Meet Bio S&T A foreword by our CEO Upcoming conferences BAC library expertise 3 options BAC sequencing RFQ cDNA and DNA libraries Lambda cDNA/genomic Plasmid cDNA/genomic Normalized cDNA RFQ Gene synthesis Deliverables and guarantees RFQ Mutagenesis Deliverables and guarantees RFQ DNA sequencing Individual sequencing PCR products END EST clones Whole genome Technology portfolio Drug screening APA technology Sequencing technology APAgene GOLD Walking Kits Retroviral products Other custom services Contact info Site map   APA technology PCR amplification (APA™) represents a new generation of PCR technology developed by Bio S&T (U.S. patent pending) (download brochure). Special offer:   Discover our APAgene™ GOLD Genome Walking kits  designed to rapidly (1 day!) and reliably amplify unknown genomic DNA using our patented APA Technology. Now being offered at 25% OFF! PCR technology is widely used in molecular biology for DNA cloning, genomic typing and sequencing. A variety of PCR permutations have been developed since its conception. However, a common limitation among these variations  is the need to know the DNA sequences flanking the region of interest.Thus, DNA amplification has been limited to templates with known sequences. APA™ is designed specifically to overcome this limitation. APA™ technology is capable of selectively or randomly amplifying any unknown DNA sequences. The APA™ procedure includes two steps (see figure 1):   1st step: an asymmetrical PCR step for 10-20 cycles; 2nd step: a regular PCR step for 30-40 cycles.     In step 1, particular primer sets named degenerated random tagging (DRT) primers are involved in the reaction. Each DRT primer consists of three components respectively, each containing a 4-6 nucleotide arbitrary sequence at the 3’ end, a bi-nucleotide degenerated sequence and a tagging primer binding sequence. The functions of the DRT primers are to randomly bind denatured DNA templates, lead single strand DNA amplification, and provide a tagging primer binding position in the 5’ region. The annealing temperature during this step ranges from 35-42 degrees with an optimum of  37 degrees for many species. In step 2 of the APA™ procedure, a specific primer and tagging primer are used in a reaction performed at a higher annealing temperature (higher than 57 degrees).  One of the most effective uses of APA™ Technology is gap  filling in genome integration. Conventional gap filling is often laborious, time consuming and ineffective given that too much DNA template is utilized in each reaction. Through APA™ Technology the sequencing process to walk the BAC clones is extremely simple and reliable with the additional advantage that only a minute amount of DNA is needed for the reaction. Usually, BAC End sequencing is usually limited by two factors: 1) a large amount of DNA, usually more than 5 ug is needed for each reaction 2) the BAC vector is typically a single copy vector, making it hard to extract large amounts of DNA from a BAC clone. Our revolutionary APA™ Technology has overcome these limitations. With this absolutely fresh approach, only 2-5 ng of template DNA are needed for each reaction, almost 1000 times less than required by the conventional procedure (see figure 2)!   APA™ technology's best applications are i) the generation of full-length cDNA transcripts and ii) facilitating 5’ and 3’ RACE (see figure 3). These confer a unique advantage in understanding gene regulatory pathways.                One of the path breaking achievements of biotechnology in the past decade has been the advent of transgenic technology. However, the success of this technology is tempered by the difficulties encountered in post transgenic analysis. APA™ enables the direct sequencing of transgene(s) and flanking host sequences. Undoubtedly, we believe this to be an extremely useful tool in high throughput gene expression and transfection analyses. Special offer:   Discover our APAgene™ GOLD Genome Walking kits  designed to rapidly (1 day!) and reliably amplify unknown genomic DNA using our patented APA Technology. Now being offered at 25% OFF!