3′ Cycle-Labeled Oligonucleotides with Predictable Length for Primer Extension and Transgene Analysis

Efficient labeling of short oligos at their 3′-ends was achieved through polymerase chain reaction. The length of cycled-labeled oligos can be accurately predicted by omitting one or more dNTPs in the labeling step. Thus, labeled oligos can be simply column-purified, eliminating the need for tedious gel purification. We demonstrated the effectiveness of this technique in determining the transcription start site of a given gene and in transgene analysis to differentiate the transcript of an endogenous gene from that of an introduced homologous gene. This technique could be widely extended to other molecular biology applications in which labeled oligos are employed. Efficient labeling of short oligos with α-32P-labeled radionucleotides at their 3′-ends was achieved through polymerase chain reaction (PCR) based on the working principle of cycle sequencing (1). By omitting one or more dNTPs during the cycle labeling step, 3′-ends of labeled oligos could be accurately defined which obviates the need for tedious gel purification. The use of α-32P radionucleotides in the labeling step also eliminates the exclusive requirement of γ-32P radionucleotide used in the 5′-end-labeling reactions. Moreover, radiolabeled primers can be synthesized directly from plasmid templates with this method. Using this technique, the labeled oligos can be used to hybridize to target RNA for primer extension through reverse transcription after a simple column purification to remove unincorporated nucleotides. Sequencing ladders can also be prepared using the same reagent/ enzyme system with the same labeled oligos without purification. This further avoids the employment of additional enzymes, such as T7 DNA polymerase or sequenase. Using this protocol, the transcription start site (TSS) of aspen (Populus tremuloides Michx.) caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT) transcript was successfully mapped.