1. Enzymes in Genetics Engineering

2. Restriction Enzymes & Ligase 1. Restriction Enzymes Bacterial enzymes that cut at specific restriction site sequences Cut DNA by breaking phosphodiester bonds Yield restriction fragments that can be used to clone 2. DNA Ligase Enzyme reforms phosphodiester bond between adjacent nucleotides These are used to make recombinant DNA. Two important first steps in cloning require two enzymes.

3. Types of restriction enzymes Type I Recognize specific sequences·but then track along DNA (~1000-5000 bases) before cutting one of the strands and releasing a number of nucleotides (~75) where the cut is made. A second molecule of the endonuclease is required to cut the 2nd strand of the DNA –e.g. EcoK. –Require Mg2+, ATP and SAM (S-adenosyl methionine) cofactors for function Type II Recognize a specific target sequence in DNA, and then break the DNA (both strands), within or close to, the recognition site –e.g. EcoRI –Usually require Mg2+ Type III Intermediate properties between type I and type II. Break both DNA strands at a defined distance from a recognition site –e.g. HgaI –Require Mg2+ and ATP

4. Restriction enzyme type II Type II is the most frequent used in biology molecular techniques. Hundreds of restriction enzymes have been isolated and characterised Enables DNA to be cut into discrete, manageable fragments Many are now commercially available Each restriction enzyme will recognize its own particular site Some recognize more than one sequence Many Type II restriction endonucleases recognize PALINDROMIC sequences Restriction enzymes do not discriminate between DNA from different organisms

5. The phosphodiester bond is cleaved between specific bases, one on each DNA strand

6. 5'-G A A T T C-3' 3'-C T T A A G-5' Generate 5' overhangs – eg: EcoRI Palindromic sequence Generate 3' overhangs – eg: PsfI Generate blunt end, eg: SmaI

10. Examples of restriction enzymes and the sequences they cleave Source microorganismEnzymeRecognition Site Ends produced Arthrobacter luteusAlu IAG  CTBlunt Bacillus amyloiquefaciens HBam HIG  GATCCSticky Escherichia coliEco RIG  AATTCSticky Haemophilus gallinarumHga IGACGC(N) 5  Sticky Haemophilus infulenzaeHind IIIA  AGCTTSticky Providencia stuartii 164Pst ICTGCA  GSticky Nocardia otitiscaviarunsNot IGC  GGCCGCSticky Staphylococcus aureus 3ASau 3A  GATCSticky Serratia marcesansSma ICCC  GGGBlunt Thermus aquaticusTaq IT  CGASticky

12.  Restriction endonucleases are a natural part of the bacterial defence system  Part of the restriction / modification system found in many bacteria  These enzymes RESTRICT the ability of foreign DNA (such as bacteriophage DNA) to infect / invade the host bacterial cell by cutting it up (degrading it)  The host DNA is MODIFIED by METHYLATION of the sequences these enzymes recognise  Methyl groups are added to C or A nucleotides in order to protect the bacterial host DNA from degradation by its own enzymes

14. Ligation When sticky ends are created on the vector and the rDNA, the ends are compatible and complementary Can be added as linkers or adapters DNA Ligase Seals single stranded nicks between adjacent nucleotides in a duplex DNA chain (catalyze the formation of a phosphodiester bond between adjacent 3’ hydroxyl and 5’ phosphate termini in DNA)

17. Joining DNA by Ligase

20. Generating DNA Probe

23. T4 polymerase DNA polymerase activity A very active single- stranded 3'->5' exonuclease Lacks a 5'->3' exonuclease activity

24. Dephosphorylation and phosphorylation of DNA

29. Other useful DNA modification enzymes used for manipulating DNA Alkaline phosphatase Removes phosphate groups from 5' ends of DNA (prevents unwanted re-ligation of cut DNA) DNA Ligase Joins compatible ends of DNA fragments (blunt/blunt or complementary cohesive ends). Uses ATP DNA polymerase I Synthesises DNA complementary to a DNA template in the 5'-to-3'direction. Starts from an oligonucleotide primer with a 3' OH end Exonuclease III Digests nucleotides progressiviely from a DNA strand in the 3' -to-5' direction Polynocleotide kinase Adds a phosphate group to the 5' end of double- or single-stranded DNA or RNA. Uses ATP RNase ANuclease which digests RNA, not DNA Taq DNA polymerase Heat-stable DNA polymerase isolated from a thermostable microbe (Thermus aquaticus) Terminal transferase An enzyme that can add nucleotide on to 3-end of the DNA molecule. For radiolabelling DNA phosphorylase An enzyme that can remove phosphate group from DNA molecules