|
Principle - ACP™-PCR
The ACP™ system requires a two-stage PCR (ACP™-based
PCR) amplification to maximise the functions of each portion as
follows :
(1) 1st stage PCR for generating a specific PCR product
The regulator is not favourable in annealing to the template
under the conditions that the 3'-end core portion anneals to a site
of the template at a first annealing temperature, such that the
regulator is capable of interrupting the annealing of the 5'-end
portion and restricting a primer annealing portion to the 3'-end
portion. The strength in which the specific annealing of the 3'-end
portion sequence occurs is relatively stronger than the strength
in which the non-specific annealing occurs under the first annealing
temperature, which results in the improvement of primer annealing
specificity. In this regard, the effect or performance of the regulator
on the 5' and 3'-end portions of ACP is one of the key features
of ACP™ Technology.
(2) 2nd stage PCR for amplifying 1st PCR product
Only the resultant product generated from annealing and
extension of the 3'-end portion sequence of ACP in step (1) can
be amplified close to the theoretical optimum of a two-fold increase
of product for each PCR cycle by priming the ACPs at 3'- and 5'-
ends under high stringency conditions in which the sequence of the
3'-end core portion alone is not allowed to anneal to the template.
In this step, only the sequences at the 3'- and 5'-ends of the 1st
PCR product act as donors of priming sequences for the amplification,
which results in the amplification of only a target product as a
results; this is another key feature of ACP™ Technology.
The effect of ACP is clearly proven by comparison with other conventional
short and longer primers for the purpose of the amplification of
a target sequence. The ACPs having a 10 target nucleotide sequence
(10-mer) at their 3'-end core portions produced an expected target
product, whereas the conventional short primers produced no products
and the conventional longer primers generated many other non-specific
products as well as a target product. Although the tailing of the
conventional longer primers were replaced with other universal sequences,
non-specific products were generated in a different pattern. This
clearly shows that the ACPs are exclusively involved in specific
primer annealing, resulting in only a target product in such way
PCR specificity is maximised.
Advantages
The advantages to be obtained from the use of ACP™ system
in PCR amplification are as follows.
- The regulator bridging the 3'- and 5'-end portions restricts
a primer annealing portion to the 3'-end core portion during the
1st stage PCR, so that the annealing sequence of a primer can
be precisely controlled, when make it possible to design a primer
with a desired number of annealing sequence. It is particularly
useful when an annealing portion of a primer has to be limited
(e.g., single nucleotide polymorphism (SNP) genotyping, DNA microarray
screening, and detection of differentially expressed genes).
- The regulator bridging the 3'- and 5'-end portions interrupts
the annealing of the 5-end portion to the template, and eventually
the 5'-end portion not involved in the annealing provides the
3'-end portion with primer annealing specificity.
- The specificity of primer annealing is highly sensitive enough
to detect even a single base mismatching. It is particularly useful
for the identification of a nucleotide variation in a target nucleic
acid including, single nucleotide polymorphisms and point mutations.
- Non-specific annealing is excluded from the amplification of
the 1st primed PCR product by using only the sequences at the
3'- and 5'-ends of the 1st PCR product as donors of priming sequences.
- The ACP™ system utilises a two stage PCR (ACP™-based
PCR) which allow the products to be excluded from non-specific
amplifications.
|
