Restric(on enzymes IMBB 2013

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1 Restric(on enzymes IMBB 2013

2 This presenta(on was adapted from h6p://ppge.ucdavis.edu/ h6p://web.fuhsd.org/pamela_chow/apbio/unit %20resources/unit_2_2012.html

3 Restric(on Enzymes: Molecular Scissors Restric(on enzymes (endonucleases) cut DNA at specific sequences What kinds of bonds are broken when restric(on enzymes cut? Covalent bonds Hydrogen bonds (between strands) are also broken as a result of the strands coming apart Hydrogen bond Covalent bonds Image taken from h6p://

4 Restric(on Enzymes Also called restric(on endonucleases Naturally found in different types of bacteria and archaea Bacteria use restric(on enzymes to protect themselves from foreign DNA (provide a defense mechanism against invading viruses) Bacteria have mechanisms to protect themselves from the ac(ons of their own restric(on enzymes Have been isolated and sold for use in lab work: 3,000 enzymes have been iden(fied, more than 200 have unique proper(es, and at least 600 are available commercially. A vital tool in molecular cloning and gene(c analysis

5 Restric(on Enzymes Named for bacterial genus, species, strain, and type Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1

6 Restric(on enzyme sites Restric(on enzymes recognize a specific sequence of nucleo(des and produce a double- stranded cut in the DNA. The recogni(on sequences usually vary between 4 and 8 nucleo(des Many of the recogni(on sites have symmetry (palindromic) Do geese see God? EcoRI site: 5 -GAATTC-3 3 -CTTAAG-5

7 Examples of Restric(on Enzymes Enzyme Organism source Recognized Sequence EcoRI Escherichia coli 5' GAATTC 3 3' CTTAAG 5 TaqI Thermus aqua=cus 5' TCGA 3 3' AGCT 5 HindIII Haemophilus influenzae 5'AAGCTT 3 3'TTCGAA 5 BamHI Bacillus amyloliquefaciens 5' GGATCC 3 3' CCTAGG 5 AluI Arthrobacter luteus 5' AGCT 3 3' TCGA 5

8 Restric(on Endonucleases Enzymes recognize specific 4-8 bp sequences Some enzymes cut in a staggered fashion - > s(cky ends EcoRI 5 GAATTC 3 3 CTTAAG 5 " Some enzymes cut in a direct fashion > blunt ends PvuII 5 CAGCTG 3 3 GTCGAC 5 "

9 Credit: of U. S. Department of Energy

10 RE site frequencies The frequency of restric(on enzyme recogni(on depends on the number of nucleo(des used in the site recogni(on. Each posi(on of the recogni(on site has four possible bases and therefore the probability of finding any one base is 1/4. If the length of the recogni(on site is 4 then the probability of finding that site is 1/4 * 1/4 * 1/4 * 1/4 = 1/256. For a site with six bases in the recogni(on site the probability would be (1/4) 6 = 1/4096. Therefore we would expect to see cuts in a completely random sequence, on average, every 4096 bases for a six base recogni(on enzyme.

11 Uses of restric(on enzymes: Recombinant DNA Recombinant DNA is constructed using restric(on enzymes Important: In order to join 2 pieces of DNA together they have to be cut by the same restric(on enzyme Why? Otherwise, the s(cky ends won t match DNA can t bind together

12 Recombinant Plasmids Step 1: Gene of interest and plasmid are cut with the same restric(on enzyme Step 2: mix together Step 3: Add DNA Ligase to seal DNA back together

13 Puong It All Together Create recombinant plasmid Transform plasmid into E. coli

14 Uses of restric(on enzymes: Restric(on Analysis Using restric(on enzymes to find out informa(on about a piece of DNA We can use restric(on enzymes to find out The size of a plasmid, or the size of an insert in a plasmid If there are any restric(on sites for a par(cular enzyme on a piece of DNA How many restric(on sites for a par(cular enzyme Where the restric(on sites are located

15 Restric(on Fragment Length Polymorphisms (RFLP) In molecular biology, restricaon fragment length polymorphism, or RFLP (pronounced rif- lip ), is a technique that exploits varia(ons in homologous DNA sequences. PCR + Restric(on diges(on is used to discover and detect sequence variants of par(cular genes based on variant RE sites (PCR- RFLP) Essen(ally, PCR- RFLP is a tool for genotyping What other methods of genotyping do you know? Are they based on PCR?

16 PCR- RFLP 1 2

17 PCR- RFLP at the workshop

18 PCR- RFLP at the workshop With your tutor, decide on the best restric(on enzymes to try on your PCR products (there are six 4- base cu6ers to choose from) Why are we using enzymes with 4- base recogni(on sequences? Run diges(on products on 2% gel (also run undigested controls). Are there any RFLPs between samples of your group? Presence of RFLPs will indicate differences in the DNA sequences We will confirm the presence of RE sites & RFLPs during the bioinforma(cs analysis of your sequences next week

19 Thank you