1. Cloning and Vector Chapter 3 Instructor : Prof. Myoung-Dong Kim T: 6458, mdkim@kangwon.ac.kr Room 411, Ag. Bld #3

2. Gene Cloning

3. Cloning - a definition  From the Greek - klon, a twig  An aggregate of the asexually produced progeny of an individual;a group of replicas of all or part of a macromolecule (such as DNA or an antibody)  An individual grown from a single somatic cell of its parent & genetically identical to it  Clone: a collection of molecules or cells, all identical to an original molecule or cell

4. DNA CLONING A method for identifying and purifying a particular DNA fragment (clone) of interest from a complex mixture of DNA fragments, and then producing large numbers of the fragment (clone) of interest.

5. Gene cloning  When DNA is extracted from an organism, all its genes are obtained  In gene (DNA) cloning a particular gene is copied (cloned)

6. Why Clone DNA?  A particular gene can be isolated and its nucleotide sequence determined  Control sequences of DNA can be identified & analyzed  Protein/enzyme/RNA function can be investigated  Mutations can be identified, e.g. gene defects related to specific diseases  Organisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.

7.  1) Chromosomal DNA  2) RNA converted to cDNA  3) PCR-amplified DNA Sources of DNA for Cloning

8. PCR-amplified DNA

9. Cloning Tools  Restriction endonucleases  Ligase  Vectors  Host  Methods for introducing DNA into a host cell

10. Cutting DNA  Restriction endonucleases (restriction enzymes) sticky ends sticky ends blunt ends blunt ends  Nomenclature EcoRI EcoRI E = genus (Escherichia) E = genus (Escherichia) co = species (coli) co = species (coli) R = strain R = strain I = # of enzyme I = # of enzyme

11. Blunt & Sticky ends

12. Pasting DNA  Complementary ends (sticky ends) H-bond  Ligase forms phosphodiester bond to seal strands together.

13. Vectors

14. Cloning vectors Allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level. 11 Plasmid vectors 1 22 Bacteriophage vectors 2 33 Cosmids 3 44 BACs & YACs 4

15. Plasmid vectors  Advantages: Small, easy to handle Straightforward selection strategies Useful for cloning small DNA fragments (< 10kbp)  Disadvantages: Less useful for cloning large DNA fragments (> 10kbp) Plasmid vectors are double-stranded, circular, self- replicating, extra-chromosomal DNA molecules.

16. Plasmid vectors  Plasmids are circular DNA molecules present in the cytoplasm of the bacteria  Capable of autonomous replication  Can transfer genes from one cell to other  Act as vectors in genetic engineering.  Can also present in Yeasts

17. Plasmid vectors  may encode genetic information for properties 1 Resitance to Antibiotics 2 Bacteriocins production 3 Enterotoxin production 4 Enhanced pathogen city 5 Reduced Sensitivity to mutagens 6 Degrade complex organic molecules T.V.Rao MD

18. Plasmid vector for cloning 1.Contains an origin of replication, allowing for replication independent of host’s genome. 2.Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening 3.Contains a multiple cloning site (MCS)MCS 4.Easy to be isolated from the host cell.

19. Plasmid vectors

20. Bacteriophage vectors  Advantages: Useful for cloning large DNA fragments (10 - 23 kbp) Inherent size selection for large inserts  Disadvantages: Less easy to handle

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

22. Bacteriophage

24. Cosmid vectors  Advantages: Useful for cloning very large DNA fragments (32 - 47 kbp) Inherent size selection for large inserts Handle like plasmids  Disadvantages: Not easy to handle very large plasmids (~ 50 kbp) Combine the properties of plasmid vectors with the useful properties of the l cos site

26.  ZAP

27. BACs and YACs  Advantages: Useful for cloning extremely large DNA fragments (100 - 2,000 kbp) This is very important for genome sequencing projects  Disadvantages: Not easy to handle extremely large DNA molecules BACs : Bacterial Artificial Chromosomes YACs : Yeast Artificial Chromosomes

28. BAC vector  oriS and oriE mediate replication  parA and parB maintain single copy number  Chloramphenicol R marker

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

30. What determines the choice vector?  insert size  vector size  restriction sites  copy number  cloning efficiency  ability to screen for inserts  what down-stream experiments do you plan?

31. Expression vector

32. Expression vector pSE420 Polylinker: insert desired DNA Polylinker: insert desired DNA Amp resistance Amp resistance trc promoter trc promoter lacO (operator) lacO (operator) Shine-Dalgarno (S/D) site Shine-Dalgarno (S/D) site (ribosome binding) (ribosome binding) T1, T2 transcription terminators T1, T2 transcription terminators lacI (lac repressor) lacI (lac repressor) growth inducer added cloned gene expressed; product produced

33. insertion of foreign DNA at BamHI site insertion of foreign DNA at BamHI site tet resistance gene inactivated tet resistance gene inactivated transformants carrying foreign DNA are amp resistant but tetracycline sensitive transformants carrying foreign DNA are amp resistant but tetracycline sensitive transformation: transfer of genetic information via free DNA Btech6 How to clone DNA

34.  Isolation of cloning vector (bacterial plasmid) & gene- source DNA (gene of interest)  Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase  Introduction of cloning vector into cells (transformation by bacterial cells)  Cloning of cells (and foreign genes)  Identification of cell clones carrying the gene of interest

35. Screening of the clone  The medium in this petri dish contains the antibiotic Kanamycin  The bacteria on the right contain Kan r, a plasmid that is resistant to Kanamycin, while the one on the left has no resistance  Note the difference in growth

36. Blue/White Color Screening lacZ insert functional enzymenonfunctional enzyme X-galproductX-galproduct

37. Selecting Colonies with Recombinant Plasmids

38. Colony hybridization  DNA probe available? part of same gene orthologue from another species synthetic oligonucleotide

39. Bacteriophage lambda as a cloning vector (Fig. 10.44, p. 311, Madigan et al.) transduction: transfer of host genes from one cell to another by a virus

40. Other methods for introducing DNA Electroporation: the use of an electric pulse to enable cells to take up DNA Millisecond-length pulses open small pores in cell membranes Millisecond-length pulses open small pores in cell membranes DNA can move into/out of the cells via pores DNA can move into/out of the cells via pores cellplasmidtransformant plasmid donor desired transformant microprojectile “gun”

41. Transgenic plants may be produced with binary vector system in Agrobacterium tumefaciens (a) generalized plant cloning vector ends of T-DNA (red) ends of T-DNA (red) ori (E. coli), ori (A. tumefaciens) ori (E. coli), ori (A. tumefaciens) resistance markers (kan, spec) resistance markers (kan, spec) (b) can clone in E. coli; transfer to A. tumefaciens by conjugation (c) D-Ti = engineered Ti (to remove pathogenesis genes) (d) D-Ti will mobilize T-DNA of vector → plant cells grown in tissue culture (e) whole plants can be regenerated from recombinant cell