1 / 42

RECOMBINANT DNA TECHNIQUES and PROTEIN ENGINEERING

RECOMBINANT DNA TECHNIQUES and PROTEIN ENGINEERING. Enzymes used in molecular cloning. Restriction endonucleases (REases) Methylases Ligases Polymerases Other nucleases Kinases Phosphatases Topoisomerase. Biotech companies. New England Biolabs http://www.neb.com/nebecomm/default.asp

Download Presentation

RECOMBINANT DNA TECHNIQUES and PROTEIN ENGINEERING

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. RECOMBINANT DNA TECHNIQUESandPROTEIN ENGINEERING Enzymes used in molecular cloning

  2. Restriction endonucleases (REases) • Methylases • Ligases • Polymerases • Other nucleases • Kinases • Phosphatases • Topoisomerase

  3. Biotech companies • New England Biolabs http://www.neb.com/nebecomm/default.asp • Fermentas http://www.fermentas.com • Promega http://www.promega.com • Stratagene http://www.stratagene.com • Merck Biosciences (Novagene) http://www.merckbiosciences.co.uk/home.asp • BD Biosciences (Clonetech, Pharmingen) http://www.bdbiosciences.com • Sigma-Aldrich http://www.sigmaaldrich.com • GE Healthcare (Amersham Biosciences, Pharmacia) http://www.gehealthcare.com/usen/index.html

  4. Fermentas is a Lithuanian company

  5. RESTRICTION AND MODIFICATION EXPERIMENTS (DUSSOIX & ARBER, 1962) • E coli K and C strains • Bacteriophage lambda (l) 1010 pfu/1ml Phage does not absorb to „new” host – false Phage DNA does not enter into „new” host – false Phage DNA rapidly degraded in „new” host – TRUE RESTRICTION ENDONUCLEASES METHYLASES

  6. Restriction endonucleases http://www.typei-rm.info/index.htm http://www.typei-rm.info/index.htm

  7. EcoR124I recognises GAAn6RTCG where R = purine and n = any nucleotide

  8. Assembly into an anti-codon nuclease (ACNase) The bacteriophage T4 encodes a small (26 amino acid) polypeptide called Stp, which can interact with the EcoprrI restriction endonuclease as an anti restriction determinant The EcoprrI restriction endonuclease interacts with another host protein, PrrC, to produce a latent anti-codon nuclease (ACNase). The fully functional ACNase is able to cleave tRNALys at the codon triplet. however, to prevent cleavage of host (bacterial) tRNA the enzyme is latent and shows no such activity. Infection of the bacteria by bacteriophage T4 introduces the Stp polypeptide as an early gene and this polypeptide can activate the ACNase resulting in cleavage of the bacteriophage tRNALys. This prevents growth of the bacteriophage

  9. Restriction endonucleases

  10. Restriction endonucleases

  11. Type II restriction endonucleases • >3500 type II enzyme,(750 strains) • Rebase enzyme list: >10,000 • 292 sequenced recognition seqence • REBASE(New England Biolabs): • http://rebase.neb.com/rebase/rebase.charts.html • Nomenclature: • HindIII Haemophilus influenzae dserotype, 3. enzyme • SmaI Serratia marcescens 1. enzyme

  12. Type II restriction endonucleasesMore detailed classification

  13. Type II restriction endonucleases • Recognition sequence: • Usually palindrome • Terta-, penta-, hexa-, octanucleotide • HpaIICCGG • HinfIGANTC • BamHIGGATCC • NotIGCGGCCGC • Interrupted palindrom • BstXI CCANNNNNTGG

  14. Type II restriction endonucleases • Cleavage within the recognition sequence • In the middle: blunt • EcoRV GATATC • Asymetrically: sticky • 5’ overhang EcoRIGAATTC • 3’ overhang KpnIGGTACC • length of overhang: 1, 2, 3, 4, (5) • Frequency of recognition sites • Ptetra=4-4 =1/256 • Phexa=4-6 =1/4096

  15. Type II restriction endonucleases

  16. Type II restriction endonucleases W=A,T M=A,C R=A,G S=C,G Y=C,T K=G,T V=A,C,G H=A,C,T D=A,G,T B=C,G,T

  17. EcoRI Type II restriction endonucleases

  18. KpnI Type II restriction endonucleases

  19. Type II restriction endonucleases recongize and cut only double stranded DNA • HindIII co-crystalized with CCAAGCTTGG • the strands are not separated • „indirect readout” • pattern in the major groove read by the enzyme

  20. Type II restriction endonucleases • Isoschizomers • identical recognition and cleavage sites MboI, Sau3AIGATC • Neoschizomers • identical recognition, different cleavage SmaI CCCGGG XmaI CCCGGG • Compatible overhangs MboIGATC BamHGGATCC BglIIAGATCT BclITGATCA

  21. Type II restriction endonucleases • Effect of methylation • Methylases in E. coli: • DammethylaseGmN6ATC GmN6ATC MboI – sensitive Sau3AI, Bsp143I – cleaves TCTAG mN6A XbaI – sensitive if overlapping dam site • DcmmethylaseCm5CWGG Cm5CGG HpaII – sensitive MspI – cleaves DpnI – cleaves only methylated GGGCCm5C ApaI – sensitive if overlapping dcm site • CpGmethylation in eukaryots SmaI - sensitive

  22. Conditions for digestion Optimal buffer (pH, I, composition) Temperature (not always 37oC!) Star activity (relaxation of specificity) High glycerol conc. Excess enzyme Low ionic strength, high pH etc. Site preferences Cleavage rates differing by an order of magnitude Activity: 1unit cleaves 1 g in 60 min under optimal conditions (in 50 l) Cleavage close to the end (see tables in catalogs!) Double digestion (see optimal buffers in catalogs!) Type II restriction endonucleases

  23. Cleavage close to the end http://www.fermentas.com/techinfo/re/restrdigpcrii.htm

  24. Double digestion http://www.fermentas.com/doubledigest/index.html

  25. Methylases EcoRI methylase

  26. Ligases

  27. Ligases

  28. Ligases

  29. T4 RNA ligase

  30. DNA polymerases DNA pol I Klenow enzyme T4 polymerase T7 polymerase Thermostable Taq polymerase Pfu polymerase Reverse transcriptase

  31. 3’ 5’ nuclease activity basicly proofreading activity – improves fidelity in the absence dNTP-s slowly removes correct base pairs too T4 polymerase active on 3’ overhang: generates blunt end: „polishing”

  32. 5’->3’ nuclease activity Nick translation!

  33. Reverse transcriptase

  34. RNase activity associated with RT

  35. T7 T3 Sp6 RNA polymerase run off synthesis

  36. Template independent polymerases • DNA • Terminal deoxyribonucleotidyl transferase • Template: single stranded DNA • 3’ overhang • Co2+ : blunt, 5’ overhang, RNA • Homopolymer tailing • RNA • Poly(A) polymerase • Labeling 3’ ends of RNA

  37. Terminal transferase

  38. Polynucleotide kinase

  39. Polynucleotide kinase: exchange reaction

  40. Phosphatases Cut phosphate ester bonds • Bacterial alkaline phosphatase (BAP) • Calf intestinal phosphatase (CIP) • Shrimp phosphatase

More Related