| Tail PCR | | 点击： 作者：51protocol收集 来源： 时间： 2007-03-29 本站论坛 |
|  | Tail PCRAmplification of flanking sequences To determine flanking sequences of RDA subtraction products in the genome, a two-step PCR reaction technique can be applied using a biotinylated primer and degenerate primers for amplification. Amplified biotinylated DNA fragments were isolated with a Dynabeads M-280 Streptavidin system (Sorensen et al., 1993). Figure 1 shows the procedure applied. All PCR reactions were carried out using the PCR protocol outlined under “Polymerase chain reaction” but with 42 cycles of amplification and primer annealing at 62°C. Genomic DNA was used as template in the first PCR reaction, whereas an aliquot from the first PCR reaction was used as a DNA template in the second PCR reaction. Isolation of amplification products After the first PCR reaction using a biotinylated primer, amplified, biotinylated DNA fragments were isolated by mixing 40 ml of the PCR mixture with 40 ml of 200 mg pre-washed Dynabead M280-streptavidin as recommended by the supplier (Dynal Biotech, Norway). Biotinylated DNA fragments were removed from the mixture using a Dynal magnetic particle concentrator. All binding and washing steps were done in the presence of a binding and washing buffer consisting of 10 mM Tris-HCl (pH 7.5), 1 mM EDTA and 2 M NaCl. After incubation for 15 min to remove the biotinylated DNA fragments from the mixture and washing in buffer, the Dynabead-bound DNA fragments were “melted” in 8 ml of 100 mM NaOH for 10 min. The supernatant containing the non-biotinylated strands was then neutralised with 4 ml of 200 mM HCl and 1 ml 1 M Tris-HCl, pH 8. After filling up to 30 ml with sdH2O, 2 ml of the mixture was used as a DNA template in a second PCR reaction using a specific primer pair for amplification. Amplified and agarose gel-purified DNA fragments bands were finally cloned into the vector pMOSBlue and the sequence of the cloned DNA fragments was finally analysed on an automated DNA sequencer. Figure 1: Isolation of flanking DNA sequences in the genome by a two-step polymerase chain reaction and amplification of unknown DNA sequences (thin line) flanking a known DNA region (broad line). Arrows indicate biotinylated primer (primer 1), specific sequence primers (primers 2 and 3) and degenerated primers (primer FP). Green circle indicates biotin coupled to the 5’ of the primer and both blue square and half moon indicate beads with streptavidin covalently bound to their surface. Sorensen A.B., Duch M., Jorgensen P. and Pedersen F.S. 1993. Amplification and sequence analysis of DNA flanking integrated proviruses by a simple two-step polymerase chain reaction method. American Society of Microbiology 67: 7118-7124.
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