DNA Cloning


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DNA Cloning

  2. 2. Outline 1. 2. 3. 4. 5. 6. 7. Source of DNA Vector Restriction enzyme Ligation Bacteria host Transformation Selection of recombinants
  4. 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. 5. Fig. 20-2 Cell containing gene of interest Bacterium 1 Gene inserted into plasmid Bacterial Plasmid chromosome Recombinant DNA (plasmid) Gene of interest DNA of chromosome 2 Plasmid put into bacterial cell Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Copies of gene Basic Protein harvested 4 Basic research and various applications research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Basic research on protein Human growth hormone treats stunted growth
  6. 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. 7. Gene cloning, genetic engineering, recombinant DNA technology They‟re 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. 8. Why genetic engineering ? Medical & health applications Production of novel and important proteins
  9. 9. Insulin.. See chapter 1
  10. 10. Agricultural applications e.g. GM crops „golden rice‟ - Inserting the gene for synthesis of carotene (Vitamin A) into rice
  11. 11. Cloning genes for scientific studies
  12. 12. Basic of DNA Cloning
  13. 13. The basics of cloning You need : 1) Source of DNA - to be cloned 2) Choice of vectors – to carry, maintain and replicate cloned gene in host cell 3) Restriction enzymes - to cut DNA 4) DNA ligase - to join foreign and vector DNA  recombinant DNA 5) Host cell – in which the recombinant DNA can replicate
  14. 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. 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. 16. Different types of cloning vectors •plasmids •bacteriophage l, M13 •Cosmids, phagemids •Artificial chromosomes BAC, YAC, MAC etc.
  17. 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. 18. pBR322 – a high copy number plasmid Important 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. 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. 20. Restriction enzymes cut DNA at very specific sequences • HindIII PstI • EcoRI FatI • SexAI SspI Recognition sites – always palindromic -Formation of hairpin loops
  21. 21. How REs cut DNA Sticky ends can re-anneal by base-pairing
  22. 22. Sticky ends has complementary overhangs - allows for proper reannealing and joining of DNA molecules
  23. 23. Bacterial transformation
  24. 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. 25. Selecting for transformants carrying recombinant DNA No vector or recombinant DNA – will not grow on media + ampicillin Vector only – will grow on media + ampicillin Recombinant DNA (vector + insert) – will grow on ampicillin This 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. 26. The goal of any cloning experiment is to obtain transformants carrying cloned insert DNA. There are several strategies to maximise these 1. Directional cloning Use 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 EcoRI BamHI EcoRI BamHI
  27. 27. 3. Dephosphorylation of vector -both the 3‟OH group and 5‟PO4 group are required for ligation -if the 5‟PO4 groups on the vector ends are removed – cannot self-ligate -Using a phosphatase enzyme -e.g calf intestinal phosphatase etc. P P
  28. 28. Blue white selection – lacZ complementation The vector contains a portion of the E.coli LacZ gene. A multiple cloning site (MCS) sequence is inserted into the LacZ‟ fragment
  29. 29. The LacZ gene codes for the b-galactosidase enzyme The b-gal enzyme hydrolyses lactose into glucose and galactose
  30. 30. The LacZ gene can be broken into two parts, a and b - each part encoding a fragment of the b-galactosidase enzyme LacZb’ Inserted into plasmid vector LacZa b- fragment
  31. 31. A fully active enzyme can be reconstituted from both fragments LacZb’ Inserted into plasmid vector LacZa b- fragment The b-gal enzyme can also hydrolyse a colorless substance called X-Gal into glucose and a blue color pigment
  32. 32. To do blue white selection, the gene of interest is cloned into the MCS Gene you want to clone Transformants 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. 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. 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. 35. Just to remind you the basic steps….
  36. 36. Sometimes, a simple cloning vector is not good enough We might want to ask the bacteria cell to make proteins using information on the cloned gene We need to use an expression vector
  37. 37. Expression vector - clone foreign gene AND make foreign protein - requires extra DNA elements Promoter – 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. 38. Recombinant Insulin – not as easy as it looks The insulin molecule as coded by DNA
  39. 39. Active insulin molecule C-peptide is removed Disulfide bonds formed between Peptide A & B Not done by bacterial cell !
  40. 40. Production of recombinant insulin – „Humulin‟ in E.coli DNA 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-galactosidase Both DNA‟s 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. 41. Expression driven by the LacZ promoter Fusion proteins are purified from the cells The B-gal part is then cleaved off by reacting with cyanogen bromide which cleaves methionine The peptide and then purified and chemically reacted to form disulfide bonds What is the problem of this approach ?