Chemical methods for 5’ non- isotopic labelling of PCR probes and primers

Abstract

Abstract Synthetic oligonucleotides are now accessible to most laboratories conducting molecular biology experiments. The decreases in preparation time, costs, and the expertise required have enabled the annual production and use of several million oligonucleotides (1). PCR undoubtedly consumes more of these sequences than any other application. Many PCR experiments require the covalent attachment (labelling, tagging, conjugation) of small molecules to perform ancillary functions, in addition to base-specific annealing to complementary nucleic acid and 3’ chain extension by polymerase. The labels (reporter groups) conduct or facilitate detection, quantification, isolation, sizing, and location of the amplified sequence. Covalent attachment of molecules, such as fluorescent dyes, biotin, and proteins, can be made at virtually any site on the oligonucleotide, including the 5’- and 3’-termini, through the sugars and bases, and by internucleotide phosphate modification (2). The sequence- and base-specific hybridization properties must be preserved, as well as the chain extension capacity for PCR primers. The methods described in this chapter will focus only on examples of specific, chemical 5’-labelling of oligonucleotides, for use as PCR primers and probes of PCR products (3). Post-synthesis protocols such as cleavage/deprotection, analysis, purification, and 5’-labelling operations are described. Enzymatic labelling methods conducted before, during, or after PCR have been reviewed elsewhere (4).