1. Restriction Enzyme Digest October 8, 2015

2. Restriction Enzymes

3. Also known as restriction endonucleases Scan the DNA sequence Find a very specific set of nucleotides Make a specific cut Used to construct recombinant DNA plasmids

4. Palindromes in DNA sequences Genetic palindromes are similar to verbal palindromes. A palindromic sequence in DNA is one in which the 5’ to 3’ base pair sequence is identical on both strands. 5’5’ 5’5’ 3’3’ 3’3’

5. Restriction enzymes recognize and make a cut within specific palindromic sequences, known as restriction sites, in the DNA. This is usually a 4- or 6 base pair sequence.

6. Restriction Endonuclease Types Type I- multi-subunit, both endonuclease and methylase activities, cleave at random up to 1000 bp from recognition sequence Type II- most single subunit, cleave DNA within recognition sequence Type III- multi-subunit, endonuclease and methylase about 25 bp from recognition sequence

7. Hae III HaeIII is a restriction enzyme that searches the DNA molecule until it finds this sequence of four nitrogen bases. 5’ TGACGGGTTCGA GGCC AG 3’ 3’ ACTGCCCAAGGT CCGG TC 5’

8. Once the recognition site is found Hae III will cleave the DNA at that site 5’ TGACGGGTTCGA GGCC AG 3’ 3’ ACTGCCCAAGGT CCGG TC 5’

9. These cuts produce “blunt ends” 5’ TGACGGGTTCGA GGCC AG 3’ 3’ ACTGCCCAAGGT CCGG TC 5’

10. The names for restriction enzymes come from: the type of bacteria in which the enzyme is found the order in which the restriction enzyme was identified and isolated. EcoRI for example R strain of E.coli bacteria I as it is was the first E. coli restriction enzyme to be discovered.

11. “blunt ends” and “sticky ends” Hae III produced a “blunt end”? EcoRI makes a staggered cut and produces a “sticky end” 5’ GAATTC 3’ 3’ CTTAAG 5’ 5’ GA ATTC 3’ 3’ CTTA AG 5’ 5’ GAATTC 3’ 3’ CTTAAG 5’

12. More examples of restriction sites of restriction enzymes with their cut sites Hind III: 5’ AAGCTT 3’ 3’ TTCGAA 5’ Bam HI: 5’ GGATCC 3’ 3’ CCTAGG 5’ Alu I: 5’ AGCT 3’ 3’ TCGA 5’

13. Separating Restriction Fragments

14. Our digests:

17. …

18. Run digest on gel to check fragment sizes miR-29c construct ~ 5kB plasmid – BglII/EcoRI digest ~250bp fragment PRKCBP1 construct ~9kB plasmid – AsiSI/BglII digest ~1.8kB fragment

19. Uses of restriction enzyme analysis- forensics http://science.howstuff works.com/dna- profiling1.htm

20. Uses of restriction enzyme analysis- Medical diagnosis http://www.ncbi.nlm.nih.gov/probe/docs/techrflp/

21. Uses of restriction enzyme analysis- Inheritance and genetic counseling http://www.ncbi.nlm.nih.gov/probe/docs/techrflp/

22. https://en.wikipedia.org/wiki/Sanger_sequencing#/media/File:Sanger-sequencing.svg Sanger sequencing:

23. Cost to sequence DNA has dropped dramatically Hayden, Nature 2014

24. Next-generation sequencing: Metzker, Nat Rev Gen 2010