Primer fabrication using polymerase mediated oligonucleotide synthesis.
BACKGROUND
Custom solid phase oligonucleotide synthesis is an important foundation supporting nearly every aspect of current genomics. In spite of the demand for oligonucleotide primers, their synthesis remains relatively expensive, time consuming and in many circumstances a wasteful process. In this methodology, described as polymerase mediated oligonucleotide synthesis (PMOS), a DNA polymerase is used to increase the hybridization affinity of one oligonucleotide by using another as a template for DNA synthesis. This self-assembly process provides an opportunity to instantly generate a very large number of useful gene-specific primers from a small library of simple precursors. PMOS can be used to generate primers directly in the end-users laboratory within the context of any DNA polymerase chemistry such as in PCR or sequencing reactions


RESULTS
To demonstrate the utility of PMOS, a universal 768-member oligonucleotide library (UniSeq) was designed, fabricated and its performance optimized and evaluated in a range of PCR and DNA sequencing reactions. This methodology used to derive specific 11-mers, performed well in each of these activities and produced the desired amplification or sequencing analysis with results comparable to primers made by time consuming and expensive custom synthesis.


CONCLUSIONS
On the basis of these experiments, we believe this novel system would be broadly applicable and could in many circumstances replace the need for conventional oligonucleotide synthesis.
Cairns
Murray J
MJ
Schizophrenia Research Institute, Sydney, NSW 2006, Australia. murray.cairns@newcastle.edu.au
Thomas
Torsten
T
Beltran
Carolina E
CE
Tillett
Daniel
D
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015723:Gene Library; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods
D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase