copies of the target DNA are synthesized

19. Cloning Cellular DNA Fragments
Figure 14.10

20. 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
each fragment’s migration rate is inversely proportional to the log of its molecular weight

21. Gel Electrophoresis of DNA
Figure 14.11

22. Cloning Vectors and Creating Recombinant DNA
there are four types of cloning vectors
plasmids (most commonly used)
phages and viruses
cosmids
artificial chromosomes

23. Recombinant DNA Cloning Vectors
Table 14.3

24. 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

26. 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.

27. Construction of Genomic Libraries
used when gene of interest is on a chromosome that has not been sequenced
the library is constructed by cleaving the genome and then cloning the fragments into vectors
the libraries are screened for the genes of interest in a variety of ways

28. Gene library Collection of genes from an organism
Mouse gene library, yeast gene library
Biotechnology company
Yeast gene library
DNA – fragment – restriction enzyme – fragments are inserted into plasmids – introduced into a bacterial cell
Each bacterial cell having the recombinant plasmid is a clone
Large # clones – a clone for each gene that exists in the yeast cell

29. Hexokinase
Malate
Dehydrogenase
pheromone
Helps the cells
To come together
And mate

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

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

32. Table 14.4

33. Agriculture

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

35. 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