1. Recombinant DNA and Other Topics in Biotechnology
Recombinant DNA Technology
Preparing Recombinant DNA
Amplification of DNA by the Polymerase Chain Reaction
Applications of Recombinant DNA Technology

2. The potential
The area of recombinant DNA seeks to engineer changes in an organism’s genome to perform useful tasks.
New lab methods have made DNA easy to work with. We can now cut well defined fragments and splice it into the DNA of another species.
Promising potential
Gene replacement therapy
DNA finger printing
Improved agricultural products

3. Recombinant DNA technology
Gradual increase in DNA knowledge over the last 100 years.
1868 Discovery of DNA.
1944 Confirmed as the carrier of genetic information.
1953 Determination of structure and the methods of replication, transcription, translation and regulation.
1960’s Deciphering of the genetic code.
mid 1970’s Genetic engineering.

4. Recombinant DNA DNA recombination or molecular cloning
Covalent insertion of a DNA fragment from one cell or organism into the replicating DNA of another. +

5. Basic steps
Select and isolate a DNA molecule to serve as the carrier (vector) for the foreign DNA.
Cleave the vector DNA strands with a restriction endonuclease.
Prepare and insert the foreign DNA, producing a recombinant or hybrid DNA molecule.
Introduce the hybrid into a host organism, often a bacterial cell - transformation.
Develop a means to screen and identify host cells that have accepted the hybrid DNA.

6. Major breakthroughs
Isolation of a mutant strain of E. coli that did not have restriction endonucleases - could not degrade foreign DNA.
Development of bacterial plasmids and bacteriophage DNA as cloning vectors.
Discovery of restriction endonucleases that permit selective DNA cleavage.
Discovery of DNA ligase that catalyzes the formation of phosphodiester link for final site closure.

7. Cloning vectors Bacterial Plasmids Found in most bacterial cells.
Self-replicating, extra chromosomal DNA.
Closed, circular, double-stranded.
Smaller than chromosomal DNA with only 3,000-30,000 base pairs.
Contain information for translation of specialized and protective proteins.

8. Plasmid replication Two possible modes
Stringent - only a few copies are made
Relaxed - many, many copies are made.
Some relaxed plasmids may continue to replicate forming 2, ,000 copies, accounting for 30-40% of all cellular DNA.
A typical plasmid will accept foreign DNA up to 15, 000 base pairs.

9. Ideal plasmid cloning vector properties
It should:
replicate in a relaxed fashion so that many copies are produced.
be small so it is easy to separate from chromosomal DNA and easier to handle without damage.
have only a few sites for attack by restriction enzymes.
have identifiable markers for screening.
have a single cleavage site for a given restriction enzyme - within a gene.

10. Ideal plasmid cloning vector properties
Examples:
E. coli E1
Bacterial strain that has been shown to be useful for recombinant DNA work.
pBR322
A plasmid of E. coli E. It has 4363 base pairs. It is cleaved at a single site by the restriction endonuclease EcoRI

11. Bacteriophage DNA  phage is the most widely used.
Double stranded DNA with ~50,000 base pairs.
It can produce many DNA copies within a host cell.
It acts as a package to introduce DNA by infecting host bacteria.
It is easily screened for.
DNA fragments up to 23,000 base pairs can be added.

12. Preparing recombinant DNA
Foreign double stranded DNA must be prepared for insertion.
Methods include
Chemical synthesis
Use of restriction endonucleases
Reverse transcription of mRNA

13. Preparing recombinant DNA
Fragments prepared by restriction endonuclease have either “sticky” or blunt ends.
Sticky or cohesive ends
Blunt ends

14. Preparing recombinant DNA
Unpaired cohesive ends, up to 5 nucleotide bases, can be joined to a plasmid that was cleaved by the same endonuclease.
It can lead to regions that may not be stable.

15. Preparing recombinant DNA
Blunt ends can be attached using DNA ligase.
5’ end must have phosphate groups and the 3’ end must have a free hydroxyl group.
ATP,

16. Use of homopolymer tails
This is a more widely used approach.
Segments of poly-A or poly-G are added to one fragment - typically the plasmid.
Equal length segments of poly-T or poly-C are added to the other fragment.
Both are accomplished using deoxynucleotidyl transferase.
They are then allowed to incubate together to allow hydrogen bonding to bring them together.

17. Use of homopolymer tails
n-dTTP
n-dATP

18. Use of homopolymer tails
Foreign DNA with
poly T tails +
Linearized plasmid
with poly A tails

19. Getting the hybrid into the host
Transformation
Introduction of the hybrid DNA into host cell.
Current methods are inefficient with only 1 molecule in 10,000 being successfully replicated. +

20. Transformation of E. coli cells
One can promote the introduction of hybrid plasmid using CaCl2
Wash cells with CaCl2 solution.
Incubate with a solution of hybrid DNA.
If DNA is incorporated, it is replicated.
Selective markers can be tested for.
Cloning of bacteriophage DNA is done by infecting the host bacteria.

21. Screening and separation
pBR322 example.
This plasmid has resistance for two antibiotics - ampicillin and tetracycline.
The foreign sequence is added within the gene that imparts tetracycline resistance.
The resistance is destroyed.
Sequential testing using both antibiotics can identify hybrid plasmids.

22. Screening and separation
Yellow = Ampicillin treated
Green = Tetracycline treated
identical transfer
Circled colonies are
ampicillin immune
and tetracycline
sensitive. They
must be colonies
of cells containing
the hybrid plasmid.
incubate

23. Restriction enzyme maps
Mapping is valuable in the selection of plasmids for cloning and characterization of DNA fragments.

24. Isolation and cloning of a single gene
A human gene can contain 40, ,000,000 base pairs.
That represents only about 0.03% of the entire genome.
We have the goal of identifying all human genes - Human Genome Project.
The first step is to construct a genomic library - brute force, hit or miss approach.

25. Constructing a genomic library
Cut DNA into thousands of fragments using restriction enzymes yielding random, overlapping pieces.
Separate fragments based on molecular size using methods like gel electrophoresis.
Add homopolymer tails and insert into vectors and then into host cells.
Clone the cells. This results in large population of cells, each containing a different DNA fragment.
Hope that all original genomic DNA is represented.

26. Constructing a genomic library
Now one must screen to find which cells contain a gene of interest.
Grow each cell on an agar plate.
Each colony has a different recombinant DNA.
Sample cells by blotting to identify which colonies have the sequence of interest.

27. Blotting
Apply nitrocellulose paper to the plate to produce an imprint.
Treat the paper with dilute NaOH to lyse the cells, releasing the DNA which stays on the paper.
Add a radiolabeled hybridization probe - complementary DNA or RNA molecule.
Labeled spots on the paper identify which colonies have the sequence of interest.

28. Amplification of DNA Polymerase chain reaction (PCR)
Method is used to make multiple copies of DNA without cloning.
It requires that at least part of the sequence for a DNA fragment be known.
The advantage is that the process can be repeated many times by altering temperature.
The amount of DNA increases exponentially.

29. Amplification of DNA Requirements for PCR
Two synthetic oligonucleotide primers of approximately 20 base pairs. They must be complementary to the ‘flanking sequences.”
Heat stable DNA polymerase.
All four deoxyribonucleotides as triphosphates.

30. Polymerase chain reaction
Target
sequence
Separate strands by
heating to 95oC.

31. Polymerase chain reaction
Hybridize primers
cooling to 54oC.

32. Polymerase chain reaction
Synthesize DNA by
extending primers at 72oC.

33. Application of recombinant DNA technology
Recombinant protein products
Bacteria can serve as factories for various proteins like human insulin.
Bacteria can’t be used for many eukaryotic genes since they are unable to remove introns.
Genetically altered organisms
Alter DNA to remove defects or improve quality/quantity of normal products.

34. Examples
Bacteria
Modified Pseudomonas are being constructed to degrade hazardous waste.
Plants
Flavr Savr tomato developed by Calgene. It was altered to inhibit rotting and allow it to ripen longer on the vine.
Animals
The rat growth hormone, somatotropin, was added to a plasmid and added to mouse eggs. Resulted in mice twice normal size. This was transgenic since it was transferred to subsequent generations.

35. Examples Human gene therapy
There are approximately 4,000 known genetic diseases.
There are several clinical trials ongoing or planned. They involve:
Removal of somatic cells from a patient.
Insertion of a normal gene
Reintroduction of the cells
Bone marrow, skin or liver cells are typically used.