1. Lecture# 2 Recombinant DNA technology
Dr. Shah Rukh Abbas
ASAB, NUST.

3. I. DNA is the genetic material of most organisms (from bacteria to humans)
Plasmid
Chromosome: Most bacteria have one circular DNA chromosome ranging in size from 1,000 to 8,000 kilo base pairs.
Plasmid: Extra chromosomal genetic element also made of a circular DNA molecule.
Bacterial Genome: The collection of all of the genes present on the bacteria’s chromosome or its extra chromosomal genetic elements.

4. Basics: Nucleotides are the building blocks of DNA

5. Chemical structure of DNA & RNA

6. Chemical structure of dsDNA

7. Chemical structure of dsDNA

8. II. Restriction Endonucleases (RE) enzymes
Enzymes that act as scissors to cut the DNA at specific sites.
1962: “molecular scissors” discovered in bacteria.
E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA’s sugar phosphate back bone.
Restriction Endonucleases recognize specific sequence with the DNA molecules
The recognized sequence are usually four to six base pair and are palindromic.
Named for bacterial genus, species, strain, and type. Example: EcoR1
Genus: Escherichia, Species: coli Strain: R Order discovered: 1
Bam H1 site:
5’-GGATCC-3’
3’-CCTAGG-5’

9. Types of RE enzymes
Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences.
Type II Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for routine DNA analysis and gene cloning.
Type III enzymes cleave DNA from 24 to 26 bp away from the recognition site.
Type IV  enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrr systems of E. coli.

10. Restriction Enzymes

11. Restriction Enzymes Cutting Sites
Blunt Ends
AluI
HaeIII

12. Sticky Ends
EcoRI
HindIII
- OH 3’
5’ P -
- P 5’
3’ OH -

13. Why don’t bacteria destroy their own DNA with their restriction enzymes?
What are exonucleases?
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzingreaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs.

14. Uses for Restriction Enzymes
RFLP analysis (Restriction Fragment Length Polymorphism)
DNA sequencing
DNA storage – libraries
Transformation
Large scale analysis – gene chips

15. Restriction Enzymes for RFLP_ +
DNA is negatively charged from the phosphate backbone
Visualize DNA with ethidium bromide – fluoresces ONLY when bound to DNA

16. III. Pasting DNA using DNA ligase
Complementary ends (sticky ends) H-bond
Ligase forms phosphodiester bond to seal strands together.

17. III. Vectors: Bacterial Plasmids
Allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.
TYPES:
1 Plasmid vectors
2 Bacteriophage vectors
3 Cosmids
4 BACs & YACs
Must have:
Ori of replication and in case of expression vector gene expression mechanism (operon etc).
Marker- to indicate recombinant
Multiple cloning site

18. 1. Plasmids
Plasmids - circular DNA duplexes of 1 to 200 kb that contain the requisite genetic machinery (replication origin) necessary for autonomous replication in bacteria or yeast.
Types of plasmids are determined by their copy number.
Stringent control- one to a few copies per cell.
Relaxed control- 10 to 700 copies per cell-if protein synthesis is inhibited by an antibiotic (chloramphenicol), the plasmid will continue to replicate up to copies.
Plasmids used in molecular cloning are
relatively small
replicate under relaxed control
carry genes for antibiotic resistance
number of restriction sites (polylinker) for inserting DNA segments.
Cannot be used to clone DNAs longer than ~10 kb.
Blue-white screeing.

19. 1. Plasmids Plasmid vectors are ≈1.2–3kb and contain:
Replication origin (ORI) sequence
A gene that permits selection,
Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin.
Exogenous DNA can be inserted into the bracketed region .

20. Selective Marker
Selective marker is required for maintenance of plasmid in the cell.
Because of the presence of the selective marker the plasmid becomes useful for the cell.
Under the selective conditions, only cells that contain plasmids with selectable marker can survive
Genes that confer resistance to various antibiotics are used.
Genes that make cells resistant to ampicillin, neomycin, or chloramphenicol are used

21. Origin of Replication
Origin of replication is a DNA segment recognized by the cellular DNA-replication enzymes.
Without replication origin, DNA cannot be replicated in the cell.

22. Multiple Cloning Sites
Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment with several unique sites for restriction endo- nucleases located next to each other
Restriction sites of the polylinker are not present anywhere else in the plasmid.
Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector

23. Plasmid vectors Small, easy to handle
Advantages:
Small, easy to handle
Straightforward selection strategies
Useful for cloning small DNA fragments
(< 10kbp)
Disadvantages:
Less useful for cloning large DNA fragments
(> 10kbp)

25. 2. Bacteriophage vectors
Advantages:
Useful for cloning large DNA fragments
( kbp)
Inherent size selection for large inserts
Disadvantages:
Less easy to handle

26. l vectors Left arm: head & tail proteins Right arm: DNA synthesis
regulation
host lysis
Deleted central region:
integration & excision

27. l Vector (M-13

28. 3. Cosmid vectors Advantages: Disadvantages:
Combine the properties of plasmid vectors with the useful properties of the l cos site
Advantages:
Useful for cloning very large DNA fragments
( kbp)
Inherent size selection for large inserts
Handle like plasmids
Disadvantages:
Not easy to handle very large plasmids
(~ 50 kbp)

30. lZAP

31. 4. BACs and YACs BACs : Bacterial Artificial Chromosomes
YACs : Yeast Artificial Chromosomes
Advantages:
Useful for cloning extremely large DNA fragments
( ,000 kbp)
This is very important for genome sequencing projects
Disadvantages:
Not easy to handle extremely large DNA molecules

32. BAC vector oriS and oriE mediate replication
parA and parB maintain single copy number
ChloramphenicolR marker

33. YAC vector Capable of carrying inserts of 200 - 2000 kbp in yeast
telomere
centromere
URA3
ARS
HIS3
replication
origin
markers
large
inserts
Capable of carrying inserts of kbp in yeast