Selection strategy and the design of hybrid oligonucleotide primers for RACE-PCR: cloning a family of toxin-like sequences from Agelena orientalis
© Pan et al; licensee BioMed Central Ltd. 2007
Received: 30 October 2006
Accepted: 11 May 2007
Published: 11 May 2007
the use of specific but partially degenerate primers for nucleic acid hybridisations and PCRs amplification of known or unknown gene families was first reported well over a decade ago and the technique has been used widely since then.
here we report a novel and successful selection strategy for the design of hybrid partially degenerate primers for use with RT-PCR and RACE-PCR for the identification of unknown gene families. The technique (named PaBaLiS) has proven very effective as it allowed us to identify and clone a large group of mRNAs encoding neurotoxin-like polypeptide pools from the venom of Agelena orientalis species of spider. Our approach differs radically from the generally accepted CODEHOP principle first reported in 1998. Most importantly, our method has proven very efficient by performing better than an independently generated high throughput EST cloning programme. Our method yielded nearly 130 non-identical sequences from Agelena orientalis, whilst the EST cloning technique yielded only 48 non-identical sequences from 2100 clones obtained from the same Agelena material. In addition to the primer design approach reported here, which is almost universally applicable to any PCR cloning application, our results also indicate that venom of Agelena orientalis spider contains a much larger family of related toxin-like sequences than previously thought.
with upwards of 100,000 species of spider thought to exist, and a propensity for producing diverse peptide pools, many more peptides of pharmacological importance await discovery. We envisage that some of these peptides and their recombinant derivatives will provide a new range of tools for neuroscience research and could also facilitate the development of a new generation of analgesic drugs and insecticides.
Toxins and toxin-like molecules are present and used widely throughout the animal kingdom. Among the arthropods, which constitute over 90% of the animal kingdom and include bees, wasps, ants, spiders, scorpions and many other various taxa, many are well known for their predacity and toxic venoms. These have evolved to yield complex and highly specialised toxins which are now successfully used by these predaceous animals to either protect themselves or attack their prey. Despite being considered the most successful animals on Earth (over one million species known and up to 6–9 million species predicted to exist in total) and the massive research effort so far, only a tiny proportion of arthropods has been studied in detail. Spiders evolved from an arachnid ancestor around 400 million years ago and currently comprise over 100,000 different species. Spiders are the most successful predatory animals, in evolutionary terms, and they maintain by far the largest pool of toxic peptides. There are over 39,000 catalogued species , with an even larger number still awaiting characterisation . It has been calculated, based upon a conservative estimate of some 80,000 species and approximately 50 peptides per species , that there are in the region of 4 million distinct spider-venom polypeptides in existence  although of these only a few venoms have been characterised. Only a few hundred of known toxins or toxin-like genes have so far been reported (worldwide) from arthropods or other venomous creatures such as snails or snakes. The precise composition of spider venoms varies significantly between different species. Spider toxins are thought to have derived from a small number of gene super-families with many peptide toxins sharing structural features, conserved amino acids and consensus sequences. This allows them to interact with specific targets such as related classes of cellular receptors. The wide array of peptides may be associated with spiders being general predators, i.e. they do not focus on one specific prey species. It has been suggested that the generation of peptide toxin diversity in spiders has probably been achieved via a similar process to that of cone snails (a group of predatory marine snails which produce an array of neurotoxic peptides collectively named conotoxins), i.e. via extensive gene duplication followed by key hyper-mutations of the pro-peptide and mature-toxin segments. The resultant pool of genes has subsequently been subjected to the pressures of adaptive evolution and this has culminated in the vast arrays of species-specific combinatorial peptide libraries which now exist . It is by the very nature of these processes that an opportunity to discover new peptides of pharmacological importance has become possible using techniques such as PCR-based cloning incorporating degenerate primers, and EST-based cloning.
One of the few successful "molecular" approaches to molecular cloning of polypeptide toxins was reported recently by Kozlov et al. [5, 6]. There an EST (expressed sequence tag) high throughput cloning strategy was employed which yielded nearly 50 novel toxin and toxin-like sequences from Agelena orientalis following the sequencing of 2166 individual EST clones . There are two main advantages to using the EST based approach: the discovery of novel genes and their mRNAs/cDNAs does not require any prior knowledge of the nucleic acid or amino acid sequence information (open system) and if applied to non-normalised cDNA libraries (or pools) it often yields quantitative information on the individual transcripts abundance. This allows, at least in principle, the discovery and expression profiling of all genes expressed in the cell/tissue/etc. without any pre-selection. This useful property is widely relied upon in pharmacogenomics, drug discovery, biomedical and plant sciences (for reviews see [7–13]). For the same reasons, and unless an EST cloning approach is used to explore fully normalised cDNA libraries, the outcome will be biased towards the highly abundant transcripts whilst lower abundance molecular species will likely escape detection. Not surprisingly, EST sequencing of Agelena orientalis revealed that over 70% of all of the EST clones sequences (1497 out of 2166) encoded the same transcript Agelenin (see ), whilst most of the other newly identified genes (47 altogether) were represented by just a few or in most cases by a single EST . EST based cloning is a generic and universal approach to the discovery and expression profiling of genes, but despite its wide applicability it has a few drawbacks, such as the above discussed bias towards highly abundant transcripts and the requirements for lab automation, which are not universally available. This chapter aims to illustrate an alternative approach to the discovery of novel families of toxin or toxin-like polypeptides.
The use of specific but partially degenerate or hybrid primers for nucleic acid hybridisations and PCRs amplification of known or unknown gene families was first reported well over a decade ago (see e.g.  for the use of degenerate primers in hybridisations and [29, 30] for PCR applications) and further mastered by Rose et al. (see [31, 32] and references therein). The technique has been used widely since then. Introducing degeneracy into the primer sequence aims to compensate for codon degeneracy (if only the amino acid sequence is known) or amino acid degeneracy (if the aim is to amplify a family of DNAs encoding proteins having multiple amino acids at a position in the alignment). The most common problems of degenerate primer design include:
i. too high a degeneracy leading to elevated non-specific amplification (due to an abundance of irrelevant sequences) and absence of specific amplification (due to low relative concentration of the correct primer sequence and early depletion of that pool)
ii. too high a degeneracy leading to the formation of secondary structures and self-annealing, detrimental to a PCR amplification
iii. high variability in the Tm within the pool of degenerate primers, making it impossible to choose optimal annealing temperatures
iv. a further potential problem of the approach is the inability to define suitable regions of high sequence conservation and thus an inability to design any primers at all, e.g when the sequences analysed are too dissimilar and/or when a large number of sequences are being aligned (often resulting in no homologous regions altogether).
Both primer design [31–33] and the choice of conserved sequence fragments are key issues for the success of any such amplification. Although easy in principle, practical applications of degenerate primers are often limited to the amplification of gene/protein families having a high degree of sequence similarity and containing low degeneracy codons in the positions corresponding to the PCR primers. Successful application of degenerate or hybrid primers also depend on the particular application, target abundance and target sequence(s) variation. A PCR/RT-PCR employing partially degenerate primers occupies a place somewhat in-between the other PCR approaches mentioned above, see (Figure 1) and its applications range from the use of low degeneracy "best guess" primers (allowing typically for a more specific annealing and amplification) to hybrid or even fully degenerate primers which have a tendency to amplify more fragments, which are often more non-specific fragments, and to a lower degree. The range of PCRs with degenerate primers is schematically indicated on Figure 1 with grey arrows.
Results and discussion
Multiple alignment of a large number of existing toxin and toxin-like sequences yields no universal consensus sequence
The design of hybrid partially degenerate primers
Short subsets of toxin and toxin-like sequences, regardless of their origin (i.e. species), sequence/structure (i.e. short/long, cysteine pattern, presence of any secondary structure elements) or function (i.e. functional toxin or simply toxin-like sequence), were considered but only if they resulted in any alignment suitable for primer design. Although sequence similarities between the small number of highly related sequences are not likely to identify real consensus sequences the use of these was often the only possibility. However, trying hundreds of such fragments would be impractical. Therefore only those were chosen which satisfy the criteria listed below.
The next criterion was to allow the highest degeneracy in the middle of PCR primers (this was to guarantee 100 % sequence matching and hence the maximum hybrid stability). Furthermore, the 5' end of the oligonucleotide primers was often designed to have a "best guess" sequence (based on both amino acid degeneracy at the position and the codon usage frequencies). This often significantly reduced the overall sequence degeneracy, at a small price of possibly introducing mismatches at the very 5' end (these would not destabilise the hybrid compared to any mismatches in the middle of the primer). Figure 3C shows the very different distribution of degenerate bases for the 22 oligonucleotides of identical length (20-mers) out of the 44 oligonucleotides designed in this study. The maximum degeneracy is reached in the middle of the primer unlike the CODEHOP primers (Figure 3A).
PaBaLiS oligonucleotide primers designed in this study.
We can summarise the key advantages of PaBaliS over CODEHOP as follows:
i. highest specificity of annealing and amplification due to low (or absence) of degeneracy at 3' end,
ii. less stringent requirements for the length and composition of the protein consensus sequence, which in PaBaLiS is at the 3' end and is generally shorter than the equivalent "non-degenerate consensus clamp" 5' region in CODEHOP.
iii. PaBaLiS design allows avoidance of dimer formation (through non-specific annealing) since 3' ends are unique non-degenerate sequences and can be easily designed not to dimerise: in contrast to CODEHOP, where all primers will find their complementary 3' pair and will tend to dimerise (leading to quick depletion of the primer pool, non specific priming and poor amplification).
iv. PaBaLiS primers have lower degeneracy overall, and even primers with a few mismatching positions will be able to anneal specifically and continue amplification at later stages of the amplification, when the 100% matching primers are exhausted
v. PaBaLiS primer design yields a larger fraction of primers with no mismatches at the 3' end (meaning that all primers have specific 3' ends, which is most important for the specificity and efficiency of amplification).
vi. PaBaLiS primers have fewer mismatches in the middle of the primer (since degeneracy is allowed) and therefore higher overall hybrid forming stability (unlike CODEHOP where mismatches are possible in the middle of the primer, thus destabilising the hybrids).
The limitations of the CODEHOP approach were highlighted in a recent report by Gray and Coates  where the authors had to use two completely nested sets of CODEHOP RT-PCR primers to amplify a highly-conserved 168 amino acid long region (only to design RACE primers for the next amplification round), whilst PaBaLiS primers apparently work directly for RACE-PCR and can be designed to amplify sequences with just a few amino acids homology, and which do not need to be between consecutive amino acids (small gaps allowed).
Annealing temperature matching
The constraints described above reduced the number of suitable regions significantly in our case and would undoubtedly do so in any other similar cloning approach. One other important criterion of the primer design is in a reduction of the range of annealing temperatures for highly degenerate primers and matching these for the pairs of PCR primers. We soon realised that no suitable 5' and 3' PCR primer pairs (especially with the matching annealing temperatures) could be obtained. Therefore the decision was to opt for RACE-PCR. This is likely to be the only available option in any similar cloning project, when not enough sequence information is available for the design of more than one sequence-specific primer (not a primer pair with matching annealing temperatures). The nature of the RACE-PCR technique is in the use of one universal primer instead of one sequence specific primer. This eliminates the need for the second sequence-specific primer, but leads to a faster accumulation of the products of non-specific amplification and the faster depletion of the universal primer (the latter might require the use of higher concentrations of the universal primer, and has to be determined experimentally in each particular case). The major advantage of using RACE-PCR in addition to the requirement of the single sequence specific primer) is that the annealing temperatures can be more easily matched by redesigning the universal RACE primers (e.g. modifying their length). Table 1 lists such subsets of universal primers suitable for the RACE-PCR (both Oligo-dT and "URA" primer subsets were used successfully though we prefer the Oligo-dT primers over the "URA" adapter primers, also designed in the course of this work). The designed Oligo-dT primers had predicted Tm's of 68.8°C, 57.1°C and 50.1°C (calculated using on-line software from Sigma-Genosys, Cambridge, United Kingdom). The nearest Tm matched primer was used as the RACE primer for each different degenerate primer to ensure the best performance of RACE-PCR (thus the difference in the annealing temperatures was always kept low). Finally, as a last step of primer design all primer pairs (all universal "URA" primers vs. each individual hybrid partially degenerate primers) were checked for any possible dimer formation. Two nucleotides long overlap at the primers' 3' end was not considered as leading to dimer formation, but three and higher overlaps were disallowed. The primer design is therefore a careful balance between the need to allow for all known (and potentially unknown) degenerate positions and the requirement of limited degeneracy, matched annealing temperatures, absence of secondary structures, no self annealing, no primer dimers and the ease of subsequent cloning. Table 1 lists all the primers designed in our investigation and might serve an example of successful primer design for a similar experiment.
Optimisation of the amplification conditions
High throughout amplification and cloning
Of the 44 primers that passed our selection criteria described above, 40 have been shown to produce products (i.e. resulted in cDNA amplification). Altogether over 60 distinct cDNAs (or groups of cDNAs of similar lengths) were identified from Agelena orientalis total RNA by low stringency 3'-RACE-PCR (since many sets of primers have yielded more than one group of cDNA fragments, by their length). Figure 4B gives just a few examples of the first round of RACE amplifications. Large numbers of amplified fragments meant that we had to streamline the cloning and positive clone selection procedures. Identification of positive clones was carried out by PCR directly from colonies. The primers (PBS-F and PGM-2R) were designed based on the vector sequence (Table 1). The clones were picked using sterile tips and transferred onto a fresh new antibiotic plate first, then using the same tip directly dissolved in the mixtures of PCR reactions. The positive clones and sizes were identified by agarose gel electrophoresis. This was a much quicker and more informative method for screening positive clones than applying an IPTG (blue/white) selection procedure . This had the additional advantage of allowing positive clones to be identified within approximately one day and the sizes of cloned inserts to be simultaneously determined. This allowed us to improve the 'hit' rate with over 50% of clones chosen for further analysis being independent sequences. Traditional approaches relying on plasmid purification and restriction mapping were not suitable for our high-throughput cloning approach, since it was possible that the chosen restriction enzymes might cut within the unknown insert sequences. Figure 4C illustrates some of the positive clones with different insert sizes that were identified by PCR reactions directly form bacterial colonies.
Sequences identification and analysis
The RACE RT-PCR strategy is a useful technique for amplifying specific regions of mRNA between a defined and known internal site sequence and an unknown sequence located at either the 3' or 5' end. When RACE-RT PCR is combined with the application of degenerate primers the method becomes a very effective way to clone and identify novel cDNA fragments. Previously, such an approach has been used successfully to clone and sequence cDNAs encoding insecticidal peptides from primitive hunting spiders . The application of PCR-based cloning directly complements an EST-based approach . However, PCR-based cloning proffers a distinct advantage whereby even rare transcripts can be identified, i.e. low-abundance transcripts are similarly well represented following amplification and are not lost in preference to higher abundance molecules. This contrasts with EST cloning which can often result in multiple copies of the same abundant mRNA/cDNAs being cloned, unless normalised libraries are exploited . Another important issue is the proportional representation of individual sequences at the outcome of the experiment. EST based strategies (unless used with normalised libraries) preserve the information on relative abundance of respective cDNAs, whilst a PCR based approach might not, unless it is a truly quantitative qRT-PCR, see e.g. . The latter is especially valid if no truly universal oligonucleotide primers (capable of simultaneously amplifying the whole family of mRNAs/cDNAs under investigation) are used. The case presented here is a good example of such an approach. We have not preserved any quantitative information on toxin mRNA/cDNA expression, but compared to the EST approach reported in  we were able to identify more than twofold the number of different sequences from the same material. Our oligonucleotide selection algorithm is therefore superior if the discovery of new genes is sought, but should not be used if quantitative analysis of mRNA/cDNA expression is required.
As interest in the field of neuro-modulatory molecules continues to expand, our approach contributes significantly through the provision of an effective high-throughput methodology to identify novel neurotoxin-like peptide sequences from the venom of Agelena orientalis species of spiders. The collective use of published neuro-toxin sequence information, sets of specifically designed partially degenerate hybrid primers and streamlined cloning and positive clone selection procedures have enabled us to identify a large set (~130) of toxin-like sequences from Agelena orientalis. This is an invaluable resource although in all probably it represents only a very small fraction of the overall number of functional spider toxin peptides that are still awaiting discovery. The sequences identified with RACE-PCR include 80 putative novel toxin-like sequences, in addition to the ones matching the sequences obtained independently by an EST-based approach . These sequences are indicative of the vast repertoire and/or diversity of toxins, or toxin-like proteins, present in Agelena orientalis venom. These spider venom toxins are a rich biological resource of active components and this presents an opportunity to develop further biological tools for neuroscience, in drug target discovery and possibly as effective bio-pesticides. With upwards of 100,000 species of spider thought to exist, and each showing a propensity for producing diverse peptide pools, many more neurotoxin peptides await discovery. It is likely that a high percentage of these could be exploited in a wide range of novel applications, and profit both research and commercial industries.
Oligonucleotide primers designed for RACE-PCR and for the selection of positive clones
TTT TTT TTT TTT TTT TTT TTT
TTT TTT TTT TTT TTT TTT TTT TTT
TTT TTT TTT TTT TTT TTT TTT TTT TTT
- RT-PCR :
reverse transcriptase polymerase chain reaction
- RACE-PCR :
rapid amplification of cDNA ends polymerase chain reaction
- EST :
expressed sequence tag
- LS-SSP-PCR :
low stringency single specific primer polymerase chain reaction
- AP-PCR :
arbitrarily primed polymerase chain reaction
- DD-PCR :
differential display polymerase chain reaction
- AFLP :
amplified fragment length polymorphism
- CODEHOP :
COnsensus-DEgenerate Hybrid Oligonucleotide Primers.
This work was supported by a travel grant from the Royal Society (UK) to M Soloviev.
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