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Gel Electrophoresis
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Restriction Enzymes Restriction Enzymes = (aka restriction endonucleases) cut DNA at specific places by recognizing and binding to specific sequences Found in bacteria Can be used to cut up DNA in a specific way and join them back together based on what the scientists
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Many uses of restriction enzymes…
Now that we can cut DNA with restriction enzymes… we can cut up DNA from different people… or different organisms… and compare it why? forensics medical diagnostics paternity evolutionary relationships and more…
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Comparing cut up DNA How do we compare DNA fragments?
separate fragments by size How do we separate DNA fragments? run it through a gelatin gel electrophoresis How does a gel work?
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“swimming through Jello”
Gel electrophoresis A method of separating DNA in a gelatin-like material using an electrical field DNA is negatively charged when it’s in an electrical field it moves toward the positive side DNA – + “swimming through Jello”
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“swimming through Jello”
Gel electrophoresis DNA moves in an electrical field… so how does that help you compare DNA fragments? size of DNA fragment affects how far it travels small pieces travel farther large pieces travel slower & lag behind DNA – + “swimming through Jello”
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DNA & restriction enzyme
Gel Electrophoresis DNA & restriction enzyme - wells longer fragments power source gel shorter fragments + completed gel
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fragments of DNA separate out based on size
Running a Gel fragments of DNA separate out based on size cut DNA with restriction enzymes 1 2 3 Stain DNA ethidium bromide binds to DNA fluoresces under UV light
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DNA fingerprint Why is each person’s DNA pattern different?
sections of “junk” DNA doesn’t code for proteins made up of repeated patterns CAT, GCC, and others each person may have different number of repeats many sites on our 23 chromosomes with different repeat patterns GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGCTT CGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA
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DNA patterns for DNA fingerprints
Allele 1 GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA repeats cut sites Cut the DNA GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA 1 2 3 – DNA + allele 1
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Differences between people
Person 1 cut sites cut sites GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA Person 2: more repeats GCTTGTAACGGCCTCATCATCATCATCATCATCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA 1 2 3 DNA fingerprint – DNA + person 1 person 2
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Uses: Evolutionary relationships
Comparing DNA samples from different organisms to measure evolutionary relationships turtle snake rat squirrel fruitfly – 1 3 2 4 5 1 2 3 4 5 DNA +
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Uses: Medical diagnostic
Comparing normal allele to disease allele chromosome with normal allele 1 chromosome with disease-causing allele 2 allele 1 allele 2 – DNA Example: test for Huntington’s disease +
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Uses: Forensics Comparing DNA sample from crime scene with suspects & victim suspects crime scene sample S1 S2 S3 V – DNA +
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Forensic DNA fingerprints
Comparing blood samples on defendant’s clothing to determine if it belongs to victim DNA fingerprinting
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RFLP / electrophoresis use in forensics
1st case successfully using DNA evidence 1987 rape case convicting Tommie Lee Andrews “standard” semen sample from rapist blood sample from suspect “standard” “standard” semen sample from rapist blood sample from suspect “standard”
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Electrophoresis and Forensics
Evidence from murder trial Do you think suspect is guilty? blood sample 1 from crime scene blood sample 2 from crime scene blood sample 3 from crime scene “standard” blood sample from suspect OJ Simpson blood sample from victim 1 N Brown blood sample from victim 2 R Goldman “standard”
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Uses: Paternity Who’s the father? – Mom F1 F2 child DNA +
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Dye Lab
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Practice with Dyes Gel electrophoresis is typically used to separate DNA or proteins based on charge but it is a technique that can be used to separate out any type of charged molecule. Dyes common in food products may have different charges and are different sizes This will be a good way to practice on your micropipette technique
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Tips for Electrophoresis
Buffer needs to be used to provide ions and appropriate pH to maintain your samples Be careful not to poke thru the gel A weight has been added to the dye to that it will sink to the bottom of the well Avoid air bubbles because they will cause the sample to float into the buffer
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Which plant is most closely related to Botanus curus?
Biodiversity Lab Which plant is most closely related to Botanus curus?
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Botanus curus Sequences
CAC GTG GAC TGA GGA CTC CTC DNA GUG CAC CUG ACU CCU GAG GAG RNA Val His Leu Thr Pro Glu Glu AA
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Species X Sequences CAC GTG GAC TGA GGA CTC CTC
BC CAC GTG GAC AGA GGA CAC CTC DNA GUG CAC CUG UCU CCU GUG GAG RNA Val His Leu Ser Pro Val Glu AA Val His Leu Thr Pro Glu Glu BC
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Species Y Sequences CAC GTG GAC TGA GGA CTC CTC
BC CAC GTG GAC AGA GGA CAC CTC DNA GUG CAC CUG UCU CCU GUG GAG RNA Val His Leu Ser Pro Val Glu AA Val His Leu Thr Pro Glu Glu BC
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Species Z Sequences CAC GTG GAC TGA GGA CTC CTC
BC CAC GTA GAC TGA GGA CTT CTC DNA GUG CAU CUG ACU CCU GAA GAG RNA Val His Leu Thr Pro Glu Glu AA Val His Leu Thr Pro Glu Glu BC
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Amino Acid Sequences Val His Leu Thr Pro Glu Glu
BC Val His Leu Thr Pro Glu Glu X Val His Leu Ser Pro Val Glu Y Val His Leu Ser Pro Val Glu Val His Leu Thr Pro Glu Glu Z
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