Short Technical Reports
INTRODUCTION
DNA tagging by T-DNA and transposon inrtions has become an important approach for studying functional genomics in plants. Large numbers of DNA-inrtion lines and important mutations have been created in Arabidopsis and rice using this approach. To identify the genes tagged by DNA inrtions, it is necessary to recover genomic quences flanking the inrtion tags. However, the tagged gene quences cannot be obtained simply by regular PCR procedures becau the genomic flanking quences are unknown. So far, veral PCR-bad methods, such as inver PCR (1,2), adapter-ligation-mediated PCR (3–6), and thermal asymmetric interlaced (TAIL)-PCR (7,8), have been developed for ampli-fication of unknown DNA fragments flanked by known quences. TAIL-PCR is a PCR-only method and is thus especially suitable for manipu-lating 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 ud in a variety of biological rearch in various organisms,
including large-scale determination
of T-DNA and transposon inrtion
罚球规则
sites in Arabidopsis and rice (9,10),
and isolation of upstream (promoters)
and downstream quences of the
known coding quences (11,12).
TAIL-PCR utilizes nested known
quence-specific primers with a
游乐场鬼屋
melting temperature (T m) >65°C
in concutive reactions together
with a short (15–16 nucleotides)
arbitrary degenerate (AD) primer
with a T m 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 quence.
Target product(s) are then preferen-
tially amplified over nontarget ones
that are primed by the AD primer
alone by swapping two high-strin-
gency PCR cycles with one that has
reduced-stringency (TAIL-cycling).
This is bad 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 quences. However, this
tends to produce undesired smaller
products. To achieve high success
rates in obtaining target quences
with larger sizes, we developed a
high-efficiency TAIL-PCR (hiTAIL-
PCR) procedure by using specially
designed degenerate and known-
quence-specific primers.
MATERIALS AND METHODS
PCR Primers
The primers ud for hiTAIL-PCR
are shown in Figure 1.
Reagents
Genomic DNAs were prepared
工作年限怎么填写from transgenic rice lines as
described (10), which were trans-
formed by binary vector constructs
bad on pCAMBIA1305.1 and
pCAMBIA1300 (Cambia, Canberra,
Australia). Ex Taq DNA polymera
kit with 10×PCR buffer containing
20 mM MgCl2(Takara-Bio, Dalian,
China) was ud 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 cas in which two LAD
一般将来时练习题primers were ud 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-
瑞鹧鸪fold diluted pre-amplified product.
Each condary 25-μL TAIL-PCRs
High-efficiency thermal asymmetric interlaced PCR for amplification
of unknown flanking quences
Y ao-Guang Liu and Y uanling Chen
South China Agricultural University, Guangzhou, China
BioTechniques 43:649-656 (November 2007)
doi 10.2144/000112601
Isolation of unknown DNA quences flanked by known quences is an important task in molecular biology rearch. Thermal asymmetric interlaced PCR (TAIL-PCR) is an effective method for this purpo. However, the success rate of the original TAIL-PCR needs to be incread, and it is more d
esirable to obtain target products with larger sizes. Here we pres-ent 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 quences. Using this method, we isolated genomic flanking quences of T-DNA inrtions from transgenic rice lines. In our tests, the success rates of the reactions were higher than 90%, and in most cas the obtained major products had sizes of 1–3 kb.
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 condary TAIL-PCRs were scaled up to 50 μL. The PCRs were performed using a PCT-100 PCR cycler (MJ Rearch, Waltham, MA, USA) with thermal conditions shown in Table 1. The amplified products were analyzed on 1.0% agaro gels, and single fragments were recovered from the gels and purified using a DNA purification kit.
RESULTS AND DISCUSSION
Primer Design
Four relatively longer AD (LAD) primers of 33 or 34 nucleotides were designed for hiTAIL-PCR, which contained four fixed nucleo-tides at the 3′ ends, followed by degenerate nucleotides at the six or ven positions (2304- or 6912-fold degeneracy) (Figure 1). Of the degenerate nucleotide positions, two or three were designed to have three rather than four nucleotides, to avoid complete lf-pairing between the 3′ ends. The arbitrary 3′ four-ba sites have a moderate frequency (on average 256 bp a time) to anchor the LADs to the target quences. A Not I restriction site was prent in the primers as a choice for cloning of the target TAIL-PCR products in plasmid vectors containing a unique Not I site and other unique blunt-end restriction site(s). The different LAD primers shared a common quence in the 5′ half. A 16-mer primer (AC1) specific to this quence was prepared, which had a T m of 52°C as calculated by the formula T m = 69.3 + 41 × GC% - 650/L (L = primer length) (13). Since the quences of the LAD primers including their 3′ ends were arbitrarily designed, and especially, the four-ba sites cannot form specificity for given genomes of organisms, the LAD primers are universally applicable for various organisms.
To isolate T-DNA inrtion flanking quences from transgenic
plants
Figure 1. Primers ud 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 quence adjacent to RB. RB-0b, RB-1b, and RB-2b are specific to pCAMBIA-1300. In the quence tag of RB-1a (RB-1b), two bas (underlined) were designed to differ from the 3′ end of AC1 in order to avoid the priming of AC1 on this quence tag in the condary TAIL-PCR, and in quencing of the primary TAIL-PCR products from the AC1-end with AC1 as the quencing primer.
hiTAIL PCR, high-efficiency thermal asymmetric interlaced PCR.
transformed with the pCAMBIA binary vectors, nested specific primer ts (RB-0a/RB-1a/RB-2a, RB-0b/RB-1b/RB-2b) with T m >68°C were designed according to the quences adjacent to the T-DNA right border (RB) (Figure 1). In RB-1a (RB-1b) for the primary TAIL-PCR, an additional quence (21 nucleotides) that was identical (except for two bas) to the 5′ half of the LAD primer was tagged to the 5′ end, in order to produce a suppression-PCR effect (14). As a comparison, a specific primer (RB-1ac) without this quence tag was prepared for the primary TAIL-PCR. A distance of 88 bp (or 74 bp) between RB-1a and RB-2a (or RB-1b and RB-2b) was t to facilitate the confirmation of the product specificity, if necessary, by the differential shift on agaro gels.Rationale of hiTAIL-PCR
The RB-0a (RB-0b) and a LAD primer (or pooled LAD primers) are applied to the pre-amplification reaction. After 10 cycles of linear amplification of target quences primed by RB-0a (RB-0b), which increas the copy number of target molecules, a single cycle with a low annealing temperature (25°C) is carried out. The higher degree of the primer degeneracy and the low annealing temperature allow the LAD primer to bind to the target quence with a higher probability, thus efficiently creating one or more annealing sites for the AC1 primer. Then the primary TAIL-PCR is carried out using the nested specific primer RB-1a (RB-1b) and AC1. Since the DNA strands of specific products primed by RB-1a (RB-1b) and AC1 contain complementary ends (with a Melting temperature of 63°C), the relatively small ones (<500 nucleotides) tend to form a hairpin structure, which suppress the PCR amplification, while amplification of larger products is less affected, becau the longer distances between the complementary ends decrea the potential for the hairpin structure to form. The amplification of nontarget products primed by AC1 alone is of low efficiency during the TAIL-Figure 2. Amplification of T-DNA flanking quences from transgenic rice plants by high-efficien-cy thermal asymmetric interlaced PCR (hiTAIL-PCR). (A) Pre-amplification of a transgenic line (I-1) using RB-0a combined with LAD1-1 (lane 1), LAD1-2 (lane 2) and LAD1-4 (lane 3), respectively. (B) Primary TAIL-PCRs of I-1 with RB-1a (1a) or RB-1ac (1ac), showing the effect of using RB-1a on suppressing the amplification of smaller target products. The LAD primers f
or the pre-amplification are indicated. (C) Analysis of the primary and condary TAIL-PCR products obtained from the transgenic lines using RB-1b (1b) and RB-2b (2b) for the reactions, respectively. The corresponding primary and condary products show the expected differential shift (74 bp). (D) hiTAIL-PCRs using pooled LAD primers in the pre-amplification reactions.
Figure 3. Characterization of T-DNA tagged sites in the rice genome. Diagrams indicate the T-DNA inrtion sites in the rice genome of transgenic rice lines (A) I-1, (B) I-3, (C) II-2, and (D) II-4 as deter-mined by quencing of primary hiTAIL-PCR products using primers RB-2a (panels A and B) or RB-2b (panels C and D). The filled triangles indicate the T-DNA inrtions, and the numbers show the positions (in ba pairs) of the inrtion sites and the predicted open reading frames in the bacterial artificial chro-mosome clones (BAC) [OSJNba0093F12 (panel A) and OSJNBa0029P07 ( panel D)] or P1 artificial chromosome (PAC) clones [P0663C08 (panel B) and P0470B03 (panel C)]. The accession no. of the corresponding rice gene for each clone is indicated to the left of each diagram. The T-DNA quences of the right border side from pCAMBIA1305.1 (panels A and B) or pCAMBIA1300 (panels C and D) are given in capital letters and the genomic flanking quences in lower-ca letters. Overlines show the retained nucleotides of the right border.
A
B
C
D
A
B
C
D
cycling (7,8). Nontarget products from nonspecific priming by the specific primer RB-0a (RB-0b) alone, if any, are diluted and cannot be amplified in the following TAIL-PCRs using the nested specific primers. On the other hand, new nontarget products cannot be generated and amplified to visible levels from such diluted (approxi-mately 1000-fold) templates by RB-1a (RB-1b) alone in the primary TAIL-PCR.
Effectiveness of hiTAIL-PCR
The hiTAIL-PCR procedure was ud to isolate T-DNA inrtion flanking quences from trans-genic rice lines. We first tested the efficiency of using single LAD primers in hiTAIL-PCRs. Six lines transformed with pCAMBIA1305.1 (I-1 to I-6) and nine lines with pCAMBIA1300 (II-to I 9), which had single T-DNA inrtion, were lected for the test in combination with the four LAD primers. The pre-ampli-fication reactions produced smear bands (Figure 2A), which contained a large number of randomly amplified products from the rice genomic DNA. In some cas, the pre-amplified products were not detectable on the agaro gels (not shown), but target products could be obtained in the primary TAIL-PCR. In most hiTAIL-PCR, the obtained major target products had sizes of about 1–3 kb, and no products smaller than about
0.5 kb were produced (Figure 2, B–
D), whereas the original and modified TAIL-PCR procedures usually produce fragments of approximately 0.2–1.5 kb (7–9). In comparison, the control reactions without the suppression-PCR effect produced some smaller products (Figure 2B). Of the 60 reactions, 56 produced target products, giving an average success rate of 93.3%. This is much higher than that (50%–70%) of the original TAIL-PCR procedure (8). All the four LAD primers worked well in our tests. Most primary TAIL-PCR reactions produced detectable target products. Therefore, in general the condary TAIL-
PCR can be omitted. In a few cas the primary TAIL-PCR products were at relatively low levels (data not shown); however, they could
be amplified to high concentrations in
the condary TAIL-PCRs.
We also ud LAD primer pools,
LAD1-1/LAD1-3 and LAD1-3/LAD1-
4, in the pre-amplification reactions
to test if the amplification efficiency
can be further incread. The results
showed that 37 (97.4%) of the 38
reactions tested succeeded to produce
target products. The reactions using
the pooled LAD primers produced an
average of 2.5 products, while tho
using single LAD primers gave an
average of 1.8. The sizes of the products
were similar to or somewhat smaller
than tho obtained by using single
LAD primers, but tho smaller than
about 0.5 kb were blocked effectively
(Figure 2D). Therefore, it is a good
choice to u the LAD primer pools in
the application of hiTAIL-PCR.
As described in the original TAIL-
PCR (7,8), the differential shift
between the primary and condary
products on agaro gels (Figure
2, C and D) is a good indicator of
the product specificity. The speci-
ficity also was tested in some cas
by control (primary or condary)
reactions with RB-1a (RB-1b) or
RB-2a (RB-2b) alone and AC1 alone,
which did not generate detectable
products (data not shown). We further
purified veral primary hiTAIL-
PCR products for direct quencing.
The result showed that all of the
quenced products contained the
inrted T-DNA and its flanking
genomic quences of the rice
genome. Several examples of the T-
DNA tagged sites in the rice genome
are shown (Figure 3).
Conclusion
We described a substantially
improved TAIL-PCR procedure,
hiTAIL-PCR, by special design of
the arbitrary degenerate and specific
primers. This method combined
the advantages of the TAIL-cycling
and suppression-PCR, thus greatly
incread the success rate but avoided
to produce small target fragments.
This new version of TAIL-PCR
thus can replace the original one in
molecular biology rearch of various
organisms.
ACKNOWLEDGMENTS
This work was supported by the
Ministry of Science and Technology of
China.
COMPETING INTERESTS
STATEMENT
The authors declare no competing
interests.
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Received 8 June 2007; accepted 11 September 2007.
Address correspondence to Yao-Guang Liu, Key Laboratory of Plant Functional Genomics and Biotechnology of Education Department, Guangdong Province, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. e-mail: ygliu@scau.edu
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