The use and validation of a strategy that allows a universal set of bar-coded sequencing primers to be appended to an amplified PCR product is described. The strategy allows a modular approach, in that the same bar code can be used with two or more target-specific primer sets, even simultaneously.

To date, the majority of molecular genetic studies in algae have utilized a fairly limited range of markers such as the plastid rbcL gene and spacer, the mitochondrial cox2-3 spacer or the nuclear ribosomal DNA and spacers. The lack of available markers has been particularly problematic in studies of within-species variation. Whilst microsatellites are now being developed in many algal species,...

1.Atcheson, C.L., B. DiDomenico, S. Frackman, R.E. Esposito and R.T. Elder. 1987. Isolation, DNA sequence, and regulation of a meiosis-specific eukaryotic recombination gene. Proc. Natl. Acad. Sci. USA 84:80358039. 2.Cha, J., W. Bishai and S. Chandrasegaran. 1993. New vectors for direct cloning of PCR products [published erratum appears in Gene 1994; 141:149]. Gene 136:369-370. 3.Clark, J.M. 19...

9 PCR amplification of pure bacterial DNA is vital to the study of bacterial interactions 10 with corals. Commonly used bacterial-specific primers 8F and 27F paired with universal 11 primer 1492R amplify both eukaryotic and prokaryotic rDNA. An alternative primer set, 12 63F/1542R, is suggested to resolve this problem. 13

PCR amplification of pure bacterial DNA is vital to the study of bacterial interactions with corals. Commonly used Bacteria-specific primers 8F and 27F paired with the universal primer 1492R amplify both eukaryotic and prokaryotic rRNA genes. An alternative primer set, 63F/1542R, is suggested to resolve this problem.