Restriction Analysis of Plasmid DNA Restriction Analysis Pitt Kit 4/14/2017 Restriction Analysis of Plasmid DNA

Restriction Enzymes

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. BamHI …NNNGGATCCNNN… …NNNG GATCCNNN… …NNNCCTAGGNNN… …NNNCCTAG GNNN… HindIII …NNNAAGCTTNNN… …NNNA AGCTTNNN… …NNNTTCGAANNN… …NNNTTCGA ANNN…

A “restriction-modification” system. If bacteria produce restriction enzymes, why doesn’t their own DNA get cut up? Restriction Enzyme Modification Enzyme Cuts 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 protected A “restriction-modification” system.

Restriction Enzymes Create Either “Blunt” Ends or “Sticky” Ends with Overhangs BamHI 5’…NNNGGATCCNNN…3’ 5’…NNNG GATCCNNN…3’ 3’…NNNCCTAGGNNN…5’ 3’…NNNCCTAG GNNN…5’ blunt end SmaI 5’…NNNCCCGGGNNN…3’ 5’…NNNCCC GGGNNN…3’ 3’…NNNGGGCCCNNN…5’ 3’…NNNGGG CCCNNN…5’ 3’ overhang PstI 5’…NNNCTGCAGNNN…3’ 5’…NNNGCTGCA GNNN…3’ 3’…NNNGACGTCNNN…5’ 3’…NNNG ACGTCNNN…5’

Ends Produced By The Same Enzyme Can Be Rejoined By Ligation EcoRI 5’…CCCGAATTCCCC…3’ AATTCCCC…3’ 3’…GGGCTTAAGGGG…5’ GGGG…5’ Base Pairs Re-Form EcoRI 5’…AAAGAATTCAAA…3’ 5’…AAAG 3’…TTTCTTAAGTTT…5’ 3’…TTTCTTAA After Ligation with DNA Ligase 5’…AAAGAATTCCCC…3’ 3’…TTTCTTAAGGGG…5’

All blunt ends are cohesive. Cohesive Ends Produced By Different Enzymes Can Be Rejoined By Ligation BglII 5’…CCCAGATCTCCC…3’ GATCTCCC…3’ 3’…GGGTCTAGAGGG…5’ AGGG…5’ These sticky ends share the same overhang sequence BamHI 5’…AAAGGATCCAAA…3’ 5’…AAAG 3’…TTTCCTAGGTTT…5’ 3’…TTTCCTAG After Ligation with DNA Ligase 5’…AAAGGATCTCCC…3’ 3’…TTTCCTAGAGGG…5’ All blunt ends are cohesive.

Average Frequency of Recognition Sites Along a DNA Molecule 4-nucleotide recognition sequence HaeIII GGCC occurs once every 44 = 256 bp 6-nucleotide recognition sequence EcoRI GAATTC occurs once every 46 = 4,096bp 8-nucleotide recognition sequence NotI GCGGCCGC occurs once every 48 = 65,536bp

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

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… 1.2kb fragment 0.2kb fragment 1.4kb fragment

Session 1/day 1: Restriction Digest Reactions *Molecular Biology Final *Begin Here After Biotech PP and Electrophoresis activities

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. BamHI …NNNGGATCCNNN… …NNNG GATCCNNN… …NNNCCTAGGNNN… …NNNCCTAG GNNN… HindIII …NNNAAGCTTNNN… …NNNA AGCTTNNN… …NNNTTCGAANNN… …NNNTTCGA ANNN…

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)

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

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 1,120th base pair starting at “12:00”)

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. 3755 bp 784 bp

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

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

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

pKAN •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 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

Review: 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?

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?

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)

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)

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 LID

Restriction Analysis Pitt Kit 4/14/2017 This is our Goal which we will complete one step at a time: Prepare the Restriction Digest Reactions Reaction Component Volume to Add Your Plasmid DNA Sample (0.1µg/µl) 5µl H2O 9µl 5X Restriction Buffer 4µl BamHI + HindIII Restriction Enzyme mix 2µl Total Volume 20µl

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. DNA 1…12 5μl P1 TDH #3

Add Water From the tube labeled H2O, measure 9μl of water and transfer it to your Restriction Digest tube. H2O 9μl P1 TDH #3

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. 5X RE Buffer 4μl P1 TDH #3

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. BamHI + HindIII 2μl P1 TDH #3

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.

Session 2/ Day 2: Gel Electrophoresis

Prepare Your Samples for Loading Restriction Analysis Pitt Kit 4/14/2017 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.

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.

Restriction Analysis Pitt Kit 4/14/2017 Write your team initials or team number below the well into which you loaded your sample. Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 1kb ladder Period ____ 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.

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

Analysis of Gel Results

Restriction Mapping Can Be Used To Identify Unknown DNAs 3755 bp 2332 bp 784 bp 1875 bp

Restriction Analysis Pitt Kit 4/14/2017 1 2 3 4 5 6 7 8 9 10 11 12 13 Restriction Fragment Sizes pAMP: 3755, 784 pKAN: 2332 1875 Promega BenchTop 1kb Ladder Period #1 Teacher: 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

What Questions Do YOU Have?

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)