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
Cool PCR Learning Tools: hCp://learn.gene1cs.utah.edu/content/labs/pcr/
hCp://www.dnalc.org/resources/anima1ons/pcr.html
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
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.
Figure 4.1 Polymerase chain reac1on: first cycle. Figure 4.2 Polymerase chain reac1on: second cycle.
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.
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
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
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
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?
hCp://www.ncbi.nlm.nih.gov/nuccore/251831106
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
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/