1. STBP2023 – Recombinant DNA Technology I Restriction Enzymes Instructor: M. Firdaus Raih Room 1166, Bangunan Sains Biologi Tel: 03-89215961 / Email: firdaus@mfrlab.org

2. Pre-Session Questions What are restriction enzymes and why are they called restriction enzymes? What is the function of restriction enzymes? What reactions do restriction enzymes catalyze? How is a restriction enzyme digestion done? Can DNA be digested with multiple restriction enzymes? How can the results of a restriction digestion be observed? What is a restriction map? What is RFLP and what can it be used for? How can restriction enzymes be applied in recombinant DNA technology?

3. Learning objectives Must know and understand: What restriction enzymes and their functions are; What reactions REs catalyze; including basic mechanism of reaction. Must know and understand: How a restriction enzyme digestion can be carried out in the lab; How the results of a restriction digestion can be observed in the lab; The applications of restriction enzyme digestion in recombinant DNA technology; –Applications associated with visualization of restriction fragments –Direct application of digested DNA fragments

4. What are restriction enzymes? Why are they called restriction enzymes? What are they ‘restricting’? Where are they sourced from?

5. What are restriction enzymes? Restriction enzymes (RE) are nucleases: –They recognize specific recognition sequences and cut DNA (ss or ds) to leave cohesive or blunt ends. –Also called restriction endonuclease. –The specific sequences they recognize are called restriction sites. –Over 100 are now known. REs are believed to be defence mechanisms: –The defend a bacterial host from foreign viral (phage) DNA. –By chopping up foreign DNA to be non-functioning they restrict the replication of such DNA. REs are found in bacteria and archaea: –They have 3 letter names based on the genus, species (sometimes followed by a letter for the strain) of the bacterium from which they were isolated and a Roman numeral to indicate the enzyme’s identity in the case one species has more than one enzyme. eg. EcoRI from Escherichia coli strain RY13 1 st enzyme isolated.

6. What are restriction enzymes?

8. How do REs digest DNA? What is the function of REs? What reactions do they catalyze? How is a RE digestion done in the lab?

9. How do REs digest DNA? What is the function of REs? –Natural function  defence against phage DNA (Think: What about the host DNA? Does it not get digested too?) –Can this function be manipulated for use in-vitro and for what? What reactions do they catalyze? –REs recognize specific nucleotide sequences – 4,5 or 6 nucleotides long, then cut both DNA strands, cleavage of phosphodiester bond 3 possible cuts – staggered cut to leave either a 5’ overhang or a 3’ overhang OR a cut leaving a blunt end. GGATTC CCTAGG G 3’ 5’ GATTC CCTAG 5’ 3’ G (5’ overhang) CAGCTG GTCGAC CAG 3’ 5’ CTG GTC 5’ 3’ GAC (blunt end) GGTACC CCATGG CGTAC 3’ 5’ C C 5’ 3’ CATGG (3’ overhang)

10. How do REs digest DNA? If REs are a defence against phage DNA, what about the host DNA? Does it not get digested too? Answer: NO, host DNA is not cleaved. But why not? Restriction enzymes are part of - restriction and modification system. They act together with methylases which methylates DNA. DNA which is methylated cannot be cleaved by the RE. In the simplest system, two different proteins which recognize the same site act independently of each other: (i) a methylase which marks the bacterial DNA and (ii) a RE which digests phage DNA EcoRI is an example of such a restriction modification system.

11. How do REs digest DNA? What reactions do they catalyze? (cont.) –REs are classified into 3 groups (Types I, II, III) based on: (i)Composition and enzyme co-factor requirements (ii)Target sequence (iii)Position of cleavage site relative to target sequence. –The ones used in the lab will be Type II REs Type II REs bind at a recognition site (ie. GAATTC), then cleave the molecule by cutting the DNA backbones somewhere within this sequence of bases. Each RE has a single, specific recognition sequence, and cuts the DNA molecule at a specific site.

12. How do REs digest DNA? What reactions do they catalyze? (cont.) –REs are classified into 3 groups (Types I, II, III) based on: (i)Composition and enzyme co-factor requirements (ii)Target sequence (iii)Position of cleavage site relative to target sequence. –The ones used in the lab will be Type II REs Type II REs bind at a recognition site (ie. GAATTC), then cleave the molecule by cutting the DNA backbones somewhere within this sequence of bases. Each RE has a single, specific recognition sequence, and cuts the DNA molecule at a specific site. 5’-NNNNNNNGAATTCNNNNNNNNN-3’ 3’-NNNNNNNCTTAAGNNNNNNNNN-5’ 5’-NNNNNNNG-3’ 5’-AATTCNNNNNNNNN-3’ 3’-NNNNNNNCTTAA-5’ 3’-GNNNNNNNNN-5’ EcoRI + Mg 2+

13. How do REs digest DNA?

14. PDBID 1qps – EcoR1

15. How do REs digest DNA? PDBID 1qps – EcoR1 Metals

16. How do REs digest DNA? PDBID 1qps – EcoR1 Cut 5’ overhang ‘Sticky end’ 5’-NNNNNNNG-3’ 5’-AATTCNNNNNNNNN-3’ 3’-NNNNNNNCTTAA-5’ 3’-GNNNNNNNNN-5’

17. How is a restriction enzyme digestion done? Protocols are provided in the lab manual for this course with further instructions during the practical sessions (Protocol 6, page 15). Digestions can use a single RE or multiple REs. The digestion reaction contains: –DNA to be analyzed –A restriction enzyme Commercially available restriction enzymes usually have activities at 10-20 units/μl. A "unit" is usually defined as the amount of enzyme needed to digest 1 μg of bacterial virus lambda DNA in 1 hour in a 50 μl reaction. Generally use about 10-20 units (1 μl) of restriction enzyme per reaction. –A restriction enzyme buffer mix. Each restriction enzyme prefers a particular restriction enzyme buffer mix. This buffer mix, commonly supplied at a 10X concentration, contains a buffering agent (usually Tris) to maintain constant pH, salt (usually NaCl or KCl) to provide the correct ionic strength for the digest, and Mg++ (from MgCl2) as a necessary cofactor for enzyme activity.

18. How is a restriction enzyme digestion done? Single Digest RE (1) EcoRI Single Digest RE (2) HindIII Double Digest RE (1) + RE (2) EcoRI + HindIII 1. DNA sample2.0μl (1.0μg) 2. 10X Buffer (1)1.0μl- 3. RE (1) [10 Units]1.0μl- 4. 10X Buffer (2)-1.0μl 5. RE (2) [10 Units]-1.0μl 6. Deionised H 2 O6.0μl 14μl - Total volumes10μl 20μl - Incubation (37 o C)1hour 30mins A sample digestion set up (extracted from the course’s protocols manual).

19. How is a restriction enzyme digestion done? Keep watch for: –Star activity – non specific cleavage Can occur during double or multiple digests due to non-optimum buffer conditions Amongst strategies to avoid this occurrence is to if possible carry out multiple digestions using enzymes sharing the same buffer conditions or similar buffer conditions. –What happens if no digestion is observed for DNA which is known to have the corresponding RE sites? Some steps… Check enzyme Check buffers Possible contamination of buffers / cofactors Chelators affecting RE activity? – use sterile ddH 2 O, appropriate concentration of cofactors Test with other fragments If double digest, then do 2 separate digestions as controls

20. What are the outcomes of a RE digestion? After digestion  fragments of DNA –In the case of EcoRI, the fragments have ‘sticky ends’. The fragments of DNA will have different molecular weights (different sizes) depending on where and how many sites for the restriction enzyme used is present in the uncut DNA. How can these fragments of DNA be visualized for analysis? What are potential applications for these fragments of DNA which correlates it to recombinant DNA technology? (CLUE: Think back to the 1978 Nobel Prize news release)

21. How is a RE digestion visualized? AA The results of a RE digestion can be visualized by agarose gel electrophoresis. Different fragment sizes are separated via different rates of migration through agarose

22. How is a RE digestion visualized? Each point represents the mobility (distance traveled from well) by a DNA fragment of the given size. The calibration curve was used to estimate the size of the DNA fragment in the bottom band of lane 2. The mobility of the band (54 mm) was projected (vertical dotted line) onto the calibration curve and then onto the Y-axis (horizontal dotted line), giving an estimated size of 1360 bp. From here a map of the fragments can be built.

23. What can be done with restriction fragments? Analyse the fragments. –Restriction maps –RFLPs Put them back together. –DNA ligation  recombinant DNA

24. What is a restriction map? Restriction map is a map of known restriction sites within a sequence of DNA.

25. What is a restriction map? Data of fragment sizes are compared to make a restriction map. First get the total size of your DNA. How can this be done in your case? Then map out the fragments from single digests. This is followed by mapping out the fragments of the multiple digestions. Combine all the maps into a single restriction map; they can be represented as circular maps if your DNA is a plasmid. Is there a quicker / easier way? Try this exercise.

26. What is a restriction map?

27. What is RFLP? RFLP = Restriction Fragment Length Polymorphism (often prounounced ‘ri flip’)

28. What is RFLP? RFLP = Restriction Fragment Length Polymorphism (often prounounced ‘ri flip’) RFLP is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination. How can RFLP be applied?

29. Genetic testing can be done using RFLP because RFLP is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP was an important tool for: –Genome mapping –Genetic fingerprinting –Testing for diseases –Paternity testing The applications for RFLP have been mainly superseded by PCR and DNA sequencing technologies.

30. How can RFLP be applied? Genetic testing can be done using RFLP because RFLP is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP was an important tool for: –Genome mapping –Genetic fingerprinting –Testing for diseases –Paternity testing The applications for RFLP have been mainly superseded by PCR and DNA sequencing technologies.

31. How can RFLP be applied? Genetic testing can be done using RFLP because RFLP is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP was an important tool for: –Genome mapping –Genetic fingerprinting –Testing for diseases –Paternity testing The applications for RFLP have been mainly superseded by PCR and DNA sequencing technologies.

32. RE and Recombinant DNA? What is the connection between REs and recombinant DNA technology?

33. RE and Recombinant DNA? What is the connection between REs and recombinant DNA technology? Sticky ends are complementary. Fragments with complementary sticky ends can therefore stick together by base pairing. This enables recombination of different fragments resulting in a recombinant DNA molecule. This topic and the lab practicals will be covered in STBC2033.

34. Comprehension Quiz Agarose gel electrophoresis reveals that a 2kb covalently closed circular plasmid is converted to a linear 2kb when digested with BamHI but appears as a 1kb band when digested with EcoRI. Explain this observation. (Answer)(Answer) The restriction enzyme BamHI cuts the sequence 5’-GGATCC-3’. The enzyme MboI cuts the sequence 5’-GATC-3’. (a) Which enzyme has more sites in a typical viral double stranded DNA? (b) Which enzyme will generate more fragments on average? (Answer)(Answer) A linear DNA is cut with the endonuclease EcoRI generating 4 fragments, EcoRI cuts this DNA how many times? (Answer)(Answer)

35. Comprehension Quiz Agarose gel electrophoresis reveals that a 2kb covalently closed circular plasmid is converted to a linear 2kb when digested with BamHI but appears as a 1kb band when digested with EcoRI. Explain this observation. Answer: The EcoRI digestion has resulted in 2 fragments of roughly 1000 base pairs each which is migrating at about the same rate through the gel thereby resulting in the visual observation that the digestion has resulted in a single 1kb band from what is known to be a 2kb fragment of DNA. The restriction enzyme BamHI cuts the sequence 5’-GGATCC-3’. The enzyme MboI cuts the sequence 5’-GATC-3’. (a) Which enzyme has more sites in a typical viral double stranded DNA? (b) Which enzyme will generate more fragments on average? Answer: (a) MboI (b) MboI MboI recognizes 4 bases, which is therefore less specific and in a genome, the probabilities of GATC occurring are more numerous than GGATCC. Furthermore, the recognition site GGATCC will also be recognized by MboI. A linear DNA is cut with the endonuclease EcoRI generating 4 fragments, EcoRI cuts this DNA how many times? Answer: 3 times.

36. Recap and Summary Must know and understand: What restriction enzymes and their functions are; What reactions REs catalyze; including basic mechanism of reaction. Must know and understand: How a restriction enzyme digestion can be carried out in the lab; How the results of a restriction digestion can be observed in the lab; The applications of restriction enzyme digestion in recombinant DNA technology; –Applications associated with visualization of restriction fragments –Direct application of digested DNA fragments

37. Self Study and Self Assessment The electronic materials made available are for self assessment and self study use and their use DOES NOT contribute to your final grades for this course. Also explore the references and texts listed in the course information file and other resources. Recommended reading: