In the name of God

1. In the name of God

2. Summer School, 2013 Influenza Lab Research Pasteur Institute of Iran

3. Gene Cloning and Recombinant DNA Technology By: F. Fotouhi

4. Key concepts of Molecular Cloning • What does the term cloning mean? • What is gene cloning? How does it differ from cloning an entire organism? • How is gene cloning accomplished ? • Why is gene cloning done? Summer School

5. Key concepts of Molecular Cloning • What does the term cloning mean? “Cloning” is a loaded term that can be used to mean very different things. • Cutting a piece of DNA from one organism and inserting it into a vector where it can be replicated by a host organism. • Using nuclear DNA from one organism to create a second organism with the same nuclear DNA Summer School

6. Key concepts of Molecular Cloning • What is gene/ molecular cloning? How does it differ from cloning an entire organism? • Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vitro. Cloning is frequently employed to amplify DNA fragments containing genes, but it can be used to sequence promoters, non-coding sequences, chemically synthesized oligonucleotides and randomly fragmented DNA • Molecular cloning is distinct from cell cloning and whole animal cloning in that the objective of the process is to allow the analysis and understanding of structure, function and regulation of individual genes. Summer School

7. Whole organisms are cloned too but differently Summer School

8. Key concepts of Molecular Cloning • How is gene cloning accomplished ? • Technique involving the insertion of a fragment of foreign DNA into a vector capable of replicating autonomously in a host cell (usually Escherichia coli). Growing the host cell allows the production of multiple copies of the inserted DNA for use in a variety of purposes. Summer School

9. Key concepts of Molecular Cloning • One of the main problems in gene analysis is the relatively uniform nature of the DNA molecule. • Human genome is 6.4 × 109 base pairs of DNA and is composed of only four different nucleotides. • To investigate the function of a single gene it is essential that the gene be studied in isolation, freed from the rest of its native genome. • The discovery of enzymes able to cleave DNA at specific sequences, restriction enzymes, led to the award of the 1978 Nobel Prize to Werner Arber, Daniel Nathans and Hamilton O. Smith. Summer School

10. 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 • Organisms can be engineered, insect resistance, etc. • The primary objective of gene for specific purposes, e.g. insulin production, cloning or genetic engineering is to express the cloned gene in a selected host organism, Development of New medicine • Biotechnology: The New industry Summer School

11. Basic Steps of Gene Cloning Summer School

12. Basic Steps of Gene Cloning Summer School

13. protein expression at a glance Summer School

14. Requirements for Molecular Cloning Foreign DNA Host organism Vector DNA for cloning Means of inserting foreign DNA into the vector Method of placing the in vitro modified DNA into the host cell Methods for selecting and/or screening cells that carry the inserted foreign DNA Summer School

15. Requirements for Molecular Cloning Foreign DNA PCR product genomic DNA complementary DNA (cDNA) Host organism bacterial host – E. coli eukaryotic host – yeast (Saccharomycescerevisiae) other hosts – other yeasts, insect cells, etc. Summer School

16. Requirements for Molecular Cloning Vector DNA for cloning DNA molecule that functions as a “molecular carrier” that carry the DNA of interest into the host cell & facilitates its replication. • Plasmids – used in cloning small segments of DNA (10-15 kb) • Bacteriophageλ – used in cloning larger segments of DNA(~20 kb) • Cosmids – plasmids containing DNA sequences (cos) from bacteriophageλ used to clone larger fragments of up to 45 Kb • Viral vectors - Adenovirus, Retrovirus, Baculovirus • Artificial chromosomes – BAC, YAC, PAK Summer School

17. Enzymes used in molecular cloning Polymerases • DNA Polymerase – catalyzes the polymerization of deoxyribonucleotides along the template strand DNA Pol I, Klenow fragment, T4 DNA Pol , T7 DNA Pol, Taq DNA Pol • DNA-dependent RNA Polymerase, Reverse Transcriptase AMV, MMLV • RNA Polymerases Bacterial, T7, T4 Summer School

18. Enzymes used in molecular cloning Nucleases • Enzymes capable of cleaving the phosphodiester bonds between nucleotide subunits of nucleic acids • Endonucleases verses Exonucleases • DNase I, nonspecific, Mg ++ and Ca ++ • Rnase A, Rnase H Summer School

19. Enzymes used in molecular cloning Ligases: forms phosphodiester bonds to join two pieces of DNA, utilizes ATP in the presence of Mg++ Kinases: transfers phosphate groups from donor molecules to acceptor molecule, phosphorylation Phosphatases: catalyzes the removal of 5’-phosphate residues, dephosphorylation Restriction Endonucleases: Summer School

20. Restriction Endonucleases Restriction enzymes (also called Restriction Endonucleases) are proteins that cleave DNA molecules at specific sites, producing discrete fragments of DNA. The main advantage of restriction enzyme is the ability to cut a DNA reproducibly in the same place; this is the basis of many techniques used to analyze genes. Nomenclature: Restriction Enzymes are named for the bacterial species and strain from which they are derived. For instance EcoRI is isolated from E. coli R strain and it is the first RE named from that strain. E: Genus Escherichia co: Species coli R: Strain RY13 I: The order in which the enzyme was isolated from the bacterial strain Summer School

21. Restriction Endonucleases • Why the DNA of an organism producing a restriction enzyme is not itself attacked by the restriction enzyme? Restriction- modification systemR-M system • Almost all restriction nucleases are paired with methylases that recognize and methylate the same DNA sites. Summer School

22. Bacterial Restriction–Modification Systems Summer School

23. The steps involved in DNA binding and cleavage by a type II restriction endonucleases Summer School

24. Restriction Endonucleases Summer School

25. Restriction Endonucleases There are three types of REs: • Type I & III: EcoK is a type I RE. • Require ATP, S-adenosylmethionine (SAM) and magnesium ions (Mg2+) for activity. • Recognize a specific DNA sequence but cleavage occurs in a random fashion several kilobases away from the recognition site. • Contains different subunits • Complex, expensive Summer School

26. Restriction Endonucleases Type II: EcoRI, HindIII, BamHI. They act as homodimer and recognize symmetrical (4-8 bp) DNA sequences called Palindrome • EcoRI: 5’- GA A T T C-3’ 3’-C T T A A G-5’ • BamHI: 5’- G G A T C C-3’ 3’-C C T A GG-5’ • Endonuclease and methylase are separate, single-subunit enzymes Summer School

27. Cleavage patterns of some common restriction endonucleases

29. DNA Cleavage by Type II Restriction EnzymesSticky (cohesive) VS. Blunt Ends Summer School

30. DNA Cleavage by Type II Restriction Enzymes Some REs generate compatible sticky ends. BamHI: G/GATCC BglII: A/GATCT BamHI: G G A T C C C C T A G G BglII:A G A T C T T C T A G A Summer School

31. DNA Cleavage by Type II Restriction Enzymes Isoschizomers and Neoschizomers: Isoschizomers: Restriction Endonucleases that recognize the same sequence but obtained from different organisms. SmaI: CCC/ GGG XmaI: CCC/ GGG Neoschizomers: Isoschizomers which cut differently. SmaI: CCC/ GGG XmaCI: C/ CCGGG Summer School

32. Restriction Endonucleases and methylation Cleavage by a RE can be blocked or impaired when a particular base in Recognision site is methylated. Prokaryotic Methylases: Dam: Methylation of the adenine in the sequence GATC. Dcm: Methylation at the C5 position of cytosine in the sequences CCAGG and CCTGG. Summer School

33. Restriction Endonucleases and methylation If your RE is M-sensitive then: 1- Choose an Isoschizomer that is not M-sensitive. 2- Transform your plasmid into an E.coli strain that is Dcm and Dam negative. GM2929, JM110 Summer School

34. Star Activity of Restriction Endonucleases • Cleavage of sequences similar to the defined recognition sequence for a restriction enzyme is called "star activity". • Low ionic strength, high pH of the reaction buffer, excess concentration of enzyme, excess incubation time, excess concentration of glycerol • Single versus double digest Summer School

35. Star Activity of Restriction Endonucleases Summer School Star Activity

36. Isolation of gene Foreign DNA, target gene, the gene of interest • genomic DNA (Library) • Plasmid DNA(cut& paste) • PCR product • complementary DNA (cDNA) Summer School

37. Plasmid DNA(cut& paste) Summer School

38. Gene isolation using PCR PCR Method for rapid Exponential amplification of DNA: 1DNA molecule to 100,000,000,000 in a few hours 15 cycles =32,767 copies 20 cycles =1,048,575 copies 30 cycles =1,073,741,823 copies Nobel Prize in Chemistry (1993) to Kary Mullis Summer School

39. Summer School

40. Using RT-PCR to clone a single cDNA

41. Cleavage close to DNA fragment Ends • Cleavage close to the ends of PCR products is slower and less efficient. • When designing PCR primers containing restriction sites, add at least 2 random bases 5´ of the restriction site. • Higher quantities of enzyme and longer reaction times are also necessary. Summer School

42. PCR Product Restriction Digestion Purify your PCR product using PCR Clean-up kit before digestion because: PCR buffer is not optimal for REs. Taq Polymerase: 5’ 3’ Exonuclease activity Adds A tails to 3’ ends of PCR products. Summer School

43. Ligation of Digested DNA fragments T4 DNA Ligase is usually used in gene cloning. Catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA with hydrolysis of ATP. T4 Ligase acts on both sticky and blunt ends. Summer School

44. Ligation of Digested DNA fragments Summer School

45. Host Cell for Gene Cloning • Escherichia coli is the most favored host cell for the propagation, manipulation, and characterization of recombinant DNA. • E. coli is an enteric rod-shaped Gram-negative bacterium with a circular genome of 4.6 Mb • The cells grow quickly (generation time of 20 min.) • The genetics are well characterized. • Engineered Strains are safe to ourselves. • Almost all strains currently used in recombinant DNA experiments are derived from a single strain: E. coli K-12, isolated from the feces of a diphtheria patient in 1922. • Common lab strains of E. coli are different from their wild type counterparts, carry some mutations designed to help plasmid propagation Summer School

46. Host Cell for Gene Cloning • simply putting a piece of foreign DNA into a bacteria cell will not result in the replication of that DNA. • Typically laboratory strains have mutations in the rec A and end A1 genes. • rec A gene involved in recombination. The mutant gene limits recombination of the plasmid with the E. coli genome so that the plasmid inserts are more stable. • the end A1 mutation inactivates endonuclease I, Improves yield and quality of isolated plasmid DNA. • To incorporate blue/white selection into subcloning scheme, we need to transform E. coli carrying a lac Z∆. • The TOP10F´ carries the tetracycline resistance gene and lacIq repressor, so requires IPTG induction for blue/white color screening. DH5α™ • F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1endA1hsdR17 (rK–, mK+) phoAsupE44 λ– thi-1 gyrA96 relA1 TOP10F’ • F´{lacIq, Tn10(TetR)} mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(araleu) 7697 galUgalKrpsL (StrR) endA1nupG Summer School

47. Vector DNA - Plasmids • Plasmids are circular pieces of dsDNA molecules found naturally in bacteria. • They are circular capable of autonomous replication independent of bacterial chromosome. • molecular parasites • carry one or more genes some of which confer resistance to certain antibiotics • origin of replication (Ori), a region of DNA that allows multiplication of the plasmid within the host (Self-Replication) • Plasmids can be engineered to be useful as cloning vector. Summer School

48. Vector DNA - Plasmids Desirable properties of plasmids: • small size • known DNA sequence • high copy number • a selectable marker: Antibiotic Resistance Gene • a second selectable gene: Blue-White Screening • large number of unique restriction sites or Multiple Cloning Site (MCS) Summer School

49. Plasmids: Independent Genetic Elements in Bacterial Cells. Summer School

50. Selective Markers of Plasmids Antibiotic Resistance Gene, Ampicillin Kanamycin Non-transformed Bacterial cells die but each bacterial cell containing our plasmid forms a colony on agar plates. Summer School