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High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences
 
Yao-Guang Liu Yuanling Chen
South China Agricultural University, Guangzhou, China
BioTechniques, Vol. 43, No. 5, November 2007, pp. 649–656
Full Text (PDF)
Abstract

Isolation of unknown DNA sequences flanked by known sequences is an important task in molecular biology research. Thermal asymmetric interlaced PCR (TAIL-PCR) is an effective method for this purpose. However, the success rate of the original TAIL-PCR needs to be increased, and it is more desirable to obtain target products with larger sizes. Here we present a substantially improved TAIL-PCR procedure with special primer design and optimized thermal conditions. This high-efficiency TAIL-PCR (hiTAIL-PCR) combines the advantages of the TAIL-cycling and suppression-PCR, thus it can block the amplification of nontarget products and suppress small target ones, but allow efficient amplification of large target sequences. Using this method, we isolated genomic flanking sequences of T-DNA insertions from transgenic rice lines. In our tests, the success rates of the reactions were higher than 90%, and in most cases the obtained major products had sizes of 1–3 kb.

Introduction

DNA tagging by T-DNA and transposon insertions has become an important approach for studying functional genomics in plants. Large numbers of DNA-insertion lines and important mutations have been created in Arabidopsis and rice using this approach. To identify the genes tagged by DNA insertions, it is necessary to recover genomic sequences flanking the insertion tags. However, the tagged gene sequences cannot be obtained simply by regular PCR procedures because the genomic flanking sequences are unknown. So far, several PCR-based methods, such as inverse PCR (1,2), adapter-ligation-mediated PCR (3,4,5,6), and thermal asymmetric interlaced (TAIL)-PCR (7,8), have been developed for amplification of unknown DNA fragments flanked by known sequences. TAIL-PCR is a PCR-only method and is thus especially suitable for manipulating a large number of samples in manual or automation (7). With the advantages of simplicity and high efficiency, TAIL-PCR and its modified procedures have been widely used in a variety of biological research in various organisms, including large-scale determination of T-DNA and transposon insertion sites in Arabidopsis and rice (9,10), and isolation of upstream (promoters) and downstream sequences of the known coding sequences (11,12).

TAIL-PCR utilizes nested known sequence-specific primers with a melting temperature (Tm) >65°C in consecutive reactions together with a short (15–16 nucleotides) arbitrary degenerate (AD) primer with a Tm of about 45°C and 64–256 folds of degeneracy, so that the relative amplification efficiencies of target and nontarget products can be thermally controlled (7,8). In the primary TAIL-PCR of the original method, one low-stringency PCR cycle is conducted to create one or more annealing sites for the AD primer along the target sequence. Target product(s) are then preferentially amplified over nontarget ones that are primed by the AD primer alone by swapping two high-stringency PCR cycles with one that has reduced-stringency (TAIL-cycling). This is based on the principle that, in the high-stringency PCR cycles with high annealing temperatures (65°–68°C) only the specific primer with the higher melting temperature can efficiently anneal to target molecules. The AD primer is much less efficient at annealing due to its lower melting temperature. AD primers with higher degrees of degeneracy, or pooled AD primers (9), may have more chances to bind to the target sequences. However, this tends to produce undesired smaller products. To achieve high success rates in obtaining target sequences with larger sizes, we developed a high-efficiency TAIL-PCR (hiTAIL-PCR) procedure by using specially designed degenerate and known-sequence-specific primers.

Materials and Methods

PCR Primers

The primers used for hiTAIL-PCR are shown in (Figure 1).

Figure 1.


Primers used for high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). RB-0a, RB-1a, and RB-2a are specific to pCAMBIA binary vectors (such as pCAMBIA-1305.1) having the Nos terminator sequence adjacent to RB. RB-0b, RB-1b, and RB-2b are specific to pCAMBIA-1300. In the sequence tag of RB-1a (RB-1b), two bases (underlined) were designed to differ from the 3′ end of AC1 in order to avoid the priming of AC1 on this sequence tag in the secondary TAIL-PCR, and in sequencing of the primary TAIL-PCR products from the AC1 -end with AC1 as the sequencing primer.

Reagents

Genomic DNAs were prepared from transgenic rice lines as described (10), which were transformed by binary vector constructs based on pCAMBIA1305.1 and pCAMBIA1300 (Cambia, Canberra, Australia). Ex Taq DNA polymerase kit with 10× PCR buffer containing 20 mM MgCl2 (Takara-Bio, Dalian, China) was used for the PCR.

PCR

Pre-amplification reactions (20 µL) were prepared, each containing 2.0 µL PCR buffer, 200 µM each of dATP, dCTP, dGTP, and dTTP (dNTPs), 1.0 µM of any one of the LAD primers (in the cases in which two LAD primers were used in single reactions, each was in 1.0 µM), 0.3 µM RB-0a or RB-0b, 0.5 U Ex Taq, and 20–30 ng transgenic rice DNA. Each 25- µL primary TAIL-PCR contained 2.5 µL PCR buffer, 200 µM each of dNTPs, 0.3 µM AC1 and RB-1a (or RB-1b), 0.6 U Ex Taq, and 1 µL 40-fold diluted pre-amplified product. Each secondary 25-µL TAIL-PCRs contained 2.5 µL PCR buffer, 200 µM each of dNTPs, 0.3 µM AC1 and RB-2a (or RB-2b), 0.5 U Ex Taq, and 1 µL 10-fold diluted primary TAIL-PCR product. When necessary, the primary or secondary TAIL-PCRs were scaled up to 50 µL. The PCRs were performed using a PCT-100 PCR cycler (MJ Research, Waltham, MA, USA) with thermal conditions shown in (Table 1). The amplified products were analyzed on 1.0% agarose gels, and single fragments were recovered from the gels and purified using a DNA purification kit.

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