3Each 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.BamHI…NNNGGATCCNNN… …NNNG GATCCNNN……NNNCCTAGGNNN… …NNNCCTAG GNNN…HindIII…NNNAAGCTTNNN… …NNNA AGCTTNNN……NNNTTCGAANNN… …NNNTTCGA ANNN…
4A “restriction-modification” system. If bacteria produce restriction enzymes, why doesn’t their own DNA get cut up?Restriction EnzymeModification EnzymeCuts DNA anywhere the recognition sequence occurs. Will not cut if DNA is methylated (has –CH3 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 protectedA “restriction-modification” system.
5Restriction Enzymes Create Either “Blunt” Ends or “Sticky” Ends with Overhangs BamHI5’…NNNGGATCCNNN…3’ 5’…NNNG GATCCNNN…3’3’…NNNCCTAGGNNN…5’ 3’…NNNCCTAG GNNN…5’blunt endSmaI5’…NNNCCCGGGNNN…3’ 5’…NNNCCC GGGNNN…3’3’…NNNGGGCCCNNN…5’ 3’…NNNGGG CCCNNN…5’3’ overhangPstI5’…NNNCTGCAGNNN…3’ 5’…NNNGCTGCA GNNN…3’3’…NNNGACGTCNNN…5’ 3’…NNNG ACGTCNNN…5’
6Ends Produced By The Same Enzyme Can Be Rejoined By Ligation EcoRI5’…CCCGAATTCCCC…3’ AATTCCCC…3’3’…GGGCTTAAGGGG…5’ GGGG…5’Base Pairs Re-FormEcoRI5’…AAAGAATTCAAA…3’ 5’…AAAG3’…TTTCTTAAGTTT…5’ 3’…TTTCTTAAAfter Ligation with DNA Ligase5’…AAAGAATTCCCC…3’3’…TTTCTTAAGGGG…5’
7All blunt ends are cohesive. Cohesive Ends Produced By Different Enzymes Can Be Rejoined By LigationBglII5’…CCCAGATCTCCC…3’ GATCTCCC…3’3’…GGGTCTAGAGGG…5’ AGGG…5’These sticky ends share the same overhang sequenceBamHI5’…AAAGGATCCAAA…3’ 5’…AAAG3’…TTTCCTAGGTTT…5’ 3’…TTTCCTAGAfter Ligation with DNA Ligase5’…AAAGGATCTCCC…3’3’…TTTCCTAGAGGG…5’All blunt ends are cohesive.
8Average Frequency of Recognition Sites Along a DNA Molecule 4-nucleotide recognition sequenceHaeIII GGCCoccurs once every 44 = 256 bp6-nucleotide recognition sequenceEcoRI GAATTCoccurs once every 46 = 4,096bp8-nucleotide recognition sequenceNotI GCGGCCGCoccurs once every 48 = 65,536bp
9Using Restriction Enzymes to Genetically Engineer Recombinant DNAs Non-RecombinantChoose enzymes that yield cohesive endsCut plasmid DNARecombinantLigate with DNA LigaseCut insert DNA
10Restriction 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…1.2kb fragment0.2kb fragment1.4kb fragment
11Session 1/day 1: Restriction Digest Reactions *Molecular Biology Final *Begin Here After Biotech PP and Electrophoresis activities
12Each 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.BamHI…NNNGGATCCNNN… …NNNG GATCCNNN……NNNCCTAGGNNN… …NNNCCTAG GNNN…HindIII…NNNAAGCTTNNN… …NNNA AGCTTNNN……NNNTTCGAANNN… …NNNTTCGA ANNN…
13Before We Begin: This is a restriction Enzyme Map The circles below represent bacterial plasmids (loops of DNAfound inside prokaryotes). The orange section is a gene for the resistance of an antibiotic (either ampicillin or kanamycin)
14A restriction map identifies where restriction sites appear along the DNA plasmid BamHI cuts hereHindIII cuts hereWhat will be different between the DNA fragments produced by cutting pAMP vs. pKAN with BamHI & HindIII?
15The restriction enzymes and the location where they will cut on this particular plasmid is indicated on the map (i.e means BamH1 will cut at the 1,120th base pair starting at “12:00”)
16Cutting 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) bp - 784bp = 3755bp is size of remaining plasmid after piece cut out.3755 bp784 bp
17DNAs can be distinguished from each other by restriction mapping. 3755 bp2332 bp784 bp1875 bp1904 – 1120 = 7844539 – 784 = 3755
18The Sample you will get for this lab will be EITHER plasmid DNA pAMP or pKAN. Name of plasmid
19pAMP; 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
20pKAN •a single replication origin •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 HindIIITreatment 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
22Review: Define Plasmid. Where are plasmids found naturally? Why are they beneficial in genetic engineering?You will be getting one of two plasmids today: pAMP or pKAN. What does the AMP signify? The KAN?
235. 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?
24If 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)
25LAB TIME!- Using Restriction Enzymes!!! Glove Up! Put on a pair of lab glovesS, M, L, XL availableMost 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)
26Label 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)P1TDHLID
27Restriction Analysis Pitt Kit 4/14/2017This is our Goal which we will complete one step at a time: Prepare the Restriction Digest ReactionsReaction ComponentVolume to AddYour Plasmid DNA Sample (0.1µg/µl)5µlH2O9µl5X Restriction Buffer4µlBamHI + HindIII Restriction Enzyme mix2µlTotal Volume20µl
28Add Plasmid DNAYour 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 contains0.5μg or 500ng of DNA.DNA 1…125μlP1TDH#3
29Add WaterFrom the tube labeled H2O, measure 9μl of water and transfer it to your Restriction Digest tube.H2O9μlP1TDH#3
30Add 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.5X RE Buffer4μlP1TDH#3
31Add 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.BamHI + HindIII2μlP1TDH#3
32Incubate 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.
34Prepare Your Samples for Loading Restriction Analysis Pitt Kit4/14/2017Prepare Your Samples for LoadingDo 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.
35Load Your Sample On The FlashGel When called, bring to the FlashGel:Your DNA sampleMicropipette with tipLoad 6μl of your sample into a well.
36Restriction Analysis Pitt Kit 4/14/2017Write your team initials or team number below the well into which you loaded your sample.Lane123456789101112131kb ladderPeriod ____Teacher: Print out copies of this slide to place near each gel each period. Have students write their initials or team number in the lane into which their sample was loaded.
37Run the GelA 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
39Restriction Mapping Can Be Used To Identify Unknown DNAs 3755 bp2332 bp784 bp1875 bp
40Restriction Analysis Pitt Kit 4/14/201712345678910111213Restriction Fragment SizespAMP:3755,784pKAN:23321875Promega BenchTop 1kb LadderPeriod #1Teacher: After capturing a digital image with the Flash Gel software, return to this slide. Right-click on the gel image, choose “Change Picture…”, and browse to the gel image you just captured. That will replace this image with your new one.1.2% 200V 8min