Maura M. Devine, Mugasimangalam C. Raja, and Levy E. Ulanovsky 
CMB, Argonne National Laboratory, Argonne, IL 60439-4833 
levy@anl.gov 

Here we present a novel PCR technique termed "branched primer PCR" which eliminates the need for custom primer synthesis by combining oligonucleotide modules selected from a pre-synthesized library. A branched primer involves two oligonucleotide modules that are physically linked by annealing to each other as well as to the target, forming a three-way junction. Branched primers were developed (initially for DNA sequencing rather than for PCR) as a type of modular primer whose modules anneal cooperatively to the template. This cooperativity is provided by mutually complementary segments in the two modules that bind to each other forming what is termed a "stem" region. Before actual PCR can take place, a branched primer is extended along the template. This extension strand is then used as the template for a reverse branched primer extension. The reverse extension product is then amplified using PCR primers homologous to the stems of each branched primer. These PCR primers are universal in that the stem sequence is the same in different branched primers. In contrast, the sequences of the stretches which are complementary to the template are variable throughout the presynthesized library of the oligonucleotide modules (each in a separate tube). Additional sequence-specificity of PCR is provided by nesting. Branched primer PCR is expected to be useful for applications such as resequencing closely related genomes (e.g. rodents and primates) which require a huge number of custom PCR primers. The latter would then be conveniently replaced with a much smaller library of presynthesized oligonucleotide modules for branched primers (2,000 to 4,000 modules instead of millions of custom PCR primers).