ARABIDOPSIS THALIANA در ژنوم آراييدوپسيس T-DNA در شناسايي و تكثير توالي هاي نوكليوتيدي مجاور TALL- PCR استفاده از

Discrimination of sequences flanking T-DNA insertions using TAIL-PCR in Arabidopsis thaliana genome.

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) in which asymmetric annealing temperature is used was employed to discriminate sequences flanking T-DNA insertions in Arabidopsis thaliana. In this method, left and right border sequences of the T-DNA insertions were regarded as the two tagged positions and two series of primers differing in length and annealing temperature were used to amplify the target sequences. The technique uses a series of nested primers specific to the known sequence of the left and right borders of the T-DNA and a series of shorter arbitrary primers with lower annealing temperature. Four mutants of Arabidopsis thaliana resistant to uniconazol (a GA-biosynthesis inhibitor), due to T-DNA insertions in their genome were studied in this research. TAIL-PCR was performed in three stages which in the second and third stages, diluted PCR products of the first and second stages were used respectively as the DNA template. In each stage of the TAIL-PCR a combination of five arbitrary primers and two specific primers for left and right borders of the T-DNA were used to amplify the target sequences in the four different mutant DNA samples. In other words, forty different reaction conditions were prepared and performed simultaneously. Investigating the PCR products of the second and third stages on 2% agarose gel and comparing the mutants bands related to the two stages, specific PCR products were detected and separated from non-specific products. Selecting the suitable bands with appropriate size, Qiagene kit was used to purify the PCR products and sequenced. Conforming the obtained sequences to the T-DNA left and right borders, indicated that the method amplified the targeted sequences with a high accuracy.