Enzymes in Genetics Engineering

1. Enzymes in Genetics Engineering

2. Restriction Enzymes & Ligase These are used to make recombinant DNA. Two important first steps in cloning require two enzymes. 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

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' Palindromic sequence Generate 3' overhangs – eg: PsfI Generate 5' overhangs – eg: EcoRI Generate blunt end , eg: SmaI

10. Examples of restriction enzymes and the sequences they cleave

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