|
Abstract This experiment was conducted to identify two nontargeted bands accompanied with heterozygote in nondenatured polyacrylamide gel electrophoresis. The nontargeted and targeted bands were cut and reclaimed, and used into PCR as templates. The PCR products were electrophoresised according to different combinations and disposals. In addition, 4 synthesized single strands were applied to simulate the formation process of nontargeted and targeted bands. The results showed that 1) The mobility property of the PCR products of DNA extracted from untargeted bands was the same as that of the PCR products of genomic DNA from heterozygote. The mobility property of the PCR products of DNA extracted from either targeted band was the same as itself. 2) If the PCR products of DNA extracted from the two targeted bands were mixed and treated with denaturation and renaturation in PCR instrument, the mobility property of them was also the same as that of the PCR products of genomic DNA from heterozygote; if mixed without other treatment, there were only two targeted bands appeared. 3) In the simulation experiments, the untargeted bands were formed by the heteroduplex DNA strands and the targeted bands by the homoduplex DNA strands. These results indicated that the two nontargeted bands were not nonspecific amplicons. Because of a 3bp deletion in one of two targeted double-stranded DNAs, two heteroduplex DNAs formed in the processes of denaturation and renaturation of PCR for random pairing of single-stranded DNAs, thus, convex rings were produced consequently. The convex rings decreased the mobility of the two heteroduplex DNAs and the two delayed heteroduplex DNAs formed the two nontargeted bands.
|
|
Received: 20 March 2009
|
|
|
|
