BACKGROUND
the advent of the genomics era has had a massive impact on almost all fields of biological enquiry.
however, it is still necessary to employ chromosome walking and flanking sequence cloning procedures in order to determine full gene structure.
such methods are also required to identify t-dna insertion sites, and this is particularly relevant for species where large t-dna insertion libraries exist, eg.
rice and arabidopsis  <cit> .
currently, a number of pcr-based methods are available for these purposes, including random pcr, inverse pcr, panhandle pcr, and cassette pcr.
however, each of these methods has drawbacks when considered for wide use in the amplification of target regions.
for example, inverse pcr is rarely used for chromosome walking due to problems with the availability of restriction sites in the unknown/known regions, and/or poor circularization of the template molecule  <cit> .
ligation-mediated pcr has proved to be too inefficient for routine use in flanking sequence identification, with non-specific amplified products accounting for the major proportion of the final pcr products  <cit> .
in recent years, improved methodologies have been developed to address the problems outlined above  <cit> .
the most successful of these involve pcr procedures using random primers, notably, the thermal asymmetric interlaced -pcr method.
such methods are relatively low cost and have the advantage of being relatively direct, i.e.
there is no requirement for intermediate steps involving restriction enzyme digestion of the dna template or ligation reactions  <cit> .
consequently, tail-pcr has been widely used, notably for the amplification of junction dna at t-dna insertion sites  <cit> , the rapid isolation of promoter sequences  <cit> , and the cloning of full length functional genes  <cit> .
however, tail-pcr is a time consuming procedure that usually involves three rounds of amplification with each round consisting of multiple pcr cycles.
the method often produces nonspecific amplification products due to the short arbitrary degenerate  primers and low annealing temperatures, and may also achieve non-efficient amplification of target sequences  <cit> .
furthermore, conventional tail-pcr tends to produce small amplification products  <cit> .
tail-pcr has subsequently been adapted, but the improved high-efficiency  tail-pcr version still remains time consuming, and is expensive in terms of primer design and synthesis.
the hitail-pcr technique also poses a great challenge to the activity of dna polymerase, and so is frequently associated with inefficient amplification results.
recently, several alternative methods have emerged to perform chromosome walking or flanking sequence cloning.
however, such methods still have the drawback that they are either restriction enzyme-dependent, phi <dig> dna polymerase-dependent, or require a series of template dna purification steps  <cit> .
thus, these alternative methods are still laborious and do not display the wide application potential of conventional tail-pcr.
therefore, it is highly desirable that further novel methods are developed for rapid and efficient chromosome walking or flanking sequence cloning.
in order to acquire precise flanking fragments rapidly, we have developed a novel efficient method, namely fusion primer and nested integrated-pcr .
we believe that this method has the potential to have wide-spread application in genetics and genome walking studies, and is applicable throughout diverse organisms.
in order to demonstrate the power of fpni-pcr, we performed long pcr walking tests on various genomic targets namely, ft, tfl <dig> and soc <dig> orthologs from the spiraea cantoniensis, pyracantha fortuneana, photinia serrulata, fragaria ananassa, rosa hybrida, prunus mume and prunus yedoensis species of rosaceace,  <dig> myb genes of rosa rugosa,  <dig> promoters from mads-box transcription factors of petunia hybrida, and  <dig> flanking sequences of t-dna insertion sites in nicotiana.
in each case, successful amplification was achieved of the unknown genomic dna fragments .
in addition, comparison with genomic sequence databanks confirmed that fpni-pcr successfully amplified various lengths of the flanking regions of  <dig> members of the wuschel gene family from arabidopsis, also the flanking regions of the osft, osmads <dig> and ostuba <dig> genes from rice, and an archived flanking sequence from the pcambia <dig> vector.
furthermore, a comparative analysis with conventional tail-pcr indicated that our novel fpni-pcr method is more flexible, time saving and powerful than tail-pcr or hitail-pcr.
thus, fpni-pcr has high potential for application in identifying tagged sequences, or for genomic walking from known dna regions toward unknown regions in organisms with a large genome.
RESULTS
principle of fpni-pcr
the basic principle of fpni-pcr is outlined in figure  <dig> and  <dig>  the method centers on a series of primers encompassing sequence-specific primers , and fusion primers which contain an arbitrary degenerate  section fused to a section of determined sequence .
the fusion of a known adaptor of determined sequence to the 5'-end of an ad  is the main characteristic of fpni-pcr , and it is this trait which differentiates fpni-pcr from tail-pcr .
the fpni-pcr protocol contains three key steps.
in the first step, a large complex mixture of dna reactions is prepared using  <dig>  μl of a gene-specific primer  designed to the genomic region of known sequence, and  <dig>  μl of a combination of nine fusion arbitrary degenerate primers ; other designed primers for fpni-pcr are presented in additional files  <dig> tables s1-s <dig>  the details of cycling parameters and pcr conditions used in this study are listed in table  <dig>  this first step consists of 3- <dig> repeats of two high stringency cycles followed by a low stringency cycle.
theoretically, single stranded pcr products from the gene-specific primer are generated during the high stringency cycles, and double-stranded products utilizing the fp primers  are developed during the low stringency cycle.
after 3- <dig> repeated cycles of this pcr regime, it is predicted that the intended target products are partially synthesized and are accompanied by other, nonspecific, products .
in the second and third steps, nested pcr is conducted using  <dig> ul of target-specific primers  and fp-specific primers .
these steps are high stringency pcr using high annealing temperatures so that target products are selectively amplified.
nonspecific products are not amplified in these steps, in part due to the nested approach with different primers.
in addition, the large hairpin structure employed in some nonspecific products also contributes to prevent to further amplification .
thus, the nonspecific products generated from the first pcr step in fpni-pcr are not amplified in the second and third steps, and become substantially diluted in the final mix .
note: * = few cycles of thermal asymmetric interlaced pcr: target product generation; incorporation of suppression pcr inverted repeat elements in partial non specific product; ** standard high stringency pcr: target sequence exponential amplification; partial nonspecific products suppression pcr; *** = standard nested-pcr: completely eliminating high background or nonspecific amplification in the second pcr.
parameters in the design and optimization of fpni-pcr
we compared two types of ad primers.
in the basic form, i.e.
type i primers, the ad primer was fused to the 3' end of an adaptor of known sequence .
type ii primers included a hairpin structure at the 5'end of the single-stranded adaptor .
we compared the results from such type i and type ii primers by testing them in combination with the same gene-specific  primers and under the same cycling parameters.
the final results of amplification of the target products indicated no obvious differences between the two primer types, i.e.
they presented similar sizes of products on the agarose gels .
thus, the hairpin structure at the 5'end of the type ii single-stranded adaptors had no apparent impact on amplification of the target.
type iii primers describe arbitrarily degenerative primers with 4- <dig> fixed nucleotides at the 3' end , and these primers were also successfully used to amplify the flanking regions of known fragments, with the various sizes of the product fragments relating to the given fusion primer.
the primary round of pcr in the fpni-pcr procedure includes an optional low stringency pcr cycle , to follow the two high stringency pcr cycles .
we tested six different annealing temperatures in these low stringency cycles, using sp <dig> and various fp primers , and found an effect on the final products obtained .
however, all of the reactions yielded flanking regions and the patterns of amplified fragments after the secondary round of pcr  exhibited strong similarities despite the differences in annealing temperatures of the first pcr step.
in many cases, lower annealing temperatures in the low stringency cycles of the first pcr step yielded fewer amplified products .
this influence of annealing temperature of the low stringency cycle was confirmed by repeated experiments for the cloning of various genes and t-dna insertion loci.
however, if the low stringency cycle of the first fpni-pcr step was entirely omitted, successful amplification of the target product was still achieved .
effectiveness and accuracy of fpni-pcr
using the fpni-pcr approach, we have successfully isolated  <dig> complete genomic sequences containing the ft, tfl <dig> and soc <dig> gene orthologs from seven rosaceace species, namely spiraea cantoniensis, pyracantha fortuneana, photinia serrulata, fragaria ananassa, rosa hybrida, prunus mume and prunus yedoensis.
in addition, we have used fpni-pcr to isolate four myb genes of rosa rugosa, three promoters from the mads-box transcription factor family of petunia hybrida, and four t-dna flanking sequences in transgenic tobacco lines.
all of these reactions were conducted using a total of just nine fp primers , in conjunction with specific primers designed to the conserved/partially known genomic fragments or known t-dna sequences.
sequences have been deposited in the genbank library, as shown in table  <dig>
comparative experiments using the same specific primers showed that, in most cases , longer products could be obtained by fpni-pcr than by tail-pcr .
furthermore, fpni-pcr was found to be more reliable as a method of amplifying target genes and unknown flanking sequences.
thus, in a comparative test using several appropriate ad primers, tail-pcr frequently failed to amplify the predicted dna fragment.
figure 5a shows the results of tail-pcr in the cloning of the ft ortholog from pyracantha fortuneana using  <dig> gene-specific primers and the fp1- <dig> arbitrary degenerate primers.
in this experiment, a  <dig> bp fragment was obtained from tail-pcr with just one  ad primer.
by contrast, figure 5b shows the products generated by fpni-pcr using the same primers for the same genome walking experiment.
here, four of the fpni-pcr reactions amplified specific fragments, and the longest fragment was ca.
<dig>  kp.
genomic walking for the soc1-like gene in pyracantha fortuneana provided another situation in which we encountered "off-target" results using tail-pcr, whereas, using the fpni-pcr method we obtained two specific fragments of  <dig> bp and  <dig> bp .
sequence analysis of  <dig> amplified dna fragments resulting from the fpni-pcr technique showed that they corresponded with 100% accuracy to the target products.
efficiency of fpni-pcr
fpni-pcr facilitates the rapid identification of target genomic sequences by permitting the use of short-cuts for a number of the steps between tissue isolation and target product sequencing, thereby reducing the input of time and effort compared to other current methods of flanking sequence cloning.
for example, fpni-pcr can be employed to generate target genomic products when cell lysates are used to supply the dna templates .
for this procedure, we extracted cell lysates from the young leaves of transgenic tobacco, petunia hybrida and rosa hybrida using the rapid naoh extraction method.
we found no difference in the success of target product amplification from these cell lysates compared to that from the higher quality dna templates as purified by the ctab method.
in addition, we found no differences between the results of direct sequencing of the fpni-pcr products compared to indirect sequencing of the cloned products within the pmd18-t vector.
fpni-pcr has the advantage that, after the first step , we can proceed to the second step directly, without requiring the prior dilution of the products from the first pcr step.
when using such practices, we commonly achieved > 85% positive cloning of target products following the secondary pcr reaction.
thus, we were able to amplify target products using just 42- <dig> pcr cycles, and this was completed in less than  <dig> hours from the start of tissue extraction.
when proceeding through the third pcr and final sequencing steps, fpni-pcr was found to provide 100% positive results in our working tests of genomic cloning.
in total, the procedure was completed in just 54- <dig> cycles, taking less than  <dig> hours.
discussion
fpni-pcr involves a novel strategy of amplification
the fpni-pcr strategy is based on the following technical points.
the arbitrary degenerate oligonucleotides forming the 3' section of the fp primers  have been designed with the intention of maximizing the level of similarity with common genomic oligo sequences throughout the various taxa.
during the first pcr step, the 3'-ends of the oligos forming the arbitrary degenerate portions of the fps  need to pair effectively with selected sites of high similarity in the single-stranded dna template.
a lower annealing temperature may be used to increase mismatch sites and, thereby, enlarge the universality of the designed fusion primers.
between  <dig> and  <dig> cycles in the first pcr step are used to ensure that the desired target products are present within the amplified mix.
in the secondary and tertiary pcr steps, the target dna should be exponentially amplified via high stringency cycles, while non-target products are not amplified by the gene-specific and fp-specific primer pairs.
off-target amplification may also be suppressed by the stem-loop structure of nonspecific dna products.
the use of high stringency pcr cycles encourages the efficient and specific amplification of the target products, while off-target pcr products are progressively diluted.
this aspect of the fpni-pcr strategy represents a key difference from the tail-pcr method developed by liu and whittier  <cit> .
tail-pcr employs multiple low stringency annealing cycles and, thus, nonspecific products are readily amplified.
in tail-pcr, enrichment of the target products relies upon the difference in amplification velocity between the target and non-target products.
thus, the tail-pcr protocol involves two rounds of laborious dna dilutions and numerous  cycles of pcr  <cit> .
an improved method, named hitail-pcr, similarly involves two rounds of laborious dna dilutions and multiple low and high stringency pcr cycles, thereby still providing opportunities for nonspecific product amplification  <cit> .
in addition, hitail-pcr is uneconomical in terms of primer design, with the secondary gene-specific primers requiring the addition of >  <dig> nucleotides  <cit> .
fpni-pcr represents a very powerful tool in genomic walking and flanking sequence cloning
tail-pcr has been widely used for genome walking and flanking sequence cloning in plants and other organisms.
commonly, however, specific products appear in the second round of pcr only to disappear during the third round of pcr  <cit> .
liu and chen reported that just 60% of reactions yielded specific products  <cit> .
our own tests of the tail-pcr method found that, on some occasions, no product was observed even after three rounds of pcr.
generally, smaller and fewer products were generated by tail-pcr as compared to the corresponding reactions using fpni-pcr .
for inverse and ligation-mediated pcr-based genome-walking methods, successful amplification of target products relies on the restriction fragmentation of genomic dna.
when employing such techniques, we also encountered some "off-target" situations, possibly due to the location of the available restriction sites relative to the locus-specific primer  <cit> .
by contrast, repeated tests of the fpni-pcr method using the nine given fp primers revealed no "off-target" cases, and more than 80% of fpni-pcr reactions yielded dna fragments larger than  <dig>  kb.
thus, our results indicate that fpni-pcr is a more powerful technique for genomic walking and flanking sequence cloning studies.
in addition, the fpni-pcr method makes it feasible to amplify large sections of specific dna fragments.
this can be achieved through the control of annealing temperatures during the low stringency cycles of the first pcr step.
the control of this parameter can also be used to avoid the cloning of "off target" flanking sequences.
fpni-pcr is a simple and rapid method of genomic walking and flanking sequence cloning
methods involving inverse pcr and ligation-mediated pcr techniques suffer from the requirement for complicated manipulations, such as restriction cleavage, ligation, or tailing before pcr amplification  <cit> .
consequently, the use of such methods to successfully amplify and clone the target product usually requires a time-scale exceeding one working day.
similarly, the successful application of tail-pcr or hitail-pcr methods is also time-consuming, with the time taken to perform the necessary number of pcr cycles  requiring a full working day   <cit> .
using fpni-pcr, a total about 42- <dig> cycles  can be adequate to produce a good rate of success in target product amplification and, in our hands, a total of 54- <dig> cycles  ensured a successful result in 100% of cases.
both tail-pcr and fpni-pcr methods demand only extremely modest levels of quantity and purity of the template dna, thus, they can work well with both cell lysates and crude dna extracts.
using fpni-pcr, however, we were able to choose a wider range of annealing temperatures during the low stringency cycles of the first pcr step, thereby extending the scope of target product that could be successfully amplified.
the currently widely used genome walker kit from clontech employs restriction enzyme cleavage of dna and adaptor-ligated genomic dna construction, and also involves a nested pcr strategy.
the procedures of integrated restriction cleavage, ligation and tailing make the genome walker kit a substantially more complicated and time-consuming process than the first pcr step of fpni-pcr.
thus, we believe that in comparison to the other reported methods of genomic walking and flanking sequence cloning , fpni-pcr is technically less demanding and is more time-efficient.
fpni-pcr is highly effective and accurate
we conducted  <dig> rounds of fpni-pcr reactions using the nine given fp primers, and in each case we successfully amplified products and experienced no "off-target" results.
this contrasts to the experiences when employing ligation-dependent pcr methods, when it is often a problem to find available restriction sites and obtain ligation products  <cit> .
in addition, sequence analysis of  <dig> amplification products from fpni-pcr indicated that the accuracy of positive cloning in our experiments was 100%.
by comparison, 60-80% of reactions yielded specific products according to the original tail-pcr technical report  <cit> , and a success rate of 93% was recorded for the improved hitail-pcr procedure  <cit> .
thus, we conclude that fpni-pcr shows a substantially higher level of effectiveness than the established tail-pcr procedures.
we suggest that the high rate of positive amplification results, at least partially, from the high stringency pcr cycles in the second and third pcr stages of the fpni-pcr protocol.
CONCLUSIONS
to date, tail-pcr has been widely used for genome walking and flanking sequence cloning in molecular biology research, but it is still laborious and time-consuming.
recently, several alternative methods have been introduced  <cit> , including the genome walker kit from clontech, but these methods still involve the use of restriction enzyme-mediated digestion, phi <dig> dna polymerase-mediated amplification, and a series of template dna purification steps.
here, we report a simple and effective pcr method namely, fusion primer and nested integrated pcr .
this method is based on the use of arbitrary degenerate nucleotides reflecting natural restriction sites or site-dependent nucleotides in the genomic dna to design universal primers.
these fp primers are used to conduct the first round of pcr, which is followed by two to three rounds of nested pcr.
we undertook a large number of experiments using fpni-pcr in order to robustly demonstrate that this novel method has the capability to be more powerful, effective and accurate than the established tail-pcr method.
furthermore, the fpni-pcr method is completed in a shorter time-scale than other widely-used genome-walking techniques, and is significantly less technically complex to perform.

 the advent of the genomics era has had a massive impact on almost all fields of biological enquiry. however, it is still necessary to employ chromosome walking and flanking sequence cloning procedures in order to determine full gene structure. such methods are also required to identify t-dna insertion sites, and this is particularly relevant for species where large t-dna insertion libraries exist, eg. rice and arabidopsis. currently, a number of pcr-based methods are available for these purposes, including random pcr, inverse pcr, panhandle pcr, and cassette pcr. however, each of these methods has drawbacks when considered for wide use in the amplification of target regions. for example, inverse pcr is rarely used for chromosome walking due to problems with the availability of restriction sites in the unknown/known regions, and/or poor circularization of the template molecule. ligation-mediated pcr has proved to be too inefficient for routine use in flanking sequence identification, with non-specific amplified products accounting for the major proportion of the final pcr products. in recent years, improved methodologies have been developed to address the problems outlined above. the most successful of these involve pcr procedures using random primers, notably, the thermal asymmetric interlaced -pcr method. such methods are relatively low cost and have the advantage of being relatively direct, i.e. there is no requirement for intermediate steps involving restriction enzyme digestion of the dna template or ligation reactions. consequently, tail-pcr has been widely used, notably for the amplification of junction dna at t-dna insertion sites, the rapid isolation of promoter sequences, and the cloning of full length functional genes. however, tail-pcr is a time consuming procedure that usually involves three rounds of amplification with each round consisting of multiple pcr cycles. the method often produces nonspecific amplification products due to the short arbitrary degenerate primers and low annealing temperatures, and may also achieve non-efficient amplification of target sequences. furthermore, conventional tail-pcr tends to produce small amplification products. tail-pcr has subsequently been adapted, but the improved high-efficiency tail-pcr version still remains time consuming, and is expensive in terms of primer design and synthesis. the hitail-pcr technique also poses a great challenge to the activity of dna polymerase, and so is frequently associated with inefficient amplification results. recently, several alternative methods have emerged to perform chromosome walking or flanking sequence cloning. however, such methods still have the drawback that they are either restriction enzyme-dependent, phi dna polymerase-dependent, or require a series of template dna purification steps. thus, these alternative methods are still laborious and do not display the wide application potential of conventional tail-pcr. therefore, it is highly desirable that further novel methods are developed for rapid and efficient chromosome walking or flanking sequence cloning. in order to acquire precise flanking fragments rapidly, we have developed a novel efficient method, namely fusion primer and nested integrated-pcr. we believe that this method has the potential to have wide-spread application in genetics and genome walking studies, and is applicable throughout diverse organisms. in order to demonstrate the power of fpni-pcr, we performed long pcr walking tests on various genomic targets namely, ft, tfl and soc orthologs from the spiraea cantoniensis, pyracantha fortuneana, photinia serrulata, fragaria ananassa, rosa hybrida, prunus mume and prunus yedoensis species of rosaceace, myb genes of rosa rugosa, promoters from mads-box transcription factors of petunia hybrida, and flanking sequences of t-dna insertion sites in nicotiana. in each case, successful amplification was achieved of the unknown genomic dna fragments. in addition, comparison with genomic sequence databanks confirmed that fpni-pcr successfully amplified various lengths of the flanking regions of members of the wuschel gene family from arabidopsis, also the flanking regions of the osft, osmads and ostuba genes from rice, and an archived flanking sequence from the pcambia vector. furthermore, a comparative analysis with conventional tail-pcr indicated that our novel fpni-pcr method is more flexible, time saving and powerful than tail-pcr or hitail-pcr. thus, fpni-pcr has high potential for application in identifying tagged sequences, or for genomic walking from known dna regions toward unknown regions in organisms with a large genome.  principle of fpni-pcr the basic principle of fpni-pcr is outlined in figure and the method centers on a series of primers encompassing sequence-specific primers, and fusion primers which contain an arbitrary degenerate section fused to a section of determined sequence. the fusion of a known adaptor of determined sequence to the 5'-end of an ad is the main characteristic of fpni-pcr, and it is this trait which differentiates fpni-pcr from tail-pcr. the fpni-pcr protocol contains three key steps. in the first step, a large complex mixture of dna reactions is prepared using μl of a gene-specific primer designed to the genomic region of known sequence, and μl of a combination of nine fusion arbitrary degenerate primers; other designed primers for fpni-pcr are presented in additional files tables s1-s the details of cycling parameters and pcr conditions used in this study are listed in table this first step consists of 3- repeats of two high stringency cycles followed by a low stringency cycle. theoretically, single stranded pcr products from the gene-specific primer are generated during the high stringency cycles, and double-stranded products utilizing the fp primers are developed during the low stringency cycle. after 3- repeated cycles of this pcr regime, it is predicted that the intended target products are partially synthesized and are accompanied by other, nonspecific, products. in the second and third steps, nested pcr is conducted using ul of target-specific primers and fp-specific primers. these steps are high stringency pcr using high annealing temperatures so that target products are selectively amplified. nonspecific products are not amplified in these steps, in part due to the nested approach with different primers. in addition, the large hairpin structure employed in some nonspecific products also contributes to prevent to further amplification. thus, the nonspecific products generated from the first pcr step in fpni-pcr are not amplified in the second and third steps, and become substantially diluted in the final mix. note: * = few cycles of thermal asymmetric interlaced pcr: target product generation; incorporation of suppression pcr inverted repeat elements in partial non specific product; ** standard high stringency pcr: target sequence exponential amplification; partial nonspecific products suppression pcr; *** = standard nested-pcr: completely eliminating high background or nonspecific amplification in the second pcr. parameters in the design and optimization of fpni-pcr we compared two types of ad primers. in the basic form, i.e. type i primers, the ad primer was fused to the 3' end of an adaptor of known sequence. type ii primers included a hairpin structure at the 5'end of the single-stranded adaptor. we compared the results from such type i and type ii primers by testing them in combination with the same gene-specific primers and under the same cycling parameters. the final results of amplification of the target products indicated no obvious differences between the two primer types, i.e. they presented similar sizes of products on the agarose gels. thus, the hairpin structure at the 5'end of the type ii single-stranded adaptors had no apparent impact on amplification of the target. type iii primers describe arbitrarily degenerative primers with 4- fixed nucleotides at the 3' end, and these primers were also successfully used to amplify the flanking regions of known fragments, with the various sizes of the product fragments relating to the given fusion primer. the primary round of pcr in the fpni-pcr procedure includes an optional low stringency pcr cycle, to follow the two high stringency pcr cycles. we tested six different annealing temperatures in these low stringency cycles, using sp and various fp primers, and found an effect on the final products obtained. however, all of the reactions yielded flanking regions and the patterns of amplified fragments after the secondary round of pcr exhibited strong similarities despite the differences in annealing temperatures of the first pcr step. in many cases, lower annealing temperatures in the low stringency cycles of the first pcr step yielded fewer amplified products. this influence of annealing temperature of the low stringency cycle was confirmed by repeated experiments for the cloning of various genes and t-dna insertion loci. however, if the low stringency cycle of the first fpni-pcr step was entirely omitted, successful amplification of the target product was still achieved. effectiveness and accuracy of fpni-pcr using the fpni-pcr approach, we have successfully isolated complete genomic sequences containing the ft, tfl and soc gene orthologs from seven rosaceace species, namely spiraea cantoniensis, pyracantha fortuneana, photinia serrulata, fragaria ananassa, rosa hybrida, prunus mume and prunus yedoensis. in addition, we have used fpni-pcr to isolate four myb genes of rosa rugosa, three promoters from the mads-box transcription factor family of petunia hybrida, and four t-dna flanking sequences in transgenic tobacco lines. all of these reactions were conducted using a total of just nine fp primers, in conjunction with specific primers designed to the conserved/partially known genomic fragments or known t-dna sequences. sequences have been deposited in the genbank library, as shown in table comparative experiments using the same specific primers showed that, in most cases, longer products could be obtained by fpni-pcr than by tail-pcr. furthermore, fpni-pcr was found to be more reliable as a method of amplifying target genes and unknown flanking sequences. thus, in a comparative test using several appropriate ad primers, tail-pcr frequently failed to amplify the predicted dna fragment. figure 5a shows the results of tail-pcr in the cloning of the ft ortholog from pyracantha fortuneana using gene-specific primers and the fp1- arbitrary degenerate primers. in this experiment, a bp fragment was obtained from tail-pcr with just one ad primer. by contrast, figure 5b shows the products generated by fpni-pcr using the same primers for the same genome walking experiment. here, four of the fpni-pcr reactions amplified specific fragments, and the longest fragment was ca. kp. genomic walking for the soc1-like gene in pyracantha fortuneana provided another situation in which we encountered "off-target" results using tail-pcr, whereas, using the fpni-pcr method we obtained two specific fragments of bp and bp. sequence analysis of amplified dna fragments resulting from the fpni-pcr technique showed that they corresponded with 100% accuracy to the target products. efficiency of fpni-pcr fpni-pcr facilitates the rapid identification of target genomic sequences by permitting the use of short-cuts for a number of the steps between tissue isolation and target product sequencing, thereby reducing the input of time and effort compared to other current methods of flanking sequence cloning. for example, fpni-pcr can be employed to generate target genomic products when cell lysates are used to supply the dna templates. for this procedure, we extracted cell lysates from the young leaves of transgenic tobacco, petunia hybrida and rosa hybrida using the rapid naoh extraction method. we found no difference in the success of target product amplification from these cell lysates compared to that from the higher quality dna templates as purified by the ctab method. in addition, we found no differences between the results of direct sequencing of the fpni-pcr products compared to indirect sequencing of the cloned products within the pmd18-t vector. fpni-pcr has the advantage that, after the first step, we can proceed to the second step directly, without requiring the prior dilution of the products from the first pcr step. when using such practices, we commonly achieved > 85% positive cloning of target products following the secondary pcr reaction. thus, we were able to amplify target products using just 42- pcr cycles, and this was completed in less than hours from the start of tissue extraction. when proceeding through the third pcr and final sequencing steps, fpni-pcr was found to provide 100% positive results in our working tests of genomic cloning. in total, the procedure was completed in just 54- cycles, taking less than hours. discussion fpni-pcr involves a novel strategy of amplification the fpni-pcr strategy is based on the following technical points. the arbitrary degenerate oligonucleotides forming the 3' section of the fp primers have been designed with the intention of maximizing the level of similarity with common genomic oligo sequences throughout the various taxa. during the first pcr step, the 3'-ends of the oligos forming the arbitrary degenerate portions of the fps need to pair effectively with selected sites of high similarity in the single-stranded dna template. a lower annealing temperature may be used to increase mismatch sites and, thereby, enlarge the universality of the designed fusion primers. between and cycles in the first pcr step are used to ensure that the desired target products are present within the amplified mix. in the secondary and tertiary pcr steps, the target dna should be exponentially amplified via high stringency cycles, while non-target products are not amplified by the gene-specific and fp-specific primer pairs. off-target amplification may also be suppressed by the stem-loop structure of nonspecific dna products. the use of high stringency pcr cycles encourages the efficient and specific amplification of the target products, while off-target pcr products are progressively diluted. this aspect of the fpni-pcr strategy represents a key difference from the tail-pcr method developed by liu and whittier. tail-pcr employs multiple low stringency annealing cycles and, thus, nonspecific products are readily amplified. in tail-pcr, enrichment of the target products relies upon the difference in amplification velocity between the target and non-target products. thus, the tail-pcr protocol involves two rounds of laborious dna dilutions and numerous cycles of pcr. an improved method, named hitail-pcr, similarly involves two rounds of laborious dna dilutions and multiple low and high stringency pcr cycles, thereby still providing opportunities for nonspecific product amplification. in addition, hitail-pcr is uneconomical in terms of primer design, with the secondary gene-specific primers requiring the addition of > nucleotides. fpni-pcr represents a very powerful tool in genomic walking and flanking sequence cloning tail-pcr has been widely used for genome walking and flanking sequence cloning in plants and other organisms. commonly, however, specific products appear in the second round of pcr only to disappear during the third round of pcr. liu and chen reported that just 60% of reactions yielded specific products. our own tests of the tail-pcr method found that, on some occasions, no product was observed even after three rounds of pcr. generally, smaller and fewer products were generated by tail-pcr as compared to the corresponding reactions using fpni-pcr. for inverse and ligation-mediated pcr-based genome-walking methods, successful amplification of target products relies on the restriction fragmentation of genomic dna. when employing such techniques, we also encountered some "off-target" situations, possibly due to the location of the available restriction sites relative to the locus-specific primer. by contrast, repeated tests of the fpni-pcr method using the nine given fp primers revealed no "off-target" cases, and more than 80% of fpni-pcr reactions yielded dna fragments larger than kb. thus, our results indicate that fpni-pcr is a more powerful technique for genomic walking and flanking sequence cloning studies. in addition, the fpni-pcr method makes it feasible to amplify large sections of specific dna fragments. this can be achieved through the control of annealing temperatures during the low stringency cycles of the first pcr step. the control of this parameter can also be used to avoid the cloning of "off target" flanking sequences. fpni-pcr is a simple and rapid method of genomic walking and flanking sequence cloning methods involving inverse pcr and ligation-mediated pcr techniques suffer from the requirement for complicated manipulations, such as restriction cleavage, ligation, or tailing before pcr amplification. consequently, the use of such methods to successfully amplify and clone the target product usually requires a time-scale exceeding one working day. similarly, the successful application of tail-pcr or hitail-pcr methods is also time-consuming, with the time taken to perform the necessary number of pcr cycles requiring a full working day. using fpni-pcr, a total about 42- cycles can be adequate to produce a good rate of success in target product amplification and, in our hands, a total of 54- cycles ensured a successful result in 100% of cases. both tail-pcr and fpni-pcr methods demand only extremely modest levels of quantity and purity of the template dna, thus, they can work well with both cell lysates and crude dna extracts. using fpni-pcr, however, we were able to choose a wider range of annealing temperatures during the low stringency cycles of the first pcr step, thereby extending the scope of target product that could be successfully amplified. the currently widely used genome walker kit from clontech employs restriction enzyme cleavage of dna and adaptor-ligated genomic dna construction, and also involves a nested pcr strategy. the procedures of integrated restriction cleavage, ligation and tailing make the genome walker kit a substantially more complicated and time-consuming process than the first pcr step of fpni-pcr. thus, we believe that in comparison to the other reported methods of genomic walking and flanking sequence cloning, fpni-pcr is technically less demanding and is more time-efficient. fpni-pcr is highly effective and accurate we conducted rounds of fpni-pcr reactions using the nine given fp primers, and in each case we successfully amplified products and experienced no "off-target" results. this contrasts to the experiences when employing ligation-dependent pcr methods, when it is often a problem to find available restriction sites and obtain ligation products. in addition, sequence analysis of amplification products from fpni-pcr indicated that the accuracy of positive cloning in our experiments was 100%. by comparison, 60-80% of reactions yielded specific products according to the original tail-pcr technical report, and a success rate of 93% was recorded for the improved hitail-pcr procedure. thus, we conclude that fpni-pcr shows a substantially higher level of effectiveness than the established tail-pcr procedures. we suggest that the high rate of positive amplification results, at least partially, from the high stringency pcr cycles in the second and third pcr stages of the fpni-pcr protocol.  to date, tail-pcr has been widely used for genome walking and flanking sequence cloning in molecular biology research, but it is still laborious and time-consuming. recently, several alternative methods have been introduced, including the genome walker kit from clontech, but these methods still involve the use of restriction enzyme-mediated digestion, phi dna polymerase-mediated amplification, and a series of template dna purification steps. here, we report a simple and effective pcr method namely, fusion primer and nested integrated pcr. this method is based on the use of arbitrary degenerate nucleotides reflecting natural restriction sites or site-dependent nucleotides in the genomic dna to design universal primers. these fp primers are used to conduct the first round of pcr, which is followed by two to three rounds of nested pcr. we undertook a large number of experiments using fpni-pcr in order to robustly demonstrate that this novel method has the capability to be more powerful, effective and accurate than the established tail-pcr method. furthermore, the fpni-pcr method is completed in a shorter time-scale than other widely-used genome-walking techniques, and is significantly less technically complex to perform. 
the successful amplification of target products through fpni-pcr verified that this novel strategy is an effective, low cost and simple procedure. these arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers. these fusion primers are employed in the first step of an integrated nested pcr strategy which defines the overall fpni-pcr protocol. such methods are used to identify t-dna insertion sites and so are especially relevant for organisms where large t-dna insertion libraries have been created, such as rice and arabidopsis. in brief, a set of universal primers was designed that consisted of various 15- base arbitrary degenerate oligonucleotides. the advent of genomics-based technologies has revolutionized many fields of biological enquiry. in order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, orthologs of genes in various species of rosaceace, myb genes of rosa rugosa, promoters of transcription factors of petunia hybrida, and flanking sequences of t-dna insertion sites in transgenic tobacco lines and specific genes from sequenced genome of rice and arabidopsis.
 the advent of genomics-based technologies has revolutionized many fields of biological enquiry. however, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. such methods are used to identify t-dna insertion sites and so are especially relevant for organisms where large t-dna insertion libraries have been created, such as rice and arabidopsis. the currently available methods for flanking sequence cloning, including the popular tail-pcr technique, are relatively laborious and slow.  here, we report a simple and effective fusion primer and nested integrated pcr method for the identification and cloning of unknown genomic regions flanked known sequences. in brief, a set of universal primers was designed that consisted of various 15- base arbitrary degenerate oligonucleotides. these arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers. these fusion primers are employed in the first step of an integrated nested pcr strategy which defines the overall fpni-pcr protocol. in order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, orthologs of genes in various species of rosaceace, myb genes of rosa rugosa, promoters of transcription factors of petunia hybrida, and flanking sequences of t-dna insertion sites in transgenic tobacco lines and specific genes from sequenced genome of rice and arabidopsis.  the successful amplification of target products through fpni-pcr verified that this novel strategy is an effective, low cost and simple procedure. furthermore, fpni-pcr represents a more sensitive, rapid and accurate technique than the established tail-pcr and hitail-pcr procedures. 