An enhanced method for sequence walking and paralog mining: TOPO^® Vector-Ligation PCR

Template Preparation for TVL-PCR

DNA was isolated from unexpanded leaf tissue of Fragaria virginiana accession L2 (CFRA 1995) as described [6], except that no chloroform:octanol solution was included in the microfuge tube to which CTAB slurry was transferred. Per reaction, 400 ng of genomic DNA was digested with 20 U of Eco RI, Bam HI, or Hin dIII (New England Biolabs, Ipswich, Massachusetts) in a 40 μl reaction that was incubated overnight at 37°C. Employed restriction enzymes must produce recessed 3' (5' overhangs) or blunt ends. Digestion was verified by electrophoresis of 100 ng digested genomic DNA and undigested comparator on a 1% agarose TBE gel.

Repair of Fragment Ends and Addition of 3'A Overhang

End-repair employed 20 μl (200 ng) of each digested DNA sample with 1 μl of 10 mM dNTP mix, 1.3 μl sterile water, 2.5 μl of EconoTaq^® buffer (Lucigen, Middleton, Wisconsin) and 0.2 μl (1 U) EconoTaq DNA polymerase. Reactions were incubated at 72°C for 30 minutes to fill in recessed 3' ends of cut sites, add a 3' adenosine overhang, and heat-inactivate the restriction enzyme.

Ligation

End-repaired DNA was ligated to the pCR4-TOPO vector (Invitrogen) using 4.5 μl (36 ng) end-repaired DNA solution, with 0.5 μl (5 ng) of TOPO vector and 1 μl of supplied salt solution. The reaction was gently mixed and incubated at room temperature for one hour.

Primer Design

The design of the gene-specific primers relied on alignments comprised of Superman-like sequences from four heterologous sources (Petunia, Nicotiana, Arabidopsis, and Malus), and strawberry Superman-like sequences obtained via conventional PCR amplification using degenerate primers targeted to sites conserved among the four heterologous sequences (results not shown). Two strategies were employed. For paralog mining of new Superman-like genes, degenerate primers were targeted to genomic sites identified as conserved among the heterologous and strawberry sequences. For sequence-specific walking to extend initially obtained gene segments, sequence-specific primers were targeted to genomic sites that were not conserved among the aligned heterologous and strawberry sequences. Genomic primers were always sited at least 50 bases upstream of the transition point from known to unknown sequence, to provide a sufficient read of known sequence in the resulting TVL-PCR product to confirm sequence identity and continuity.

TVL-PCR

The thermocycler profile used for both the first and second rounds of TVL-PCR was: initial denaturation at 94°C for 1 min; then 35 cycles of 94°C for 30 sec, 52°C for 30 sec, 68°C for 4 min; followed by a final extension at 68°C for 10 minutes. For each ligated template, two first-round amplification reactions were employed for the initial step of TVL-PCR, because the genomic fragment can ligate to the vector in alternate orientations (Figure 2). In one first-round TVL-PCR reaction (Figure 1C), the distal genomic primer (G1) was paired with the M13F or T3 vector primer, while in the other the G1 primer was paired with the M13R or T7 vector primer. In the first round, use of a distal M13_ (M13F or M13R) vector primer allows for subsequent vector primer nesting, while use of a proximal T_ (T3 or T7) vector primer precludes subsequent vector primer nesting (and is therefore recommended only if incompatibility exists between M13 primers and the distal genomic primer). For the second-round of TVL-PCR (Figure 1D), each first-round product was used as a template, and the nested genomic primer (G2) was paired with the appropriate T_ vector primer (T3 or T7, respectively - see below).

The 25 μl first-round TVL-PCR reactions contained 3 μl ligation reaction template, 0.5 μl of 20 μM G1 genomic primer, 0.5 μl vector primer, 0.1 μl (0.5 U) AccuPrime™ Taq DNA Polymerase High Fidelity (Invitrogen), and 2.5 μl 10× AccuPrime PCR buffer II. The first-round product (10 μl) was visualized on a 2% agarose 1% TBE gel run at 4.1 V/cm for 80 minutes. The second-round of TVL-PCR was performed with 1 μl of first-round product as the template, and 0.5 μl of 20 μM stock of both the nested G2 genomic primer and the appropriate vector primer. If the first-round vector primer was M13F or T3, then T3 was used as the second-round vector primer. If the first-round vector primer was M13R or T7, then T7 was used as the second-round vector primer.

Cloning and Sequencing of TVL-PCR Products

The amplification products from each second round TVL-PCR reaction were cloned using the Invitrogen TOPO TA Cloning^® kit for sequencing, per the manufacturer's instructions. Transformed cells were plated on LB agar plates containing 50 μg/ml ampicillin and were grown at 37°C overnight. Colony PCR was performed using M13 primers provided with the TOPO cloning kit to confirm insert size. Plasmids were isolated from clones of interest using the Promega Wizard^®Plus SV Minipreps DNA Purification System (Promega, Madison, WI). The plasmid inserts were then sequenced bidirectionally on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA), using M13 forward and M13 reverse sequencing primers. The T3 and T7 sequencing primers cannot be used for this purpose, because their priming sites may be represented twice in the final TOPO clone: once in the cloning vector itself and once in the cloned product of second round TVL-PCR (Figure 2).

Paralog Mining

Use of degenerate primers targeted to genomic sites conserved among heterologous Superman sequences resulted in acquisition of multiple candidate clones from strawberry accession L2 (results not shown), including that of clone 143-2-1. The latter sequence provided the basis for design of site-specific primers used for sequence walking in accession L2, as described below.

Sequence Walking

Specific primers targeted to non-conserved sites in clone 143-2-1 were designed (Table 1) and used to extend this gene fragment in both the 5' and 3' directions. For walking in the 5' direction, G1 primer 14321 5'F1 paired with the M13R vector primer produced a spectrum of products in the first-round reaction using Hin dIII (H), Eco RI (E), or Bam HI (B) digested genomic DNA as template (Figure 3A - top). The second round of TVL-PCR, using the first-round products as template with nested G2 primer 14321 5'F2 and the nested T3 vector primer, produced a diminished spectrum of bands in the H and E lanes, but a proliferation of bands in the B lane (Figure 3A - bottom). After shotgun cloning of the second round TVL-PCR products, colony PCR, and sequencing of three clones selected on the basis of favorable size (> 500 bp), one clone containing a Superman-like sequence was found. Its size corresponded to the boxed band in Figure 3A (bottom). This newly acquired sequence extended upstream from the known gene sequence to the putative start codon and beyond.

Primer Name	Primer Sequence	Tm (°C) *
Vector Primers
M13F	5'-GTAAAACGACGGCCAG-3'	50.7
M13R	5'-CAGGAAACAGCTATGAC-3'	47.0
T3	5'-ATTAACCCTCACTAAAGGGA-3'	50.3
T7	5'-TAATACGACTCACTATAGGG-3'	47.5
Gene Primers
14321 5' F1	5'-AGGGTTAGGTTTAAGGTTGAG-3'	51.8
14321 5' F2	5'-GTGAGGGTACGAAAGTAGG-3'	51.9
14321 3' F1	5'-TCGCAAGTTGAACTATGTATCC-3'	52.6
14321 3' F2	5'-CAGTTTGTTCAGGACTGAGT-3'	52.2

Vector primer sequences are standard, and are provided here for convenience. Gene primers were designed by us.

*Melting temperature values were determined by the Integrated DNA Technologies OligoAnalyzer application [7], and may not be the same as those published elsewhere for M13_ and T_ primers.

For walking in the 3' direction, the first round of TVL-PCR employed G1 primer 14321 3'F1 paired only with the T3 vector primer. The product from this reaction (Figure 3B - top) was used as template for the second round of TVL-PCR using the nested G2 primer 14321 3'F2 in conjunction with the T3 vector primer. Shotgun cloning of the second round TVL-PCR products (Figure 3B - bottom), followed by colony PCR and sequencing of two appropriately sized products (> 500 bp), yielded one product that contained the targeted sequence. The respective PCR product size corresponded to the boxed gel band (Figure 3B - bottom). This product was slightly less than 2 kb in length, and extended well into the 3' UTR of the targeted gene.

Overall, a total of five clones were sequenced from shotgun cloned and size-selected TVL-PCR products, of which one provided targeted sequence extension in the 5' direction and another in the 3' direction. The remaining three sequenced clones were not the targeted sequence, but displayed a vector primer sequence at one end and a respective genomic primer sequence at the other, indicating that each arose from non-specific priming by the genomic primer.

Using methods similar to those described above, TVL-PCR was also used successfully to extend the sequence of Superman-like clone 7266 upstream far enough to surpass the putative start codon (results not shown). In addition, the use of degenerate primers resulted in the acquisition of 5' and 3' sequences from three additional Superman family sequences in strawberry. All strawberry Superman-like genes isolated from chromosome walking via TVL-PCR have been deposited in GenBank under accession numbers, GU830924: 7266 5' walk, GU830926: 14321 5' walk, and GU830925: 14321 3' walk.