More Basic Biotechnology Tools
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Transcript of More Basic Biotechnology Tools
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More Basic Biotechnology Tools
Sorting & Copying DNA
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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 gel
• agarose• made from algae
– gel electrophoresis
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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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Gel Electrophoresis
longer fragments
shorter fragments
powersource
completed gel
gel
DNA &restriction enzyme
wells
-
+
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Running a gel
1 2
cut DNA with restriction enzymes
fragments of DNAseparate out based
on size
3
• Stain DNA– ethidium bromide– We used Syber
safe stain– binds to DNA– fluoresces under
UV light
But…The human genome is toobig once you cut it with
restriction enzymes!
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Southern Blotting (Hybridization)
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Uses: Evolutionary relationships• Comparing DNA samples from
different organisms to measure evolutionary relationships
–
+
DNA
↓
1 32 4 5 1 2 3 4 5
turtle snake rat squirrel fruitfly
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Uses: Medical diagnostic
What would theGel look like for A heterozygote?
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Uses: Forensics
• Comparing DNA sample from crime scene with suspects & victim
–
+
S1
DNA
↓
S2 S3 V
suspects crime scene sample
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DNA fingerprints
• Comparing blood samples on defendant’s clothing to determine if it belongs to victim– DNA fingerprinting– comparing DNA
banding pattern between different individuals
– unique patterns
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Differences at the DNA level• 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
GCTTGTAACGGCCTGCTTGTAACGGCCTCATCATCATCATCATCATTCGCCGGCCTACGCTTTCGCCGGCCTACGCTTCGAACATTGCCGGACGAACATTGCCGGAGTAGTAGTAGTAGTAGTAAGCGGCCGGATGCGAAAGCGGCCGGATGCGAA
GCTTGTAACGGGCTTGTAACGGCATCATCATCATCATCATCATCATCATCATCATCATCCGGCCTACGCTTCCGGCCTACGCTTCGAACATTGCCCGAACATTGCCGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGGCCGGATGCGAAGGCCGGATGCGAA
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Allele Allele 11 GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
repeats
DNA patterns for DNA fingerprints
cut sitescut sites
GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTTCGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA
1 2 3
DNA →– +allele 1
Cut the DNACut the DNA
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Allele Allele 11 GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
Differences between people
cut sitescut sites
DNA →– +allele 1
Allele 2: more repeatsAllele 2: more repeatsGCTTGTAACGGCCTCATCATCATCATCATCATCCGGCCTACGCTTCGAACATTGCCGGAGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA
DNA fingerprintDNA fingerprint
allele 2
1 2 3
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RFLPs• Restriction Fragment Length Polymorphism
– differences in DNA between individuals
change in DNA sequence affects restriction enzyme “cut” site
creates different fragment sizes & different band pattern
Alec Jeffries 1984
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Polymorphisms in populations• Differences between individuals at
the DNA level– many differences accumulate in “junk” DNA
-
-
-
+
+
+
restriction enzymecutting sites
single base-pairchange
sequenceduplication
2 bands
1 band
2 different bands
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RFLP / electrophoresis use in forensics• 1st case successfully using DNA evidence
– 1987 rape case convicting Tommie Lee Andrews
“standard”
“standard”
“standard”
“standard”
semen sample from rapist
semen sample from rapist
blood sample from suspect
blood sample from suspect
How can you compare DNA from
blood & from semen?RBC?
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Electrophoresis use in forensics
• Evidence from murder trial– Do you think suspect is guilty?
“standard”
blood sample 3 from crime scene
“standard”
blood sample 1 from crime scene
blood sample 2 from crime scene
blood sample from victim 2
blood sample from victim 1
blood sample from suspect OJ Simpson
N Brown
R Goldman
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Uses: Paternity
• Who’s the father?
+
DNA
↓
childMom F1 F2–
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Uses:Gene Therapy
Not assimple
as it looks
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Pharm Plants and Animals
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Environmental Cleanup
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GMO’sQuickTime™ and a
decompressorare needed to see this picture.
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Making lots of copies of DNA
But it would be so much easier if we didn’t have to use bacteria every
time…
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Copy DNA without plasmids? PCR!
• Polymerase Chain Reaction– method for
making many, many copies of a specific segment of DNA
– only need 1 cell of DNA to start
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PCR process• It’s copying DNA in a test tube!• What do you need?
ThermocyclerThermocycler
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PCR primers• The primers are critical!
– need to know a bit of sequence to make proper primers
– primers can bracket target sequence• start with long piece of
DNA & copy a specified shorter segment
• primers define section of DNA to be cloned
20-30 cycles3 steps/cycle30 sec/step
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Design primers…• Design four nucleotide long primers for the
following DNA sequence:• 5- CCTTGACGATCGTTACCG -3• 3- GGAACTGGTAGCAATGGC -5
Forward primers: 5- CCTT -3Reverse primers: 5 -CGGT -3
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PCR process• What do you need to do?
– in tube: DNA, DNA polymerase enzyme, primer, nucleotides
– denature DNA: heat (90°C) DNA to separate strands
– anneal DNA: cool to (54°C) hybridize with primers
– etension: heat to (70 °C) to allow DNA pol to extend.
What does 90°Cdo to our
DNA polymerase?
DNA PCR Animation
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The polymerase problem• Heat DNA to denature (unwind) it
– 90°C destroys DNA polymerase– have to add new enzyme every cycle
• almost impractical!• Need enzyme that can
withstand 90°C…– Taq polymerase
• from hot springs bacteria–Thermus aquaticus
PCR20-30 cycles3 steps/cycle30 sec/step
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Kary Mullis• development of PCR technique
– a copying machine for DNA
1985 | 1993
I’m wacky
!How do scientistsIsolate specific
sequences of DNA using
Southern Blot and PCR?
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How PCR Works:
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Silly PCR Song #1
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Silly PCR Song #2
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I’m a-glow!
Got any Questions?
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Review Questions
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• 1. The segment of DNA shown in the figure below has restriction sites I and II, which create restriction fragments A, B, and C. Which of the gels producedby electrophoresis shown below would represent the separation and identity of these fragments?
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• 2. Which of the following statements is consistent with the results below? *
A. B is the child ofA and C.
B. C is the child ofA and B.
• D is the child ofB and C.
• A is the child ofB and C.
• A is the child ofC and D.
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3. What is the order of the steps involved in the Polymerase Chain Reaction?
– I. Priming– II. Heat– III.Extension
A. I, II, IIIB. II, I, III• III, I, II• II, III, I • None of the above