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Amplifying DNA. The Power of PCR. View the animation at http://www.dnalc.org/resources/animations/pcr.html (may require Flash player or Shockwave player). http:// www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553. PCR Ingredients.

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the power of pcr
The Power of PCR

View the animation at http://www.dnalc.org/resources/animations/pcr.html

(may require Flash player or Shockwave player)

http://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553

pcr ingredients
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
agarose gel electrophoresis
Agarose Gel Electrophoresis

Molecular Weight Standard

(DNA of Known Sizes)

Samples of DNA

Lanes:

1 2 3 4 5 6

2000 bp

1000 bp

750 bp

agarose gel electrophoresis1
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

genetic researchers developed primers for barcoding
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.

fish dnabarcode primers
Fish DNAbarcode Primers

Primer mix: 2 forward, 2 reverse

  • 5’- TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC-3’
  • 5’- TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3’
  • 5’- CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA-3’
  • 5’- CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA-3’
bioinformatics
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.
electronic pcr
“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
exercise a
Exercise A
  • Identify the targets of our barcoding primers (Day 1, Tuesday)
  • Determine length of amplicon DNA (Day 2, Wednesday)
slide11
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”
exercise b
Exercise B
  • Find differences in DNA (SNPs)
  • Extract haplotypes
  • Construct haplotype phylogenetic tree
exercises c d
Exercises C & D
  • Conduct Exercise B electronically
  • Compare and contrast results from C & D with those from B
dna sequencing

1. DNA “template”

  • Your PCR fragment, purified
  • 2. Taq Polymerase
  • Heat-stable DNA polymerase
  • 3. Deoxynucleotides (dNTPs) and Dideoxynucleotides
  • Building blocks of DNA; regular and altered
  • 4. Primers
  • Specific for your gene of interest
  • 5. Buffer and water

DNA Sequencing

View the animation at

http://www.dnalc.org/resources/animations/cycseq.html

(may require Flash player or Shockwave player)

http://www.scq.ubc.ca/genome-projects-uncovering-the-blueprints-of-biology/

homework
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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