amplifying dna. the power of pcr http:// view the animation at
TRANSCRIPT
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Amplifying DNA
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The Power of PCR
http://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553
View the animation at http://www.dnalc.org/resources/animations/pcr.html
(may require Flash player or Shockwave player)
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PCR Ingredients• 1. DNA “template” Your purified DNA sample
• 2. DNA Polymerase Special DNA polymerase enzyme that is heat stable
• 3. Deoxynucleotides (dNTPs) Building blocks of DNA
• 4. Primers Small pieces of DNA that match the flanks of your
gene or DNA region of interest
• 5. Buffer and water Environment necessary for DNA Polymerase to work; mimics conditions in nucleus
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Agarose Gel ElectrophoresisMolecular Weight
Standard (DNA of Known Sizes)
1 2 3 4 5 6
Samples of DNA
2000 bp
1000 bp750 bp
Lanes:
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Agarose Gel Electrophoresis
• 1. Prepare agarose gel
• 2. Prepare your sample
• 3. Load your sample on the gel
• 4. Run gel
• 5. Stain & view gel
http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate.html
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Genetic Researchers Developed Primers for Barcoding
Pool COI-2: mammals, and insects
Pool COI-3: fish
Ivanova et al. 2007. Universal primer cocktails for fish barcoding. Mol Ecol Notes.
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Fish DNAbarcode Primers
Primer mix: 2 forward, 2 reverse•5’- TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC-3’•5’- TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3’ •5’- CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA-3’•5’- CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA-3’
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Bioinformatics
• The type of computer sciences that aids biological researchers.
• Using informatics to decipher and elucidate the information entailed in biological molecules, structures, organisms and populations.
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“Electronic PCR”
• Searching databases for sequences• Using primer sequences as search terms• No amplification• Common database: NCBI’s GenBank
– National Center for Biotechnology Information
• Common search tool: BLAST– Basic Local Alignment Search Tool
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Exercise A
• Identify the targets of our barcoding primers (Day 1, Tuesday)
• Determine length of amplicon DNA (Day 2, Wednesday)
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SNPs
• Polymorphism: A genetic locus that exists in different forms• Pointmutation: A change in a single nucleotide (alteration,
deletion or insertion)• SNP: Single nucleotide polymorphism
– A pointmutation that occurs in at least 0.5% of the population
• Haplotype: A bunch of SNPs that are connected in one strand of DNA– SNPs that do not separate by crossover form a “Haplotype”
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Exercise B
• Find differences in DNA (SNPs)• Extract haplotypes• Construct haplotype phylogenetic tree
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Exercises C & D
• Conduct Exercise B electronically• Compare and contrast results from C & D with
those from B
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DNA Sequencing
http://www.scq.ubc.ca/genome-projects-uncovering-the-blueprints-of-biology/
• 1. DNA “template”Your PCR fragment, purified
• 2. Taq PolymeraseHeat-stable DNA polymerase
• 3. Deoxynucleotides (dNTPs) and DideoxynucleotidesBuilding blocks of DNA; regular and altered
• 4. PrimersSpecific for your gene of interest
• 5. Buffer and water
View the animation at
http://www.dnalc.org/resources/animations/cycseq.html
(may require Flash player or Shockwave player)
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Homework
• Work through Study questions• Register for your own account on DNA Subway (Google it)• Review Bioinformatics readings (Binder)• Review all 3 barcoding-related papers (Pre-readings)• Bonus: Work through the HHMI Seashell Phylogeny exercise
using DNA– download Word doc from
www.hhmi.org/biointeractive/activities/shells/Shell-DNA-0-6.docx).– If you don’t wish to install the software indicated in the document you
could use BioServers/Sequence Server or, alternatively, the Blue Line in DNA Subway to conduct alignments and generate phylogenetic trees.
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