Amplifying dna
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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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Amplifying DNA

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Amplifying dna

Amplifying DNA


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 PolymeraseSpecial DNA polymerase enzyme that is heat stable

  • 3. Deoxynucleotides (dNTPs) Building blocks of DNA

  • 4. PrimersSmall pieces of DNA that match the flanks of your gene or DNA region of interest

  • 5. Buffer and waterEnvironment 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)


Amplifying dna

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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