Bio382-F14 Lab #3 PCR NEW - MDCUNE · Bio382-F14 Lab #3 PCR NEW.pptx Author: Keller, Lani C. Prof....
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Polymerase Chain Reac1on
Bio382-‐F14 Lab #3
Dr. Lani Keller
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Cool PCR Learning Tools: hCp://learn.gene1cs.utah.edu/content/labs/pcr/
hCp://www.dnalc.org/resources/anima1ons/pcr.html
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Enzyma1c amplifica1on of a specific DNA fragment, using repeated cycles of denatura1on, primer annealing, and chain extension.
Polymerase Chain Reac1on (PCR)
hCp://www.karymullis.com/
• DNA cloning • DNA sequencing • Disease gene tes1ng • Forensics • Paternity tes1ng • Gene mutagenesis • Evolu1onary gene1cs
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PCR Replica1on Cycles
Several Repe**ve Steps: 1. 94-‐98°C denaturing “mel1ng” DNA into two single
strands. 2. 50-‐65°C annealing of primers to complementary
sequences (by hydrogen binding) on either side of the target sequence. Variable temperature.
3. 72°C extension by DNA polymerase binding and
synthesizing a cDNA strand from each primer.
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Figure 4.1 Polymerase chain reac1on: first cycle. Figure 4.2 Polymerase chain reac1on: second cycle.
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1. A thermo-‐stable DNA polymerase (Taq polymerase) Prior to this discovery, you would have to add new DNA polymerase each cycle because it would become inac1ve during the high-‐temp denatura1on step.
Two Important Innova1ons
2. Thermo-‐cyclers Before computers controlled the repe11ve temperature changes, people would literally set up three water baths and manually move the tubes.
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1. Reac1on buffer Provides suitable chemical environment.
2. Deoxynucleo1de triphosphates (dNTPs) Building blocks for DNA synthesis. Nucleo1des with triphosphates.
3. DNA template A DNA source containing target DNA to be amplified.
4. Forward and Reverse primers or “oligos” Small pieces of DNA that are complimentary to the target DNA
5. DNA polymerase A thermostable DNA polymerase (op1mum temperature ~70°C)
Five Main Components of PCR
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The Template DNA
3. Genomic RNA à cDNA • Isolate RNA, make cDNA by reverse transcrip1on
• Can look at what genes are being expressed
2. Genomic DNA • RNA contamina1on causes problems • Contains exons and introns • A lot of non-‐target DNA • Need 1ng – 1µg as template
1. Recombinant plasmid DNA • Most efficient • Need 1pg – 1ng as template
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Primer Design
Figure 4.3 Forward and reverse primers.
• 18-‐28bp of homology specific to target DNA • Op1mal annealing temps (~58°C) should be similar for both
forward and reverse primers
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1. Whole genome à NO 2. En1re genes à Not usually 3. Pieces of genes with SNPs à YES! 4. Can you have errors? à YES! 5. If you add 100-‐1mes the amount of star1ng
template DNA, do you expect to have 100-‐1mes the amplified DNA à NO
6. We amplified piece of mtDNA and will cut it with an endonuclease to examine your haplotype!
What do you actually amplify?
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hCp://www.ncbi.nlm.nih.gov/nuccore/251831106
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Order of lab today 1. Start with procedure D (restric1on digest of PCR
products). We will start diges1ons as soon as possible.
2. During 1 hour incuba1on: start on-‐line PCR module
3. Amer 1 hour incuba1on: prepare and load gels. Each person will run uncut PCR product (5 uL PCR/loading dye mixture) AND digested PCR product (30 uL digest + 5 uL loading dye )
4. While loading-‐ finish the PCR module, turn in Excel file, and take on-‐line post test
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Addi1onal PCR Resources and Anima1ons for Students
• hCp://www.dnalc.org/resources/anima1ons/pcr.html • hCp://learn.gene1cs.utah.edu/content/labs/pcr/ • hCp://www.sumanasinc.com/webcontent/anima1ons/content/pcr.html • hCp://highered.mheduca1on.com/sites/0072556781/student_view0/
chapter14/anima1on_quiz_6.html • hCp://www.lifetechnologies.com/us/en/home/life-‐science/pcr/elevate-‐
pcr-‐research/pcr-‐video-‐library/pcr-‐anima1on.html • hCp://highered.mheduca1on.com/olc/dl/120078/micro15.swf • hCp://www.promega.com/resources/mul1media/pcr/introduc1on-‐to-‐pcr/