Original Paper
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file on Synergy OPEN |
Acta Biochim Biophys
Sin 2006, 38: 571-576
doi:10.1111/j.1745-7270.2006.00194.x
optimized adaptor
polymerase
chain reaction-based method
for efficient genomic walking
Peng XU1,2,
Rui-Ying HU1,2, and Xiao-Yan DING1*
1
Laboratory of Molecular and Cell Biology and Laboratory of Stem Cell Biology, Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China;
2
Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China
Received: March 20,
2006
Accepted: April 29,
2006
This work was
supported by a grant from the National Key Project for Basic Science Research
of China (2001CB509901)
*Corresponding
author: Tel, 86-21-54921411; Fax, 86-21-54921011; E-mail,
Abstract Genomic walking is one of the most useful approaches in
genome-related research. Three kinds of PCR-based methods are available for
this purpose. However, none of them has been generally applied because they are
either insensitive or inefficient. Here we present an efficient PCR protocol,
an optimized adaptor PCR method for genomic walking. Using a combination of a
touchdown PCR program and a special adaptor, the optimized adaptor PCR protocol
achieves high sensitivity with low background noise. By applying this protocol,
the insertion sites of a gene trap mouse line and two gene promoters from the
incompletely sequenced Xenopus laevis genome were successfully
identified with high efficiency. The general application of this protocol in
genomic walking was proved to be promising.
Key words optimized
adaptor PCR; touchdown PCR; genomic walking; gene trap; promoter
In the postgenomic era, one major challenge is the functional
characterization of single gene. The gene trap method, using mouse embryonic stem
cells and a reporter vector randomly integrated into the genome, is a useful
tool to find novel genes and study their biological function [1]. To verify the
precise integration site, rapid and efficient genomic walking is required for
cloning the flanking sequences near to a gene trap vector.The traditional approach is to screen the genomic library using a
specific probe, however, this strategy is laborious and time-consuming. Several
PCR-based methods for isolating unknown DNA fragments flanking a known
sequence have been developed. They are: (1) inverse PCR [2], which uses a pair
of reversed primers to amplify DNA sequences in self-circularized genomic
fragments, but it meets the limitation of restriction site distribution; (2)
randomly primed PCR [36], which amplifies with a known sequence-specific primer
and an arbitrary primer, but the non-specific amplification blocks its
application; and (3) ligation-mediated PCR [715]. The adaptor PCR method, which
uses an adaptor ligated to a genomic fragment, was carried out with a
locus-specific primer and an adaptor-specific primer. As this involved the use
of the adaptor-specific primer, the amplification of adaptor itself leads to
the generation of a high level of background noise. Several improved protocols
have emerged to overcome the drawbacks, but they are either complicated or
inefficient.In this study, we describe an optimized adaptor PCR protocol
developed recently with the application of an optimized touchdown PCR program.
Using this protocol, the flanking sequences of a gene trap vector and two
promoters from the tetraploid animal Xenopus laevis were successfully
and rapidly isolated. The results indicated that the optimized adaptor PCR
would be valuable for genomic walking.
Materials and Methods
Isolation of genomic DNA and
restriction enzyme digestion
Genomic DNA was extracted from mouse tail or Xenopus liver
as previously described [16]. Briefly, homogenized tissues were digested by proteinase
K overnight at 55 ?C. The genomic DNA was extracted twice with
phenol/chloroform, precipitated by ethanol, then washed with 75% ethanol and
finally dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The quality
of DNA was determined by the ratio of A260/A280. One microgram of genomic DNA was digested overnight with 10 U of
restriction enzymes (New England Biolabs, Ipswich, USA) in 200 ml volume.
Oligonucleotide primers and
adaptor
The specific primers for each walking step were designed based on
the known sequence. Primers for the gene trap vector were: GT1, 5‘-CTCACCTTGCTCCTGCCGAGAAAGTATCCA-3‘;
GT2, 5‘-CGGAATTCGGGCTGACCGCTTCCTCGTGCTTTA-3‘. Primers for the Xenopus
PAPC gene were: PAPC1, 5‘-CTCAGCTGCCCATCACTCTCACGGACT-3‘;
PAPC2, 5‘-CAATTACAGTGCCAGGGGGTTCTTCTTC-3‘. The upper strand for
the BamHI-specific adaptor was 5‘-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGTctag-3‘, and the upper strand
for the HindIII-specific adaptor was 5‘-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGTtcga-3‘. The lower strand for
the adaptor was 5‘-PO4–agcttaccagccc-NH2-3‘. Adaptor
primer AP1 was 5‘–gtaatacgactcactataggc-3‘
and adaptor primer AP2 was 5‘–acaatagggcacgcgtggt-3‘.
The 5‘ end of the down strand of the adaptor was phosphorylated and an
amino group at the 3‘ end was modified. The same amount of upper strand
and down strand of the adaptor were denatured at 80 ?C for 10 min, then
annealed at room temperature for 30 min.
Generation of DNA
adaptor-linked digests
The ligation mixture contained 1 mg of digested genomic
fragment, 1 U of T4 DNA ligase (New England Biolabs) and 50 mM adaptor in T4
DNA ligase buffer. The ligation reaction was carried out overnight at 16 ?C.
The genomic fragments ligated to the enzyme-specific adaptor were purified.The ligation mixture contained 1 mg of digested genomic
fragment, 1 U of T4 DNA ligase (New England Biolabs) and 50 mM adaptor in T4
DNA ligase buffer. The ligation reaction was carried out overnight at 16 ?C.
The genomic fragments ligated to the enzyme-specific adaptor were purified.
PCR amplification and cloning
of products
The first-round PCR amplification was carried out in a 50 ml of reaction
mixture containing 5 ml of LA buffer II (Mg2+ plus), 250 mM dNTP, 10 pM GSP, 10 pM AP1, 100 ng of adaptor-ligated genomic DNA,
and 2.5 U of LA Taq polymerase (TaKaRa, Takara, Japan). After pre-denaturing at
94 ?C for 3 min, the condition of the first 5 cycles was: denaturing at 94 ?C
for 30 s, annealing at 70 ?C for 30 s with extension at 72 ?C for 4 min. The
annealing temperature was reduced by 0.5 ?C per cycle. PCR parameters for the
proceeding 30 cycles were: denaturing at 94 ?C for 30 s, annealing at 60 ?C for
1 min with extension at 72 ?C for 4 min, and the final extension at 72 ?C for 5
min.The second-round PCR was carried out in a 50 ml reaction
mixture containing 1 ml of the first-round PCR product diluted 1:100, 2.5 U of LA buffer
II (Mg2+ plus), 250 mM dNTP, 10 pM
AP1, 10 pM GSP2, and 2.5 ml of LA Taq polymerase. The PCR program was the same as indicated
above. The second-round PCR product was run on a 1% agarose gel. Each band was
isolated by Gel Cleanup (Eppendorf, Hamburg, Germany) and cloned into pGEM-T
easy vector (Promega, Madison, USA). The positive clones were sequenced with
ABI PRISM 377 by Shanghai Biotech Company (Shanghai, China).
Southern blot
To produce a Southern blot probe specific to the gene trap vector,
the neomycin resistance gene was amplified and the genomic DNA from the gene
trap mouse line was prepared and digested overnight with HindIII and EcoRI
restriction enzymes. Digested DNA (10 mg per lane) was run on a 0.8% agarose gel and
transferred to Hybond N+
nylon membrane (GE healthcare,
Piscataway, USA) and hybridized by a 32P-labeled probe using the Prime-a-Gene labeling kit (Promega). The
membrane was washed twice before autoradiographing at 70 ?C.
Sequence analyses
The sequence data were analyzed using the Editseq program from the
DNASTAR nucleotide sequence analysis package (Dnastar, Madison, USA) and searched in the mouse genome using
the BLAST program (http://www.ncbi.nlm.nig.gov/blast).
Results
Application of touchdown PCR
program for genomic walking
To analyze the precise insertion site of a gene trap vector, the genomic
walking from the gene trap vector is required. We found that all published
protocols have their disadvantages. Obviously, the traditional way of screening
the genomic library is time-consuming. Also the inverse PCR and randomly primed
PCR methods have not been generally applied to walk in uncloned genomic DNA. An
improved adaptor PCR, which used a special adaptor, was reported to walk
upstream from one exon. However the protocol was very inefficient so that its
application was limited [17]. Although several attempts were made to increase
its sensitivity, the method became increasingly complicated and the application
was also limited [7]. According to this protocol (Table 1, protocol 1)
we attempted to identify the flanking sequence of a gene trapping mouse line,
8A21, but failed to get any amplified products [Fig. 1(A), lane 1].Touchdown PCR is a method of PCR by which degenerate primers are
used to avoid amplifying nonspecific sequences [18]. It not only increases
specificity but also enhances yield. Its applications in genomic walking have
been mentioned in some published works [19]. However there is no published
report on applying the touchdown PCR to the adaptor PCR method. We then thought
to combine these two methods in order to increase the PCR efficiency. The
combination of touchdown PCR to adaptor PCR (Table 1, protocol 2) indeed
increased the yield of PCR products, but its smear pattern in agarose gel
argues the amplification specificity of this protocol [Fig. 1(A), lane
2].In the classic touchdown program, the annealing temperature is
reduced by 2 ?C per cycle, and this is the parameter we used in protocol 2.
However, we have accumulated data indicating that scaling down the touchdown
PCR parameters, in terms of slowly reducing the annealing temperature, was the
key step in keeping the specificity of the touchdown PCR program (unpublished
data). We then developed the third protocol, in which the annealing temperature
was lowered by 0.5 ?C per cycle (Table 1, protocol 3) and found it worked
perfectly in producing sharp PCR products (Fig. 1, lane 3). Three bands
of 3.4 kb (F1), 2.1 kb (F2) and 300 bp (F3) in length were obtained from the
gene trapping mouse line, 8A21. Sequence analysis showed that one of the three
specific bands, F1 [Fig. 1(A), lane 3], contained a 597 bp trapping
vector sequence in reverse to a 3.2 kb trapping vector sequence, indicating the
two trapping vectors tandemly, but reversely, integrated into the genome [Fig.
1(B), F1]. The second, F2, contained the 597 bp trapping vector sequence
together with a 1.5 kb sequence; and we searched the currently available mouse
genome database using the BLAST program, and it revealed that the trapping
vector was inserted into mouse chromosome 4 A5 loci encoding a P4 ATPase [Fig.
1(B), F2]. F3 was a non-specific amplificon. Thus, two insertion sites,
with their flanking sequences, were identified only by our optimized adaptor
PCR. Southern blot analysis was carried out using the gene trap
vector-specific probe to identify the number of insertion sites. The genomic
DNA extracted from the gene trap mouse line was digested with HindIII or
EcoRI restriction enzyme and transferred onto the nylon membrane. Two
insertion sites were characterized, as shown in Fig. 1(C). This result
supports the fact that optimized adaptor PCR can accurately identify multiple
trapping vector insertion sites.It requires only two rounds of PCR to get mouse gene trap insertion
sites, which is much quicker than all previously reported methods for cloning
flanking sequences. The rapid and efficient simultaneous cloning of multiple
insertion sites indicates that the optimized PCR would be a useful tool to
verify gene trap insertion sites.
Cloning PAPC promoter from Xenopus laevis
Encouraged by the rapid cloning of the trapping insertion site, we
then used the optimized adaptor PCR protocol to isolate the promoter of the X.
laevis PAPC gene, as the genome of X. laevis has not yet been
sequenced. Two specific primers, PAPC1 and PAPC2, were designed based on the sequence
adjacent to the Xenopus PAPC transcription start site. The
genomic DNA was isolated from the Xenopus liver and digested with a
restriction enzyme, HindIII. The purified genomic fragments were ligated
to the HindIII adaptor. Three protocols with different PCR conditions as
shown in Table 1 were also simultaneously applied for genomic walking.
After two rounds of PCR, the PCR products were analyzed by gel electrophoresis.
As shown in Fig. 2(A), a band of approximately 5 kb was amplified from HindIII-specific
adaptor-ligated genomic DNA [Fig. 2(A), lane 3]; while there was no
obvious bands in the two former protocols [Fig. 2(A), lanes 1 and 2].
The specific band F was isolated and sequenced. Sequence analysis indicated
that F was related to Xenopus PAPC promoter.We also used the same protocol to isolate the promoter of mespo,
another Xenopus gene, and got the same sharp results (data not shown).
In each case the whole procedure, from preparing tissues to obtaining
sequences, took only one week. Thus, the feasibility of this approach in
cloning sequences flanking a gene/vector from different genomes is evident.
Discussion
Three kinds of PCR strategies were developed for chromosome walking:
inverse PCR [2]; random primer PCR [36]; and ligation-mediated PCR [815].
Inverse PCR is used less for chromosome walking because of the limitation of
available restriction sites in the unknown/known region or poor circularization
of the template molecule. Ligation-mediated PCR and random primer PCR are more
popular for chromosome walking. The latter is the simplest and most popular
method for identification of T-DNA or transposon insertions, but its amplified
products are generally small (<1 kb). To obtain larger fragments (>1
kb) or to walk step by step, we usually resort to ligation-mediated PCR.
However, this is an inefficient strategy because, in most cases, non-specific
amplification accounts for the major proportion of the final PCR products. To
increase its specificity, an improved adaptor PCR using a special adaptor has
been developed [7]. However,the genomic complexity from different species and
low effiencent PCR program would make amplification efficiency lower,blocking
its applications. Several improved ligation-mediated PCRs increase the
amplification efficiency but their manipulations are complicated. Here we
combined the touchdown PCR with the adaptor PCR and scaled down the parameters,
developing an optimized touchdown PCR protocol. Slowly lowering the annealing
temperature makes primers find their targets precisely, so they yield specific
products efficiently. By simple modification of the PCR program, our optimized
touchdown PCR protocol could largely improve the application of adaptor PCR in
genome research.We believe that the optimized adaptor PCR is a powerful tool in the
following genome-related experiments: (1) identifying gene trap or other
transgenic vector insertion sites, especially multi-copy insertions; (2) isolating
flanking sequences from known sequences, such as cloning a gene promoter from
a cDNA fragment; (3) filling the gaps in genome sequencing; and (4) obtaining
non-conserved regions of genes in uncharacterized species, according to the
conserved sequences of reported genes.In our experiments, we found several key points for the successful
application of the optimized adaptor PCR. First, the touchdown rate should be
slow; in our case, the rate of 0.5 ?C worked perfectly in amplifying the target
bands. Second, genomic DNA with a very high average molecular weight should be
digested efficiently by enzyme. Finally, gene-specific primers should be highly
specific, preferably approximately 2530 nucleotides long, and hairpin
sequences and primer dimmers should be avoided.
Acknowledgements
We thank Dr. Babara I. Meyer
and Dr. Ulrike Teichmann (Department
of Molecular Cell Biology, Max-Plank Institutes of Biophysical Chemistry,
Gotingen, Germany) for kindly
providing gene trap mouse lines and Dr. Yuan-xin
Hu (Institutes of Biochemistry
and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy
of Sciences, Shanghai, China) for technical help with Southern blot. We are
also grateful to Dr. Xu-dong Zhao (Department of Medical Genetics,
Shanghai Jiaotong University School of Medicine, Shanghai, China) for
discussion. We thank Dr. Cheng-fu
Sun and Dr. Shuang-wei Li
(Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences) for their critical reading of this
manuscript.
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