bio lab pcr review

What does PCR amplification require to amplify? (4)

DNA template, PCR primers, DNA polymerase, dNTPs

Role of DNA Template in PCR

comes from organism of interest, in lab used DNA prep last lab 

Role of PCR Primers in PCR

forward and reverse primers select region that is amplified

Role of DNA polymerase in PCR 

enzyme that connects dNTPs into a strand of DNA (polymerization)

role of dNTPs in PCR amplification

building blocks for DNA

PCR termocycling steps (3)

denaturing, primer annealing, extension

Dentauring step in PCR

hydrogens that hold double stranded DNA together are broken, causing them to separate. Heating step.  

Primer Annealing in PCR

Primers bind to accessible unpaired bases in the separated DNA template strands (cooling step)

Extension in PCR

one of the primers bind DNA polymerase adds deoxynucleotides to the 3’ end of the primers making complete copies of each template strand. 

DNA Barcoding method

Species can be uniquely identified using a short section of DNA from specific gene or genes.

Which portion was amplified for DNA barcoding this lab?

A portion of the mitochondrial DNA control sequence for sockeye and atlantic salmon. 

why was this region chosen for PCR (3 reasons)?

1. a universal forward and reverse PCR primer will anneal and amplify DNA from any species in the kingdom. 

2. variable region different enough to tell species apart but similar enough to tell species are the same 

3. there are many identical copies of the region in a organisms genome providing many targets for PCR

how many copies of DNA are made after 30 PCR cycles?

1 billion

why is taq polymerase used?

heat stable and can tolerate denaturing temperatures

what do PCR primers add to sequence?

3’-OH

target region definition

area in genome interested in amplifying 

flanking region

area to the left and right of target region- where primers attach

what target region are used for amplifying the DNA?

cytochrome oxidase I (COI)

example of DNA polymerase in this lab

taq polymerase