An advanced and efficient asymmetric PCR method for microarray applications

Abstract

The sensitivity of a PCR based biochip assay relies on the efficiency of PCR amplicons in binding to the microarray spots. The essential factor determining the sensitivity is the amount of single stranded (ss) amplicons available for biochip hybridization. Asymmetric PCR can generate ss-amplicons depending on the ratio of primers used in the amplification process, but this process is often inefficient. We report a novel variant of PCR called the Asymmetric Exponential and Linear Amplification (AELA) which can overcome these issues and generate large amounts of single stranded amplicons. AELA-PCR introduces an amplification strategy that makes use of both exponential and linear amplification of the target nucleic acid. This is done by specifically designed primers and choice of adequate thermal profiles. In conventional PCR with a classical thermal profile, these specifically designed primers will work normally and contribute to an exponential increase of amplicons. A designed sequence extension of one of the primers and a very specific thermal profile, will result in a situation that the extended primer will be the only functional one for amplification, resulting in a linear phase of the amplification process. That is why during this step only one of the two strands of the target is amplified linearly and no longer exponentially. The result of the whole process is an amplification product enriched very strongly in one of the two single strands of the target. These adaptions in PCR are particularly favorable where the generation of ss-DNA/RNA is required. We demonstrate the higher biochip sensitivity of AELA-PCR compared to conventional amplification methods with an example of the Staphylococcus aureus detection on a DNA oligonucleotide microarray.

Protocol to retrieve unknown flanking DNA sequences using semi-site-specific PCR-based genome walking

“…Genome-walking is extensively used to probe unknown flanking DNA sequences. 2 , 3 , 4 , 5 , 6 , 7 , 8 Recently, we have devised Wristwatch PCR for genome-walking. 9 The Wristwatch PCR involves a set of three random walking primers that form a wristwatch-like structure with each other.…”

Recent advances in the synthesis of single‐stranded DNA in vitro

“…The principle is to use a pair of primers with unequal concentrations to produce large amounts of ssDNA. [35,36] These two primers are respectively termed the excess primer and limiting primer, whereby the absolute amount of the limiting primer is critical for the preparation of ssDNA. As shown in Figure 5A, in the first 10-15 cycles at the beginning of the amplification reaction, dsDNA was amplified guided by bilateral primers.…”

Multiplex Asymmetric PCR by Combining the Amplification Refractory Mutation System with the Homo-Tag-Assisted Nondimer System

“…Recognition of the reduced melting temperature ( T m ) of the limiting primer led to the development of linear after-the-exponential (LATE)-PCR, which renders asymmetric PCR assays as efficient as symmetric PCR assays, regardless of the primer ratio . Further improvements toward flexible designs have facilitated the establishment of different two-stage asymmetric PCR approaches, − all of which have achieved increased analytical sensitivity compared with conventional asymmetric PCR. However, the application of these improved approaches in multiplex assays remains unclear.…”