1.MIC 210 BASIC MOLECULAR BIOLOGY LECTURE 4 DNA CLONING BY SITI NORAZURA JAMAL (MISS AZURA) 03 006/ 06 483 2132
2. Outline 1. 2. 3. 4. 5. 6. 7.Source of DNA Vector Restriction enzyme Ligation Bacteria host Transformation Selection of recombinants 3. INTRODUCTION TO DNA CLONING 4. What Does It Mean: To Clone? Clone: a collection of molecules or cells, all identical to an original molecule or cell To "clone a gene" is to make many copies of it - for example, by replicating it in a culture of bacteria. Cloned gene can be a normal copy of a gene (= wild type). Cloned gene can be an altered version of a gene (= mutant). Recombinant DNA technology makes manipulating genes possible. To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning 5. Fig. 20-2Cell containing gene of interestBacterium 1 Gene inserted into plasmidBacterial Plasmid chromosome Recombinant DNA (plasmid)Gene of interestDNA of chromosome2 Plasmid put into bacterial cell Recombinant bacterium3 Host cell grown in culture to form a clone of cells containing the cloned gene of interest Gene of InterestProtein expressed by gene of interestCopies of geneBasicProtein harvested 4 Basic research and various applicationsresearch on geneGene for pest resistance inserted into plantsGene used to alter bacteria for cleaning up toxic wasteProtein dissolves blood clots in heart attack therapyBasic research on proteinHuman growth hormone treats stunted growth 6. A preview of gene cloning and some uses of cloned genes Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome Cloned genes are useful for making copies of a particular gene and producing a protein product Gene cloning involves using bacteria to make multiple copies of a gene Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA This results in the production of multiple copies of a single gene 7. Gene cloning, genetic engineering, recombinant DNA technology Theyre more or less the same It basically means : joining together DNA from different sources/organisms, forming a recombinant DNA molecule Then put this recombinant DNA into a host cell, usually bacteria The host cell will then replicate many copies of this recombinant DNA molecule Sometimes, we might want to ask the host cell to use the genetic information in the recombinant DNA to make proteins 8. Why genetic engineering ? Medical & health applicationsProduction of novel and important proteins 9. Insulin.. See chapter 1 10. Agricultural applications e.g. GM crops golden rice - Inserting the gene for synthesis of carotene (Vitamin A) into rice 11. Cloning genes for scientific studies 12. Basic of DNA Cloning 13. The basics of cloning You need : 1) Source of DNA - to be cloned2) Choice of vectors to carry, maintain and replicate cloned gene in host cell3) Restriction enzymes - to cut DNA 4) DNA ligase - to join foreign and vector DNA recombinant DNA5) Host cell in which the recombinant DNA can replicate 14. 1) Source of DNA Genomic DNA DNA extracted from cells and purified cDNA by reverse transcription of mRNA Amplified DNA using Polymerase Chain Reaction Synthetic DNA DNA made artificially using a machine 15. 2) Vector to carry the ligated foreign gene into the host cell maintain the foreign gene in the host cell Replicate pass into new cells during cell division Expressed the cloned foreign gene to make a protein 16. Different types of cloning vectors plasmids bacteriophage l, M13 Cosmids, phagemidsArtificial chromosomes BAC, YAC, MAC etc. 17. Plasmid Extrachromosomal DNA found in bacteria & fungi Close circular DNA molecules, supercoiled Can replicate autonomously, independent of chromosome Can be transfer to other cells by conjugation Can be integrated into the chromosome In nature, plasmids carry genes that are not essential under normal conditions But confers a survival advantage under extreme conditions eg. resistance to antibiotics, metabolism of unusual substrates Number of plasmid per cell - controlled by plasmid itself High copy number > 100 /cell; low copy number < 20 /cell Plasmid incompatibility the presence of one plasmid in a cell excludes other plasmids 18. pBR322 a high copy number plasmidImportant DNA elements :1. The rop (or sometimes ori) origin of replication, so that the plasmid can be maintained & replicated in the host cell 2. Antibiotic resistance marker genes (ApR for ampicillin resistance and TcR for tetracycline) so that we can select 3. Unique restrcition sites (EcoRI, PvuI etc) so that we can cut the plasmid in one place only. and insert the foreign gene we want to clone 19. 3) Restriction enzyme > Type II Restriction endonuclease Enzymes found in some microorganisms Natural role to destroy invading foreign DNA eg. bacteriophage DNA Recognizes very specific short sequences of DNA Each enzyme has its own recognition sequence/ site Sometimes two different enzymes have the same recognition sites, in which case they are known as isoschizomers Cuts DNA in very specific manner Technically one Unit of RE will completely digest 1 ug of substrate DNA in a 50 ul reaction volume in 60 minutes 20. Restriction enzymes cut DNA at very specific sequences HindIIIPstIEcoRIFatISexAISspIRecognition sites always palindromic -Formation of hairpin loops 21. How REs cut DNASticky ends can re-anneal by base-pairing 22. Sticky ends has complementary overhangs - allows for proper reannealing and joining of DNA molecules 23. Bacterial transformation 24. Inserting the recombinant DNA molecule into a Competent E.coli cell The cells must be made competent be treating with CaCl2 or very little DNA will be taken up. 25. Selecting for transformants carrying recombinant DNA No vector or recombinant DNA will not grow on media + ampicillinVector only will grow on media + ampicillin Recombinant DNA (vector + insert) will grow on ampicillinThis is the one we want ! The goal of any cloning experiment is to obtain transformants carrying cloned insert DNA. There are several strategies to maximise these 26. The goal of any cloning experiment is to obtain transformants carrying cloned insert DNA. There are several strategies to maximise these 1.Directional cloningUse two different restriction enzymes to cut each end of the vector (and also the foreign DNA you want to clone)- Generate different sticky ends cannot self ligate EcoRIBamHI EcoRIBamHI 27. 3. Dephosphorylation of vector-both the 3OH group and 5PO4 group are required for ligation -if the 5PO4 groups on the vector ends are removed cannot self-ligate -Using a phosphatase enzyme-e.g calf intestinal phosphatase etc.P P 28. Blue white selection lacZ complementationThe vector contains a portion of the E.coli LacZ gene. A multiple cloning site (MCS) sequence is inserted into the LacZ fragment 29. The LacZ gene codes for the b-galactosidase enzymeThe b-gal enzyme hydrolyses lactose into glucose and galactose 30. The LacZ gene can be broken into two parts, a and b - each part encoding a fragment of the b-galactosidase enzymeLacZb Inserted into plasmid vectorLacZab- fragment 31. A fully active enzyme can be reconstituted from both fragments LacZb Inserted into plasmid vectorLacZab- fragmentThe b-gal enzyme can also hydrolyse a colorless substance called X-Gal into glucose and a blue color pigment 32. To do blue white selection, the gene of interest is cloned into the MCSGene you want to cloneTransformants are plated onto a medium containing : o Antibiotic for selection o IPTG to induce expression of the LacZ o X-Gal to detect the presence of b-galactosidase 33. Transformants with vector only : o LacZ is expressed a fragment is produced o Complements b-fragment to form fully active enzyme o Hydrolyses X-Gal Blue color colonies 34. Transformants with recombinant DNA: o LacZ is destroyed by insertion of foreign gene no a fragment o Cannot form fully active enzyme o No hydrolysis of X-Gal White color colonies 35. Just to remind you the basic steps. 36. Sometimes, a simple cloning vector is not good enoughWe might want to ask the bacteria cell to make proteins using information on the cloned gene We need to use an expression vector 37. Expression vector - clone foreign gene AND make foreign protein - requires extra DNA elementsPromoter to initiate transcription synthesis of mRNA Terminator to stop transcription Fusion tags for making fusion proteins e.g. Histidinex6, c-myc, HA, GFP In frame MCS Other things e.g. Poly-A sites 38. Recombinant Insulin not as easy as it looks The insulin molecule as coded by DNA 39. Active insulin moleculeC-peptide is removed Disulfide bonds formed between Peptide A & B Not done by bacterial cell ! 40. Production of recombinant insulin Humulin in E.coliDNA for peptide A and Peptide B synthesized chemically Peptide A 21 amino acids 63 nucleotides + ATG + stop codon Peptide B 30 amino acids 90nucleotides +ATG +stop codon Clone into a different plasmid vector s into the gene for B-galactosidaseBoth DNAs were cloned in frame with the b-gal gene Expressed as fusion proteins Peptide (A or B) + part of b-gal This is necessary otherwise the small peptides will be quickly degraded Fusion with b-gal stabilises the peptides 41. Expression driven by the LacZ promoterFusion proteins are purified from the cells The B-gal part is then cleaved off by reacting with cyanogen bromide which cleaves methionineThe peptide and then purified and chemically reacted to form disulfide bondsWhat is the problem of this approach ?