Asymmetric PCR

PCR can also be used to generate an excess of single-stranded DNA which can then be labeled and used as DNA probes. This technique, which is called asymmetric PCR, involves using a 100-fold excess of one primer over the other. With this asymmetric ratio, double-stranded DNA will be synthesized in the first 20 to 25 cycles, at which time the primer used at a lower concentration would be consumed, leaving the primer that is in excess to preferentially synthesize single-stranded DNA over the next 5 to 10 cycles (G3). 

Asymmetric PCR, as opposed to symmetric PCR, is used in the labeling step because, although the former creates strands at a slower arithmetic rate, it will produce an abundance of labeled single strands lacking complementary strands with which to reanneal. The PCR-STR template and complementary strands are repetitive and have a high potential to reanneal. With symmetric PCR to make labeled strands, the more balanced... 

Conventional PCR uses primers that are present in equal amounts, thereby ensuring that the majority of the products are double-stranded amplicons. A variant method uses different concentrations of the two primers to generate more of one strand than of the other (asymmetric PCR). For instance, the use of primer A at 0.5 jiM and primer B at 0.005 pM produces mostly single-stranded DNA extended off the more abundant primer. This is useful for sequencing purposes or making single-stranded probes. Yield of the product, however, may be low. With less extreme ratios (e.g., primer A at 0.5 pM and primer B at 0.2 pM), the yield is mostly preserved, with one strand produced in enough excess to make it more available for probe hybridization. 

Kerman, K., Vestergaard, M., Nagatani, N., Takamura, Y. and Tamiya, E. (2006) Electrochemical genosensor based on peptide nucleic acid-mediated PCR and asymmetric PCR techniques Electrostatic interactions with a metal cation. Anal. Chem. 78, 2182-2189... 

Asymmetric PCR with the excess of the primer that is extended to aptamer candidates over the primer that is extended to complementary aptamer sequences has been used successfully in quick testing of affinity between rounds of SELEX. Asymmetric PCR is slower, but it allows us to avoid the strand separation procedure, as complementary strands are generated in small amounts and affect... 

For DNA sequencing, it is highly desirable to produce a single-stranded DNA product. To produce single strands in PCR, a simple approach called asymmetric PCR can be used. Here ordinary PCR is carried out for a few less cycles, say 20. Then one primer is depleted or eliminated, and the other is allowed to continue through an additional 10 cycles of linear PCR. The result is a product that is almost entirely single stranded. Clearly we can use whichever strand we want by appropriate choice of primer. 

Recently, a lambda Red beta protein-based genome editing method was developed in B. subtilis to inactivate target genes using ssDNA fragments obtained by asymmetric PCR [185]. The ssDNA comprised a 1081-nt disruption... 

Add the entire asymmetric PCR product sample (Subheading 3.2, step 4) to the washed beads, mix, and incubate at room temperature for 10 min. Separate the beads using the magnetic separation rack, remove and save supernatant. 

Key words Synthetic vims. Oligonucleotides, Poliovfrus, cDNA, Infectious agent. Asymmetric PCR,... 

To produce the best results in the asymmetric PCR, the overlapping region between two oligonucleotides should have (1) a melting temperature in the range of 52-58°C and (2) GC content of40-60%. 

Under limited or suboptimal reaction conditions, the onset of linear and plateau phases may occur prematurely, rendering the yield of PCR arbitrary with respect to the number of cycles. In fact, the gain can be controlled by carefully adjusting the reaction conditions, giving rise to such useful techniques as asymmetric PCR, whereby daughter strands are produced by linear amplification (2,44). 

Taq Pol can also be employed as an auxilliary enzyme which just fulfills the need to prepare ample amounts of template DNA (either ssDNA or dsDNA). Single-stranded DNA templates can be readily generated by a nonstandard PCR strategy known as asymmetric PCR. Compared with symmetric PCR, which in essence corresponds to the standard PCR, the asymmetric PCR employs one of the two primers about 100-fold more than the other primer such that, after a certain number of exponential amplification which exhausts the limiting primers, linear amplification of ssDNA is attained. 

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