Investigation On The Non-specific Bands Observed In The Process For Recovery Of In Vitro Ligated Products Of Viroid CDNA

Agarose gel electrophoresis analysis has been generally adopted in analyzing DNA in molecular biology experiments. In this study, as recovering dimmeric bands of in vitro ligated products of Hop stunt viroid cDNAs from agarose gel, monomeric and other nonspecific bands were also observed. As this phenomenon had been reported, related experiments were conducted to clarify the reasons. The results were obtained as follows:1. The mixture sample including monomer, dimmer and polymers more than two units of HSVd cDNAs were conducted by PCR and in vitro ligation, respectively, migrated on agarose gel, recovered the dimmer, and analyzed on Polyacrylamide acrylamide gel(PAGE). The results showed that monomer and other unspecific bands were also recovered besides the dimer.2. The mixture sample including monomer, dimmer and polymers more than two units of Peach latent Mosaic viroid(PLMVd) and elongation factor gene of a fungus were conducted in vitro ligation, and recovered the dimers after migrated on agarose gel. The results showed that the monomer and other unspecific bands were also recovered besides the dimer, suggesting that the unspecific reaction was occurred not only for HSVd cDNAs, but also for other type of DNAs, which is a general phenomenon.3. The mixture sample including monomer, dimmer and polymers more than two units of HSVd cDNAs was conducted by PCR, and the dimers were recovered using four kits with different brands and then analyzed on PAGE. The results showed that the monomer and other unspecific bands were also recovered besides the dimer, suggesting that the unspecific reaction had little relation with the kit brands.4. The mixture sample mentioned above was analyzed via low-melting point agarose gel and PAGE, respectively, and the dimers were recovered from the gels and analyzed on PAGE. The results showed that the monomer and other unspecific bands were also recovered besides the dimer as purified via the low-melting point agarose gel, whereas only the dimer was recovered as purified via the PAGE. It suggested that the unspecific reaction occurred during the recovering process via agarose gel: unspecific reaction occurred as the samples containing untarget DNA bands were recovered via agarose gel.5. With the mixture sample mentioned above and the dimmeric HSVd cDNAs purified via PAGE, they were migrated on agarose gel, recovered the dimeric cDNAs, and analyzed on PAGE. The results showed that non-specific bands were observed in the mixture sample, but not in the purified one, from which only the target band was observed. It suggested that nonspecific bands could be recovered via agarose gel.6. The dimeric cDNA of HSVd purified from the mixture mentioned above were cloned and identified by PCR, results showed that different-sized fragments could be detected in 24 clones that were chosen for analysis. Of which, six clones contained the monomer, nine the dimer, ten the fragments with sizes similar to the dimer, and one trimer. Three clones with sizes of the dimer were sequenced and results showed that they had sequence similarities of 99.32-99.66% with the one inserted in the template plasmid. Simultaneously, the dimeric cDNA of HSVd that were recovered from agarose gel after purified from PAGE were also cloned and identified by PCR, results showed that nine clones were positive in 24 ones, and all of them contained the dimer. Three clones with sizes of the dimer were sequenced and results showed that they had sequence similarities of 99.31-99.83% with the one inserted in the template plasmid. The progeny clones obtained via the two processes had no obvious difference.This study clarified that the unspecific reaction observed for recovery of a DNA band from in vitro ligated products of viroid cDNAs was resulted in the process using agarose gel for purification, which provides a reference for the cloning experiments that required purifying DNAs.