Amplifying DNA

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)

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 ElectrophoresisMolecular Weight

Standard (DNA of Known Sizes)

1 2 3 4 5 6

Samples of DNA

2000 bp

1000 bp750 bp

Lanes:

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

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

Primer mix: 2 forward, 2 reverse•5’- TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC-3’•5’- TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3’ •5’- CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA-3’•5’- CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA-3’

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”

• 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

• Identify the targets of our barcoding primers (Day 1, Tuesday)

• Determine length of amplicon DNA (Day 2, Wednesday)

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

• Find differences in DNA (SNPs)• Extract haplotypes• Construct haplotype phylogenetic tree

Exercises C & D

• Conduct Exercise B electronically• Compare and contrast results from C & D with

those from B

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)

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.