1. Restriction mapping Site-specific restriction endonucleases are used to identify DNA molecules

2. What are restriction endonucleases (REs)? How can REs be used to identify DNA molecules? How can I find RE recognition sites in the MET plasmids?

3. Restriction endonucleases are part of a bacterium’s defense against invaders electron micrograph by Graham Colm of bacteriophage infecting a bacterium Restriction-modification systems allow the bacterium to distinguish self from non-self DNA Restriction: bacterial endonucleases cleave both strands of foreign DNA at specific recognition sites Modification: bacteria protect their own DNA by adding a methyl group to the recognition sites in their own DNA Type II restriction enzymes are widely used in molecular biology: enzymes cleave, but do not modify, their specific recognition sites

4. REs with 6-nucleotide recognition sites (6-cutters) are widely used in molecular biology Sites would randomly be expected every 1/4096 nucleotides (1/4 6 ) Actual sizes vary widely with average of ~4000 bp REStrain of originRecognition site EcoRI E. coli (strain RY13)G A A T T C Hind III H. influenzaA A G C T T BamHIB. amyloliquefaciensG G A T C C Recognition sites are often palindromes Crystal structure 2CKQ EcoRI bound to DNA

5. GAATT C CTTAA G 3’ 5’ 3’ GAATT C CTTAA G 3’ 5’ 3’ G AATTC CTTAA G 3’ 5’ 3’ EcoRI recognition site is a palindrome with an axis of symmetry EcoRI dimer binds sequence and catalyzes double- strand cleavage Products have “sticky ends”: unpaired hydrogen bonds on nitrogen bases

6. The sticky ends generated by REs are useful in generating recombinant DNA molecules (more later........) REs are the scissors—ligases are the paste G CTTAA 5’ 3’ AATTC G 3’ 5’ GAATT C CTTAA G 3’ 5’ 3’ DNA ligase Sticky ends from two molecules form hydrogen bonds Recombinant molecule

7. What are restriction endonucleases (REs)? How can REs be used to identify DNA molecules? How can I find RE recognition sites in the MET plasmids?

8. Preparing a restriction map pBG1805-MSRA is digested with: Acc I BsaA I Hinc II MSRA (555 bp) inserted here pBG1805 (6573 bp) Restriction fragments are separated on 1% agarose gels

9. Markers Uncut Acc I BsaA I Hinc II RE Digests 21,228 Size (bp) 5148, 4973 4268 3530 2027 1904 1584 1375 947 831 564 Standards EcoRI and HindIII digest of lambda DNA RE digests of pBG1805 containing YER042W ORF Each restriction enzyme produces a distinct set of fragments

10. pBG1805 (6573 bp) S. cerevisiae ORF pYES2.1 (5886 bp) S. pombe ORF or LacZ Your task: Design a strategy to distinguish your three plasmids with restriction endonucleases

11. What are restriction endonucleases (REs)? How can REs be used to identify DNA molecules? How can I find RE recognition sites in the MET plasmids?

12. Program for finding restriction sites in DNA sequences

13. Overview Find the vector (plasmid) sequence Access the pBG1805 sequence in NCBI’s Nucleotide database The pYES2.1 sequence is available on Blackboard* Paste the sequence into NEB cutter and give the file a name Find the MET gene sequence in SGD (yeastgenome.org) Paste the MET coding sequence at the end of the vector sequence Indicate that the sequence is circular and click submit Use NEB cutter to find restriction sites for four restriction endonucleases: AccI HincII ScaI XbaI *Control pYES2.1-LacZ sequence can be pasted directly into NEB cutter