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Restriction Analysis of Plasmid DNA. RESTRICTION ENZYMES 2.

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Presentation on theme: "Restriction Analysis of Plasmid DNA. RESTRICTION ENZYMES 2."— Presentation transcript:

1 Restriction Analysis of Plasmid DNA

2 RESTRICTION ENZYMES 2

3 Each restriction enzyme cuts DNA wherever its recognition site appears. Each restriction enzyme recognizes a particular sequence of nucleotides, called its restriction site. Many recognition sites are palindromes. 3 BamHI …NNN GGATCC NNN……NNN G GATCC NNN… …NNN CCTAGG NNN……NNN CCTAG G NNN… HindIII …NNN AAGCTT NNN……NNN A AGCTT NNN… …NNN TTCGAA NNN……NNN TTCGA A NNN…

4 Restriction EnzymeModification Enzyme If bacteria produce restriction enzymes, why doesn’t their own DNA get cut up? Cuts DNA anywhere the recognition sequence occurs. Will not cut if DNA is methylated (has –CH 3 groups added) Methylases act at same recognition site as restriction enzyme Protects bacteria’s own DNA from its own restriction enzymes Foreign DNA is not protected 4 A “restriction- modification” system.

5 Restriction Enzymes Create Either “Blunt” Ends or “Sticky” Ends with Overhangs 5 BamHI 5’…NNN GGATCC NNN…3’5’…NNN G GATCC NNN…3’ 3’…NNN CCTAGG NNN…5’3’…NNN CCTAG G NNN…5’ 5’ overhang SmaI 5’…NNN CCCGGG NNN…3’5’…NNN CCC GGG NNN…3’ 3’…NNN GGGCCC NNN…5’3’…NNN GGG CCC NNN…5’ blunt end PstI 5’…NNN CTGCAG NNN…3’5’…NNN GCTGCA G NNN…3’ 3’…NNN GACGTC NNN…5’3’…NNN G ACGTC NNN…5’ 3’ overhang

6 Ends Produced By The Same Enzyme Can Be Rejoined By Ligation 6 EcoRI 5’…AAA GAATTC AAA…3’5’…AAA G 3’…TTT CTTAAG TTT…5’3’…TTT CTTAA EcoRI 5’…CCC GAATTC CCC…3’ AATTC CCC…3’ 3’…GGG CTTAAG GGG…5’ G GGG…5’ After Ligation with DNA Ligase 5’…AAA GAATTC CCC…3’ 3’…TTT CTTAAG GGG…5’ Base Pairs Re-Form

7 Cohesive Ends Produced By Different Enzymes Can Be Rejoined By Ligation 7 BamHI 5’…AAA GGATCC AAA…3’5’…AAA G 3’…TTT CCTAGG TTT…5’3’…TTT CCTAG BglII 5’…CCC AGATCT CCC…3’ GATCT CCC…3’ 3’…GGG TCTAGA GGG…5’ A GGG…5’ After Ligation with DNA Ligase 5’…AAA GGATCT CCC…3’ 3’…TTT CCTAGA GGG…5’ All blunt ends are cohesive. These sticky ends share the same overhang sequence

8 Average Frequency of Recognition Sites Along a DNA Molecule 4-nucleotide recognition sequence HaeIIIGGCC occurs once every 4 4 = 256 bp 6-nucleotide recognition sequence EcoRIGAATTC occurs once every 4 6 = 4,096bp 8-nucleotide recognition sequence NotIGCGGCCGC occurs once every 4 8 = 65,536bp 8

9 Using Restriction Enzymes to Genetically Engineer Recombinant DNAs 9 Cut plasmid DNA Cut insert DNA Choose enzymes that yield cohesive ends Ligate with DNA Ligase Non- Recombinant Recombinant

10 Restriction Fragment Length Polymorphism (RFLP) Analysis MstII recognizes the sequence CCTNAGG (“N” can be any nucleotide). The mutation that causes sickle- cell anemia eliminates a MstII recognition site. Normal …ProGluGlu… …CCTTAGG……………………………………………CCTGAGGAG………CCTTAGG… Mutant …ProValGlu… …CCTTAGG……………………………………………CCTGTGGAG………CCTTAGG… 10 1.2kb fragment0.2kb fragment 1.4kb fragment

11 SESSION 1/DAY 1: RESTRICTION DIGEST REACTIONS *MOLECULAR BIOLOGY FINAL *Begin Here After Biotech PP and Electrophoresis activities 11

12 Each restriction enzyme cuts DNA wherever its recognition site appears. Each restriction enzyme recognizes a particular sequence of nucleotides, called its restriction site. Many recognition sites are palindromes. 12 BamHI …NNN GGATCC NNN……NNN G GATCC NNN… …NNN CCTAGG NNN……NNN CCTAG G NNN… HindIII …NNN AAGCTT NNN……NNN A AGCTT NNN… …NNN TTCGAA NNN……NNN TTCGA A NNN…

13 13 Before We Begin: This is a restriction Enzyme Map The circles below represent bacterial plasmids (loops of DNA found inside prokaryotes). The orange section is a gene for the resistance of an antibiotic (either ampicillin or kanamycin)

14 A restriction map identifies where restriction sites appear along the DNA plasmid 14 HindIII cuts here BamHI cuts here What will be different between the DNA fragments produced by cutting pAMP vs. pKAN with BamHI & HindIII?

15 The restriction enzymes and the location where they will cut on this particular plasmid is indicated on the map (i.e. 1120 means BamH1 will cut at the 1,120 th base pair starting at “12:00”) 15

16 16 Cutting with Restriction Enzymes: If you are cutting with BamH1 For Example: The number 1120 represents the # of base pairs where BamH1 will cut from12:00 noon. So… If you are also cutting with HinDIII and you want to know the size of the piece you are cutting out take 1904bp – 1120bp = 784bp (size of what will be cut out). 4539bp - 784bp = 3755bp is size of remaining plasmid after piece cut out. 784 bp 3755 bp

17 DNAs can be distinguished from each other by restriction mapping. 17 1904 – 1120 = 784 784 bp 3755 bp 2332 bp 1875 bp 4539 – 784 = 3755

18 The Sample you will get for this lab will be EITHER plasmid DNA pAMP or pKAN. 18 Name of plasmid

19 pAMP; Let’s get acquainted, shall we? 4539 base pairs a single replication origin a gene (ampr)conferring resistance to the antibiotic ampicillin (a relative of penicillin) a single occurrence of the sequence 5' GGATCC 3' 3' CCTAGG 5' that is cut by the restriction enzyme BamHI a single occurrence of the sequence 5' AAGCTT 3' 3' TTCGAA 5' that is cut by the restriction enzyme HindIII Treatment of pAMP with a mixture of BamHI and HindIII produces: a fragment of 3755 base pairs carrying both the ampr gene and the replication origin a fragment of 784 base pairs both fragments have sticky ends 19

20 pKAN 4207 base pairs a single replication origin a gene (kanr) conferring resistance to the antibiotic kanamycin. a single site cut by BamHI a single site cut by HindIII Treatment of pKAN with a mixture of BamHI and HindIII produces: a fragment of 2332 base pairs a fragment of 1875 base pairs with the kanr gene (but no origin of replication) both fragments have sticky ends 20

21 21

22 Review: 1.Define Plasmid. 2.Where are plasmids found naturally? 3.Why are they beneficial in genetic engineering? 4.You will be getting one of two plasmids today: pAMP or pKAN. What does the AMP signify? The KAN? 22

23 5. Your plasmid (millions of them) will be given to you in a microtube and then you will add both Hind111 and BamH1. What are they and what will they do? 6. Will the restriction enzymes cut the plasmids differently depending on which you have? Why? 7. How many plasmid (DNA) fragments will you end up with after the restriction digest? 8. Based on yesterdays notes, in general will the fragment sizes be similar from the pAMP digest? The pKAN digest? 23

24 If these are run on a gel- pKAN will have two bands closer to top (larger) and pAMP will have one far band (smaller) and one larger band (bigger) 24

25 LAB TIME!- Using Restriction Enzymes!!! Glove Up! Put on a pair of lab gloves S, M, L, XL available Most hands will fit in M or L gloves. Try those sizes first unless you have particularly small or large hands. Made of nitrile (no latex = no allergies) 25

26 Label a Restriction Digest Tube From the jar with the white screw cap, remove one 1.5ml microtube. With a lab marker, label the lid of the microtube with your period number and the first initials of each team member- (save room to record a number) P1 TDH 26 LID

27 This is our Goal which we will complete one step at a time: Prepare the Restriction Digest Reactions Reaction ComponentVolume to Add Your Plasmid DNA Sample (0.1µg/µl)5µl H2OH2O9µl 5X Restriction Buffer4µl BamHI + HindIII Restriction Enzyme mix2µl Total Volume20µl 27

28 Add Plasmid DNA Your team was given a sample of either pAMP or pKAN plasmid DNA in a tube labeled “DNA” and a number. BE SURE TO RECORD THIS NUMBER on your restriction digest tube lid! From this tube, use your micropipette to measure 5μl of plasmid DNA and transfer it to your Restriction Digest tube. At 0.1μg/μl, this 5μl contains 0.5μg or 500ng of DNA. P1 TDH #3 DNA 1…12 5μl 28

29 Add Water From the tube labeled H 2 O, measure 9μl of water and transfer it to your Restriction Digest tube. P1 TDH #3 H2OH2O9μl 29

30 Add Restriction Reaction Buffer/Loading Dye Enzymes require a chemical environment of the right pH and concentration of ions. The 5X restriction buffer is a concentrated mix that provides the environment needed for the restriction enzymes to work properly. From the tube labeled 5X RE Buffer, measure 4μl of 5x Restriction Digest Buffer and transfer it to your Restriction Digest tube. P1 TDH #3 5X RE Buffer 4μl 30

31 Add Restriction Enzymes You will cut your plasmid DNA with two restriction enzymes: BamHI and HindIII. From the tube labeled BamHI + HindIII measure 2μl of the BamHI and HindIII mix and transfer it to your Restriction Digest tube. P1 TDH #3 BamHI + HindIII 2μl 31

32 Incubate the Restriction Digest Reaction Close the cap on your Restriction Digest tube and place it in the heating block set at 37°C. The restriction enzymes work best at 37°C. The reactions will incubate for one hour, then be stored in a freezer until you examine them using gel electrophoresis. 32

33 SESSION 2/ DAY 2: GEL ELECTROPHORESIS 33

34 Prepare Your Samples for Loading Do not have to add (was added to the buffer) Add 4µl of the 6X Loading Dye to your restriction digest sample. If your liquids are sticking separately to the side of the tube, flick the tube with your finger and tap the bottom gently on your lab bench, or spin briefly in microcentrifuge to collect entire sample at bottom of tube. 34

35 Load Your Sample On The FlashGel When called, bring to the FlashGel: Your DNA sample Micropipette with tip Load 6μl of your sample into a well. 35

36 Write your team initials or team number below the well into which you loaded your sample. Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 Lane 9 Lane 10 Lane 11 Lane 12 Lane 13 1kb ladder Period ____ 36

37 Run the Gel A power supply provides current to the electrodes and through the buffer and gel. The progress of migration through the gel is monitored with tracking dyes that are visible without the transilluminator. 1.2% Flash Gel 200 V 8 minutes 37

38 ANALYSIS OF GEL RESULTS 38

39 Restriction Mapping Can Be Used To Identify Unknown DNAs 39 784 bp 3755 bp2332 bp 1875 bp

40 Period #1 1.2% 200V 8min Restriction Fragment Sizes pAMP: 3755, 784 pKAN: 2332 1875 12345678910111213 Promega BenchTop 1kb Ladder 40

41 What Questions Do YOU Have? 41

42 Plotting a Standard Curve for the DNA Markers Plot on Semi-Log Paper X-axis = distance migrated Y- axis = DNA length in base pairs (bp) 42


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