Competitive PCR

SOURCE:
http://www.gene-quantification.com/competitive-pcr-takara.pdf
http://ajpcell.physiology.org/content/ajpcell/275/1/C68/F2.large.jpg
https://www.researchgate.net/profile/Hans_Madsen/publication/11530929/figure/fig1/AS:28181955666331
7@1444202426722/Figure-1-Competitive-PCR-method-for-residual-disease-quantification-A-competitor-
was.png

Accurate quantitation of the target is difficult with normal PCR, as the amount of amplified product
does not necessarily reflect the amount of template initially present in reaction
This is due to "plateau phase" of PCR, which is caused by:
o Deactivation of Taq DNA polymerase
o Shortage of nucleotide substrates
o Shortage of primer
o Inhibition by pyrophosphate
o Re-annealing of amplified DNAs

For a group of samples with different template concentrations, quantitation of the target template is
difficult.

Competitive PCR was developed to overcome these difficulties in quantitation.

Principle of Competitive PCR

In competitive PCR, a known amount of a DNA fragment (competitor) that can bind to the same
primers is added to the sample.

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 As both the types of templates will compete for the same set of primers, the ratio of the amounts of
the two amplified products reflects the ratio of the amounts of the target DNA and competitor.
Since the initial amount of the competitor is known, the amount of the target DNA can then be
estimated according to the Target : Competitor (T:C) ratio
When the T:C ratio = 1, the initial amount of target DNA or RNA will correspond to the amount of
competitor

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 HEPATITIS B PLANT VACCINE

Sources:
Int. J. Mol. Sci. 2013, 14, 1978-1998; doi:10.3390/ijms14011978
http://www.reuters.com/article/us-flu-vaccine-analysis-idUSKCN0HQ2YO20141001
http://nexusacademicpublishers.com/uploads/imagesfiles/Fig2_446.png
http://s32.photobucket.com/user/hpinyerd/media/tomatovaccine.jpg.html
http://www.whatisthebiotechnology.com/blog/wp-content/uploads/2013/11/112.jpg
http://www.nature.com/mi/journal/v1/n2/images/mi200722f1.jpg

A) General mechanism of plant cloning

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1

B) The potential
Plants were considered as alternative sources of vaccines, to be mainly orally administered.

"Seven to 10 years from now (2014), plants might be the dominant vaccine-production system," said Brett Giroir,
an M.D. and CEO of Texas A&M Health Science Center in Bryan.

Texas A&M has one of three U.S. facilities tasked by the government with being ready to produce and deliver 50
million doses of flu vaccine in just 12 weeks. It is working with Caliber toward that goal.

"There's no way you can produce 50 million doses in 12 weeks" with current production technology, said Giroir.
"But plant-based production could."

Plant vaccines are Cheaper: While one chicken egg can produce one or two doses of flu vaccine, one tobacco
plant can produce 50 at a fraction of the cost.

The Defense Advanced Research Projects Agency (DARPA), an arm of the Defense Department that funds
cutting-edge research, is impressed enough with the potential of tobacco-plant production systems to have
awarded multi-million-dollar grants to both Medicago and Caliber

In a 2012 DARPA challenge, Medicago, jointly owned by Philip Morris International and Mitsubishi Tanabe
Pharma Corp, produced 10 million doses of H1N1 flu vaccine in just a month in tobacco plants inside its sprawling
North Carolina greenhouses. In animal tests, experimental vaccine successfully triggered production of
protective antibodies against H1N1.

If the upstarts succeed, they could threaten pharma giants that dominate flu vaccine production

1
VLP = Virus-like particles
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 C) The Reality for HBV plant vaccine

Despite 20-year attempts, no real anti-HBV plant-based vaccine has been developed (2013).
o Immunization trials, based on ingestion of raw plant tissue and conjugated with injection or
exclusively oral administration of lyophilized tissue, were either impractical or insufficient due to
oral tolerance2 acquisition

Hepatitis B persists as a common human disease despite effective vaccines having been employed for
almost 30 years

Further Info:

2
Oral tolerance is classically defined as suppression of immune responses to antigens that have been administered
previously by oral route.
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