RESTRICTION ENZYMES & GENETIC RECOMBINATION

1. RESTRICTION ENZYMES & GENETIC RECOMBINATION BIOLOGY 2/2A Motzko

2. Saccharin (1879)

3. Corn Flakes (1899)

4. Microwave Oven (1945)

5. Penicillin (1927)

6. Werner Arber(1969)1st To Isolate Restriction Endonucleases (Enzymes)

7. Restriction Endonucleases • Enzymes that cut DNA at specific locations on the strand known as restriction sites • Naturally produced by bacteria to protect them against viruses • Restriction sites differ depending upon the sequence of nucleotides • When restriction enzymes cut the DNA strand they can leave unpaired nucleotides called “sticky ends”

9. Sticky Ends Allow Restriction Enyzmes To Assist In The Creation Of Recombinant DNA

10. Boyer & Cohen (1973) – First to create recombinant DNA using restriction enzymes

12. LAB: Genetic Recombination • Purpose: • To model the digestion of DNA strands by restriction enyzmes and the synthesis of recombinant DNA • To demonstrate how the pGLO plasmid was constructed from bacterial plasmid DNA and two genes: beta-lactamase and GFP.

13. pGLO Plasmid

14. 5’-CTGACCATGATTACGCCAAGCTTGCAAGGATCCCCGGGTA-3 Obtain initial bacterial plasmid sequence (white)

15. 5’-CTGACCATGATTACGCCAAGCTTGCAAGGATCCCCGGGTA-33’-GACTGGTACTAATGCGGTTCGAACGTTCCTAGGGGCCCAT-55’-CTGACCATGATTACGCCAAGCTTGCAAGGATCCCCGGGTA-33’-GACTGGTACTAATGCGGTTCGAACGTTCCTAGGGGCCCAT-5 Obtain initial bacterial plasmid sequence (white) Write out complimentary base pairs (cDNA) for plasmid

16. 5’-CTGACCATGATTACGCCAAGCTTGCAAGGATCCCCGGGTA-33’-GACTGGTACTAATGCGGTTCGAACGTTCCTAGGGGCCCAT-55’-CTGACCATGATTACGCCAAGCTTGCAAGGATCCCCGGGTA-33’-GACTGGTACTAATGCGGTTCGAACGTTCCTAGGGGCCCAT-5 Obtain initial bacterial plasmid sequence (white) Write out complimentary base pairs (cDNA) for plasmid Tape ends of plasmid together 5->3

17. 5’-CTGACCATGATTACGCCAAGCTTGCAAGGATCCCCGGGTA-33’-GACTGGTACTAATGCGGTTCGAACGTTCCTAGGGGCCCAT-5Gec1 HindIII BamH1 Sma1 Obtain initial bacterial plasmid sequence (white) Write out complimentary base pairs (cDNA) for plasmid Tape ends of plasmid 5’->3’ Identify the locations on the bacterial plasmid where the following four restriction enzymes will eventually cut the plasmid A) HindIII B) BamH1 C) Sma1 D) Gec1

18. 5’-CACAAGCTTAGCATAGATCTAGCTACGCAA-3’ 3’-GTGTTCGAATCGTATCTAGATCGATGCGTT-5’HindIIIEcoRVI • 5) Identify the Green Fluorescent Protein (GFP) gene (green) using the following restriction enzymes • A) HindIII B) EcoRVI

19. 5’-AATGGTACAATGCTATGCATGGCTATA-3’ 3’-TTACCATGTTACGATACGTACCGATAT-5’Sma1 • 6) Identify the β-Lactamase gene (pink) with the following restriction enzymes A) Sma1 B) Gec1

20. Cut the identified sites on all three strands using the patterns in your handout

21. Splicing The Genes Into The Plasmid • Insert the GFP and β-Lactamase Genes into the bacterial plasmid by pairing the complimentary bases in the sticky ends of each segment • Tape these complimentary base sequences together