Randomly primed and ligation-independent PCR methods : High efficiency and cost effective strategies for genome walking and flanking sequence cloning

Introduction The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still an important needed tool to isolate the unknown DNA sequences flanked by known sequences or T-DNA insertions in molecular biology research. In this article, we described and compared the principles, efficiency, and applications of the five reported technically less complex methods for chromosome walking or flanking sequence cloning, namely (i) thermal asymmetric interlaced PCR (TAILPCR), (ii)high-efficiency TAILPCR(hiTAIL-PCR),(iii)fusion primer and nested integrated-PCR (FPNIPCR),(iv) Sitefinding-PCR and (v)Selfformed Adaptor PCR (SEFAPCR).While they are all based on the randomly primed PCR without enzyme digestion and ligation procedures, they show difference in efficiency and applications due to the different principles. Methodology The principle, efficiency, and applications of the five enzyme digestion-free methods:(i) TAIL-PCR, (ii) hiTAIL-PCR,(iii) FPNI-PCR, (iv) SiteFinding-PCR and (v) SEFA-PCR are reviewed in detail. Comparative analysis is undertaken to illustrate that the newly developed FPNI-PCR method has shown to be more powerful, effective, time-saving, and accurate than the established conventional reported methods. Conclusion Randomly primed PCR methods represent simplicity, less complex manipulations and possibly will be continuously widely employed by many molecular biology research laboratories. The newly developed FPNI-PCR can be done in a shorter time-scale and it is more powerful, effective, and accurate than conventional TAIL-PCR and will be widely used for genome walking and flanking sequence cloning.


Introduction
Genomics-based technologies allow us to access information on the entire genomic DNA or partial RNA sequence of interest research organisms.However, convenient chromosome walking or flanking sequence cloning is still important and needed in many laboratories.Up to date, many chromosome walking methods based on PCR have been developed.These methods are usually used to identify the unknown regions flanked by known DNA sequences or the insertion tags, particularly in species in which large T-DNA insertion libraries have existed, e.g.Rice and Arabidopsis or those with known genomic sequences 1,11,16 .
The two former methods both rely on the suitable restriction sites in the unknown/known regions, and thus they require high quality of the DNA template and complicated manipulations, including digestion, ligation or circularization of the template molecule.Randomly primed PCR is a PCR-only method using the rationally designed sequence-specific primers and random primers.There is no requirement of intermediate steps involving restriction enzyme digestion or ligation reactions to generate the final DNA template.Randomly primed PCRs have been proved with high efficiency for routine use in flanking sequence cloning because of the simplicity, low cost and high efficiency.Of them, the thermal asymmetric interlaced (TAIL)-PCR method is the typical random PCR without the shortcomings of both of the inverse PCR and ligation-dependent PCR methods.Consequently, in past years, TAIL-PCR has been widely used 5,8,10 .
Recently, a novel efficient method, namely fusion primer and nested integrated-PCR (FPNI-PCR) has also been developed 25 .It is reported that FPNI-PCR method are more flexible, less time consuming and more powerful than TAIL-PCR, and that FPNI-PCR is successfully applied in many organisms 12,25,29,30,31 .In this article, we reviewed the principle, efficiency, and applications of the five reported less technically complicated to perform and enzyme digestion-free methods: (i) TAIL-PCR, (ii) hiTAIL-PCR, (iii)FPNI-PCR, (iv) SiteFinding-PCR and (v) SEFA-PCR.

Methodology
The authors have referenced some of their own studies in this methodology.The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.

TAIL-PCR (The thermal asymmetric interlaced PCR)
In the past years, the thermal asymmetric interlaced (TAIL)-PCR method is the typical and widely used ligation-independent and randomly primed PCR, which was developed by Liu and Whittier in 1995 7 .It relies on the three nested primers that are specific to the known region and one shorter arbitrary degenerate (AD) primer, usually containing 8-15 random nucleotide sequences (Figure 1A).This method involves three round PCR reactions, in every round PCR reaction it includes high-stringency and low-stringency mixed cycles 8 .Of TAIL-PCR, in the primary PCR, the first five high-stringency cycles increase the quantity of the DNA template linearly and the following one low-stringency PCR cycle creates one or more annealing sites for the AD primer.The "supercycles" of high-and low-stringency cycles are performed to guarantee the generation target sequence.
Moreover, the two nested specific primers used in the secondary and tertiary reactions favour the amplification of the interested product.In the primary PCR, three types of products are formed: those primed by both primers (type I), those by the specific primer alone (type II), and those by the AD primer alone (type III) 8 .In the secondary and tertiary PCR, Type II products and the nonspecific type I products can be eliminated by the nested PCR, and the type III nonspecific products (the major source of the nonspecific amplification) cannot be eliminated with the nested specific primers though its quantity is much lower than the specific type I products after the secondary and tertiary reaction.
With the merits of simplicity, efficiency and sensitivity, TAIL-PCR has been widely used in a variety of species, including large-scale determination of T-DNA and transposon insertion sites in Arabidopsis, rice and maize 11,16,17 , and isolation of upstream (promoters) and downstream sequences of the known region 2,18 .However, TAIL-PCR is a time consuming and usually "offtargets" procedure, and the most amplified products are limited to a length of < 1kb.

SiteFinding-PCR
In 2005, based on the suppression PCR 19 , another ligation-independent Sitefinding-PCR was developed by Tanetal. 20.In this method, the adaptor-specific primer contains excess 5'-base to make nonspecific PCR products that form the stem-loop structure.Sitefinding-PCR was primed by a Sitefinder at a low temperature and then the target products were amplified exponentially with genespecific and SiteFinder primers (Figure 1B).
Finally, the target products are screened out by another gene-specific primer and a vector primer 20 .The key point pertaining to this method is the design of the SiteFinder.For example the SiteFinder-1contains the oligonucleotide 5'-NNNNNNGCCT-3' at the 3' end and was utilized to find the GCCT sites on the target sequence and non-target sequence.SiteFinder-1 and SiteFinder-2 shared a common sequence in the 5' half with the length of 51 oligonucleotides including a rare restriction enzyme site for Not I, which facilitates cloning into vectors.
The SiteFinder primers SFP1 and SFP2 are specific to the 51 oligonucleotide sequence.This method consists of three main steps:

HiTAIL-PCR (high-efficiency TAIL-PCR)
TAIL-PCR has subsequently been adapted as high-efficiency TAIL-PCR (hiTAIL-PCR) as reported by Liu 9 .This PCR combines the advantages of the TAIL-cycling and suppression-PCR, thus it incorporate the special design of the arbitrary degenerated and specific primers (Figure 1C).To some extent, it can block the non-specific amplification and suppress the small target products.
It is designed with four relatively longer AD (LAD) primers of 33 or 34 nucleotides, which contain four fixed nucleotides at the 3' end and followed by degenerated nucleotide positions 9 .
All the four LAD primers shared a common sequence in the 5' half, and a 16-mer primer (AC1) specific to this sequence was prepared for the next two rounds of PCR procedures.The second specific primer SP2 also contains the sequence in the 5'end like the LAD primers except for two bases were designed to differ from the 3' end of AC1.There primers are able to suppress the PCR amplification in the secondary TAIL-PCR since both SP2 and AC1 contain complementary ends and the relatively small ones tend to form a stem-loop structure.It has been successful to isolate genomic flanking sequences of T-DNA insertions from transgenic rice 9 and strawberries 15 , and to determine the full gene structure 26,27 .
Whereas, the improved hiTAIL-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.

SEFA-PCR (Self-formed Adaptor PCR)
Wang et al. 24 developed another randomly primed PCR method, namely "self-formed adaptor PCR", which includes one cycle of DNA synthesis at a low annealing temperature with a sequence partially complementary to a known sequence, followed by linear amplification at a high temperature with a sequence-specific primer to pool and select the target product for the next panhandle formation (Figure 1D).It seems that SEFA-PCR combines the advantages of the panhandle PCR 6 in its specificity and TAIL-PCR 7 in its simplicity.In the SEFA-PCR, four primers are designed to locate sequentially on the known DNA sequences.SP1, SP2 and SP4 are specific primers and have high annealing temperatures.SP3 is a partially degenerated primer and plays a key role in the process as a walking primer.Its both ends are specific to the known sequence and its middle part is eight N oligonucleotides (Figure 1D).According to its procedure, a single cycle of PCR was firstly performed at a low annealing temperature with SP3 primer alone, thus SP3 can prime and extend at many sites on the template.Probably, a downstream site of the known DNA region where SP3 could prime and extend, thus it will create a single strand with a binding site for SP1 24 .
After the single cycle with low annealing temperature, the annealing temperature is increased corresponding to the Tm of SP1, and then SP1 is added to the reaction mixture for 25 high-stringency cycles.At the high annealing temperature, only SP1 is able to prime the target site and generates a pool of single-stranded DNA.Finally, 5-10 cycled of thermal asymmetric interlaced PCR are carried out to facilitate the stem-loop formation, thus create an adaptor which contains binding sites for SP1 and SP2.Once the adaptor has been created, the target sequences can be amplified efficiently by SP1 24 .This method takes full advantage of the suppression-PCR and usually the target products are longer than 2kb.While SEFA-PCR only can flank one side of the known region by SP3 and needs the formation of the panhandle step itself.There are also a few reports that documented the successful use of this method in microorganisms and plants 3,28 .

FPNI-PCR (fusion primer and nested integrated-PCR)
FPNI-PCR is another randomly primed PCR method developed in 2011 25 .It also takes the advantage of the suppression-PCR and TAIL-PCR.The method mainly contains a series of primers encompassing sequencespecific primers (designed on regions of known DNA sequence), and fusion primers which contain an arbitrary degenerated (AD) section fused to a section of known sequence (Figure 1E).The fusion of a known adaptor of determined sequence to the 5'-end of an AD (or other short site-dependent primer) is the main characteristic of FPNI-PCR, and it is the trait which differentiates FPNI-PCR from TAIL-PCR.There are three key steps in the FPNI-PCR protocol.In the first step, a large complex mixture of DNA reactions is prepared using a gene-specific primer (SP1) designed to the genomic region of known sequence, and a combination of nine fusion arbitrary degenerate primers 25 .This first step consists of 3-6 repeats of two high stringency cycles followed by a low stringency cycle.Theoretically, single stranded PCR products from the genespecific primer are generated during the high stringency cycles, and double-stranded products utilizing the fusion primers are developed during the low stringency cycle.
After 3-6 repeated cycles of this PCR regime, it is predicted that the intended target products are partially synthesized and accompanied by other nonspecific products.In the second and third steps, nested PCR is conducted by target-specific primers (SP2/ SP3, respectively) and specific primers developed from the fusion primers (FSP1/ FSP2, respectively).
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 25 .In the FPNI-PCR, the nonspecific products generated from the first PCR step are not amplified in the second and third steps, and are eliminated or substantially diluted in the final mix.
The recent successful amplifications of FPNI-PCR in gene isolation in other organisms suggest this method has great potential to have wide-spread applications in genetics and genome walking studies throughout diverse organisms 12,29,30,31 .

Discussion
In the past years, a number of methods are available for genomic walking and flanking sequence cloning.Methods involving inverse PCR and ligation-mediated PCR techniques suffer a limited utilization due to the requirement for complicated manipulations, such as restriction cleavage, ligation, or tailing before PCR amplification 25 .In this review, we have reviewed the reported five typical genomic walking methods based on randomly primed PCR, including some of our own previous studies.
Those randomly primed PCR strategies are more simple, efficient, less time-consuming, and inexpensive.These methods have been successfully applied to different organisms.Of these methods, Sitefinding-PCR seemed to be relatively efficient and inexpensive, the specific product can reach to 4-5kb, while it is relatively complex in the vector cloning manipulations as far as the other four methods are concerned.
Though SEFA-PCR is successfully used in some microorganisms and plants 3,28 , its major weak point is that SEFA-PCR can just flank one side of the known region and need the formation of the panhandle step itself beside the manipulation of complex.
In addition, the DNA template concentration in the primary SEFA-PCR should be high to facilitate the creation of the adaptors and SP1 should be added at a temperature above its annealing temperature to improve its specificity 24 .According to the previous reports, it is known that the TAIL-PCR method has been widely used for genome walking and flanking sequence cloning.While it is documented that only 60-80% of reactions yielded specific products according to the original TAIL-PCR technical report, which is lower than that derived from SiteFinding-PCR, hiTAIL-PCR, SEFA-PCR and FPNI-PCR, all of the later four methods achieved an average success rate of more than 90%.The TAIL-PCR method involves two rounds of laborious DNA dilutions and numerous (approx.110) cycles of PCR, it usually takes one work day to finish the whole procedure.HiTAIL-PCR, as an improved TAIL-PCR version, similarly involves two rounds of laborious DNA dilutions and multiple low and high stringency PCR cycles, thereby still providing opportunities for nonspecific product amplification 9 .In addition, hiTAIL-PCR is less cost effective in terms of primer designing.The currently developed FPNI-PCR strategy represents a new method which is different from the TAIL-PCR method.Using the FPNI-PCR, a total of 54-57 cycles (completed in less than 4 hours) ensured a successful result in 100% of cases.A large number of experiments using FPNI-PCR greatly demonstrate that this novel method has the capability to be more powerful, effective, and accurate than the established TAIL-PCR method 12,25,29,30,31 .Ligationindependent randomly primed PCR methods represent the kind of easy, simple and less complex, efficient and cost effective strategies for genome walking and flanking sequence cloning.The TAIL-PCR method had been widely used for genome walking and flanking sequence cloning in most molecular biology researches in the past.Currently, the newly developed FPNI-PCR method has shown to be more powerful, effective, time-saving, and accurate than the established conventional TAIL-PCR method.

Conclusion
Though the advent of genomics-based technologies has revolutionized many fields of biological enquiry, chromosome walking or flanking sequence cloning is still an important and needed tool.Randomly primed PCR methods represent simplicity, less complex manipulations and possibly will be continuously widely employed by many molecular biology research laboratories.The newly developed FPNI-PCR can be done in a shorter time-scale and it is more powerful, effective, and accurate than conventional TAIL-PCR and will be widely used for genome walking and flanking sequence cloning.
(a) SiteFinding reaction with the SiteFinder-1 or 2 alone under low annealing temperature to generate double-stranded target sequences with different lengths; (b) Nested PCR: the target DNA was amplified exponentially with nested genespecific (SP1 and SP 2) and SiteFinder primers (SFP1 and SFP 2), while the non-target amplification was suppressed by the stem-loop structure of the DNA; (c) Cloning target molecules and sequenced by another gene-specific primer (SP3) and a vector primer.Using this method, it successfully isolates genomic flanking sequences of T-DNA insertions from transgenic Arabidopsis 20 .

Figure 1 :
Figure 1: Schematic outline of the differences in amplification strategies of target and non-target sequences in randomly primed PCR methods.(A) TAIL-PCR; (B) SiteFinding-PCR; (C) hiTAIL-PCR; (D) SEFA-PCR ; (E) FPNI-PCR.Black and dotted lines represent the target and non-target regions, respectively.Red lines represent the region of known genomic sequence (or known sequence of T-DNA or transposon elements integrated in the genomic DNA).Each red arrow denotes a specific primer designed from the known nucleotide sequence.A blue/ green chimeric arrow denotes the fusion primers derived from the combinations of between degenerated / random primers and known designed nucleotide sequence used in the varied methods.Each blue arrow denotes a specific primer designed from the known adaptor sequence.NotI indicates a NotI enzyme identified nucleotide sequence is added in some of the fusion primers in each randomly primed PCR method.