{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T10:43:27Z","timestamp":1773657807340,"version":"3.50.1"},"reference-count":44,"publisher":"Springer Science and Business Media LLC","issue":"1","content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Molecular Biol"],"published-print":{"date-parts":[[2007,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:sec>\n            <jats:title>Background<\/jats:title>\n            <jats:p>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.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>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 <jats:italic>Agelena orientalis<\/jats:italic> 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 <jats:italic>Agelena orientalis<\/jats:italic>, whilst the EST cloning technique yielded only 48 non-identical sequences from 2100 clones obtained from the same <jats:italic>Agelena<\/jats:italic> 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 <jats:italic>Agelena orientalis<\/jats:italic> spider contains a much larger family of related toxin-like sequences than previously thought.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p>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.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1471-2199-8-32","type":"journal-article","created":{"date-parts":[[2007,5,12]],"date-time":"2007-05-12T06:13:24Z","timestamp":1178950404000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Selection strategy and the design of hybrid oligonucleotide primers for RACE-PCR: cloning a family of toxin-like sequences from Agelena orientalis"],"prefix":"10.1186","volume":"8","author":[{"given":"Zhensheng","family":"Pan","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard","family":"Barry","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexey","family":"Lipkin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mikhail","family":"Soloviev","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2007,5,11]]},"reference":[{"key":"169_CR1","unstructured":"The World Spider Catalogue. http:\/\/research.amnh.org\/entomology\/spiders\/catalog\/"},{"key":"169_CR2","doi-asserted-by":"publisher","first-page":"565","DOI":"10.1146\/annurev.es.22.110191.003025","volume":"22","author":"JA Coddington","year":"1991","unstructured":"Coddington JA, Levi HW: Systematics and evolution of spiders (Araneae). Annu Rev Ecol Syst. 1991, 22: 565-592.","journal-title":"Annu Rev Ecol Syst"},{"key":"169_CR3","doi-asserted-by":"publisher","first-page":"555","DOI":"10.1016\/j.toxicon.2004.02.007","volume":"43","author":"P Escoubas","year":"2004","unstructured":"Escoubas P, Rash L: Tarantulas: eight-legged pharmacists and combinatorial chemists. Toxicon. 2004, 43: 555-574.","journal-title":"Toxicon"},{"key":"169_CR4","doi-asserted-by":"publisher","first-page":"131","DOI":"10.1016\/j.peptides.2004.07.016","volume":"26","author":"BL Sollod","year":"2005","unstructured":"Sollod BL, Wilson D, Zhaxybayeva O, Gogarten JP, Drinkwater R, King GF: Were arachnids the first to use combinatorial peptide libraries?. Peptides. 2005, 26: 131-139.","journal-title":"Peptides"},{"key":"169_CR5","doi-asserted-by":"publisher","first-page":"20983","DOI":"10.1074\/jbc.M602168200","volume":"281","author":"SA Kozlov","year":"2006","unstructured":"Kozlov SA, Vassilevski AA, Feofanov AV, Surovoy AY, Karpunin DV, Grishin EV: Latarcins, antimicrobial and cytolytic peptides from the venom of the spider Lachesana tarabaevi (Zodariidae) that exemplify biomolecular diversity. J Biol Chem. 2006, 281: 20983-20992.","journal-title":"J Biol Chem"},{"key":"169_CR6","doi-asserted-by":"publisher","first-page":"131","DOI":"10.1002\/prot.20390","volume":"59","author":"S Kozlov","year":"2005","unstructured":"Kozlov S, Malyavka A, McCutchen B, Lu A, Schepers E, Herrmann R, Grishin E: A novel strategy for the identification of toxinlike structures in spider venom. Proteins. 2005, 59: 131-140.","journal-title":"Proteins"},{"key":"169_CR7","doi-asserted-by":"publisher","first-page":"227","DOI":"10.2217\/14622416.7.2.227","volume":"7","author":"D Bol","year":"2006","unstructured":"Bol D, Ebner R: Gene expression profiling in the discovery, optimization and development of novel drugs: one universal screening platform. Pharmacogenomics. 2006, 7: 227-235.","journal-title":"Pharmacogenomics"},{"key":"169_CR8","first-page":"123","volume":"2","author":"A Lindlof","year":"2003","unstructured":"Lindlof A: Gene identification through large-scale EST sequence processing. Appl Bioinformatics. 2003, 2: 123-129.","journal-title":"Appl Bioinformatics"},{"key":"169_CR9","doi-asserted-by":"publisher","first-page":"224","DOI":"10.1016\/S1369-5266(00)00068-6","volume":"3","author":"J Ohlrogge","year":"2000","unstructured":"Ohlrogge J, Benning C: Unraveling plant metabolism by EST analysis. Curr Opin Plan Biol. 2000, 3: 224-228.","journal-title":"Curr Opin Plan Biol"},{"key":"169_CR10","doi-asserted-by":"publisher","first-page":"497","DOI":"10.2217\/14796694.1.4.497","volume":"1","author":"F Weber","year":"2005","unstructured":"Weber F, Eng C: Gene-expression profiling in differentiated thyroid cancer\u2013a viable strategy for the practice of genomic medicine?. Future Oncol. 2005, 1: 497-510.","journal-title":"Future Oncol"},{"key":"169_CR11","doi-asserted-by":"publisher","first-page":"9","DOI":"10.2152\/jmi.53.9","volume":"53","author":"M Takahashi","year":"2006","unstructured":"Takahashi M, Teh BT, Kanayama HO: Elucidation of the molecular signatures of renal cell carcinoma by gene expression profiling. J Med Invest. 2006, 53: 9-19.","journal-title":"J Med Invest"},{"key":"169_CR12","doi-asserted-by":"publisher","first-page":"2","DOI":"10.2325\/jbcs.13.2","volume":"13","author":"K Nagasaki","year":"2006","unstructured":"Nagasaki K, Miki Y: Gene expression profiling of breast cancer. Breast Cancer. 2006, 13: 2-7.","journal-title":"Breast Cancer"},{"key":"169_CR13","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1002\/(SICI)1097-4547(20000101)59:1<1::AID-JNR1>3.0.CO;2-2","volume":"59","author":"C Colantuoni","year":"2000","unstructured":"Colantuoni C, Purcell AE, Bouton CM, Pevsner J: High throughput analysis of gene expression in the human brain. J Neurosci Res. 2000, 59: 1-10.","journal-title":"J Neurosci Res"},{"key":"169_CR14","doi-asserted-by":"publisher","first-page":"335","DOI":"10.1016\/0076-6879(87)55023-6","volume":"155","author":"KB Mullis","year":"1987","unstructured":"Mullis KB, Faloona FA: Specific synthesis of DNA in vitro via a polymerase-catalyzed chain-reaction. Methods Enzymol. 1987, 155: 335-350.","journal-title":"Methods Enzymol"},{"key":"169_CR15","doi-asserted-by":"publisher","first-page":"1350","DOI":"10.1126\/science.2999980","volume":"230","author":"RK Saiki","year":"1985","unstructured":"Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N: Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985, 230: 1350-1354.","journal-title":"Science"},{"key":"169_CR16","doi-asserted-by":"publisher","first-page":"289","DOI":"10.1016\/0959-8049(92)90436-6","volume":"28","author":"RA Eeles","year":"1992","unstructured":"Eeles RA, Warren W, Stamps A: The PCR revolution. Eur J Cancer. 1992, 28: 289-293.","journal-title":"Eur J Cancer"},{"key":"169_CR17","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1186\/1477-3155-1-3","volume":"1","author":"AC Stamps","year":"2003","unstructured":"Stamps AC, Terrett JA, Adam PJ: Application of in situ reverse trancriptase-polymerase chain reaction (RT-PCR) to tissue microarrays. J Nanobiotechnology. 2003, 1: 3-","journal-title":"J Nanobiotechnology"},{"key":"169_CR18","doi-asserted-by":"publisher","DOI":"10.1385\/159259283X","volume-title":"Methods in Molecular Biology: RT-PCR Protocols","author":"J O'Connell","year":"2002","unstructured":"O'Connell J: Methods in Molecular Biology: RT-PCR Protocols. 2002, 193: Totowa New Jersey: Humana Press Inc"},{"key":"169_CR19","doi-asserted-by":"publisher","first-page":"340","DOI":"10.1016\/0076-6879(93)18026-9","volume":"218","author":"MA Frohman","year":"1993","unstructured":"Frohman MA: Rapid amplification of complementary-DNA ends for generation of full-length complementary DNAs \u2013 thermal RACE. Methods Enzymol. 1993, 218: 340-356.","journal-title":"Methods Enzymol"},{"key":"169_CR20","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1186\/1471-2199-7-27","volume":"7","author":"RMK Yu","year":"2006","unstructured":"Yu RMK, Chen EXH, Kong RYC, Ng PKS, Mok HOL, Au DWT: Hypoxia induces telomerase reverse transcriptase (TERT) gene expression in non-tumor fish tissues in vivo: the marine medaka (Oryzias melastigma) model. BMC Mol Biol. 2006, 7: 27-","journal-title":"BMC Mol Biol"},{"key":"169_CR21","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1186\/1471-2199-6-7","volume":"6","author":"MD Pondel","year":"2005","unstructured":"Pondel MD, Mould R: Cloning and transcriptional analysis of the mouse receptor activity modifying protein-1 gene promoter. BMC Mol Biol. 2005, 6: 7-","journal-title":"BMC Mol Biol"},{"key":"169_CR22","doi-asserted-by":"publisher","first-page":"4","DOI":"10.1186\/1471-2199-7-4","volume":"7","author":"TS Finco","year":"2006","unstructured":"Finco TS, Justice-Healy GE, Patel SJ, Hamilton VE: Regulation of the human LAT gene by the Elf-1 transcription factor. BMC Mol Biol. 2006, 7: 4-","journal-title":"BMC Mol Biol"},{"key":"169_CR23","doi-asserted-by":"publisher","first-page":"1946","DOI":"10.1073\/pnas.91.5.1946","volume":"91","author":"SD Pena","year":"1994","unstructured":"Pena SD, Barreto G, Vago AR, De Marco L, Reinach FC, Dias Neto E, Simpson AJ: Sequence-specific \"gene signatures\" can be obtained by PCR with single specific primers at low stringency. Proc Natl Acad Sci USA. 1994, 91: 1946-1949.","journal-title":"Proc Natl Acad Sci USA"},{"key":"169_CR24","doi-asserted-by":"publisher","first-page":"7213","DOI":"10.1093\/nar\/18.24.7213","volume":"18","author":"J Welsh","year":"1990","unstructured":"Welsh J, McClelland M: Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990, 18: 7213-7218.","journal-title":"Nucleic Acids Res"},{"key":"169_CR25","doi-asserted-by":"publisher","first-page":"967","DOI":"10.1126\/science.1354393","volume":"257","author":"P Liang","year":"1992","unstructured":"Liang P, Pardee A: Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992, 257: 967-971.","journal-title":"Science"},{"key":"169_CR26","doi-asserted-by":"publisher","first-page":"4407","DOI":"10.1093\/nar\/23.21.4407","volume":"23","author":"P Vos","year":"1995","unstructured":"Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M: AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 1995, 23: 4407-4414.","journal-title":"Nucleic Acids Res"},{"key":"169_CR27","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1002\/jmv.1890300202","volume":"30","author":"K Porter-Jordan","year":"1990","unstructured":"Porter-Jordan K, Rosenberg EI, Keiser JF, Gross JD, Ross AM, Nasim S, Garrett CT: Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA. J Med Virol. 1990, 30: 85-91.","journal-title":"J Med Virol"},{"key":"169_CR28","doi-asserted-by":"publisher","first-page":"756","DOI":"10.1038\/313756a0","volume":"313","author":"A Ullrich","year":"1985","unstructured":"Ullrich A, Bell JR, Chen EY, Herrera R, Petruzzelli LM, Dull TJ, Gray A, Coussens L, Liao YC, Tsubokawa M, Mason A, Seeburg PH, Grunfeld C, Rosen OM, Ramachandran J: Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature. 1985, 313: 756-761.","journal-title":"Nature"},{"key":"169_CR29","doi-asserted-by":"publisher","first-page":"239","DOI":"10.1038\/341239a0","volume":"341","author":"TR Burglin","year":"1989","unstructured":"Burglin TR, Finney M, Coulson A, Ruvkun G: Caenorhabditis elegans has scores of homoeobox-containing genes. Nature. 1989, 341: 239-243.","journal-title":"Nature"},{"key":"169_CR30","doi-asserted-by":"publisher","first-page":"11751","DOI":"10.1073\/pnas.93.21.11751","volume":"93","author":"YG Yu","year":"1996","unstructured":"Yu YG, Buss GR, Maroof MAS: Isolation of a superfamily of candidate disease-resistance genes in soybean based on a conserved nucleotide-binding site. Proc Natl Acad Sci USA. 1996, 93: 11751-11756.","journal-title":"Proc Natl Acad Sci USA"},{"key":"169_CR31","doi-asserted-by":"publisher","first-page":"1628","DOI":"10.1093\/nar\/26.7.1628","volume":"26","author":"TM Rose","year":"1998","unstructured":"Rose TM, Schultz ER, Henikoff JG, Pietrokovski S, McCallum CM, Henikoff S: Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res. 1998, 26: 1628-1635.","journal-title":"Nucleic Acids Res"},{"key":"169_CR32","doi-asserted-by":"publisher","first-page":"3763","DOI":"10.1093\/nar\/gkg524","volume":"31","author":"TM Rose","year":"2003","unstructured":"Rose TM, Henikoff JG, Henikoff S: CODEHOP (COnsensus-DEgenerate hybrid oligonucleotide primer) PCR primer design. Nucleic Acids Res. 2003, 31: 3763-3766.","journal-title":"Nucleic Acids Res"},{"key":"169_CR33","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1385\/0-89603-199-3:55","volume-title":"A Laboratory Manual in Molecular Biology: Protocols in Molecular Neurobiology","author":"AN Bateson","year":"1992","unstructured":"Bateson AN, Darlison MG: The design and use of oligonucleotides. A Laboratory Manual in Molecular Biology: Protocols in Molecular Neurobiology. Edited by: Longstaff A, Revest P. 1992, 13: 55-66. Totowa New Jersey: Humana Press Inc"},{"key":"169_CR34","doi-asserted-by":"publisher","first-page":"6820","DOI":"10.1073\/pnas.96.12.6820","volume":"96","author":"TF Duda Jr","year":"1999","unstructured":"Duda TF, Palumbi SR: Molecular genetics of ecological diversification: duplication and rapid evolution of toxin genes of the venomous gastropod Conus. Proc Natl Acad Sci USA. 1999, 96: 6820-6823.","journal-title":"Proc Natl Acad Sci USA"},{"key":"169_CR35","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1186\/1471-2199-6-16","volume":"6","author":"CE Gray","year":"2005","unstructured":"Gray CE, Coates CJ: Cloning and characterization of cDNAs encoding putative CTCFs in the mosquitoes, Aedes aegypti and Anopheles gambiae. BMC Mol Biol. 2005, 6: 16-","journal-title":"BMC Mol Biol"},{"key":"169_CR36","doi-asserted-by":"publisher","first-page":"3749","DOI":"10.1093\/nar\/19.13.3749","volume":"19","author":"RT D'Aquila","year":"1991","unstructured":"D'Aquila RT, Bechtel LJ, Videler JA, Eron JJ, Gorczyca P, Kaplan JC: Maximizing sensitivity and specificity of PCR by preamplification heating. Nucleic Acids Res. 1991, 19: 3749-3749.","journal-title":"Nucleic Acids Res"},{"key":"169_CR37","doi-asserted-by":"publisher","first-page":"4008","DOI":"10.1093\/nar\/19.14.4008","volume":"19","author":"RH Don","year":"1991","unstructured":"Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS: 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991, 19: 4008-4008.","journal-title":"Nucleic Acids Res"},{"key":"169_CR38","doi-asserted-by":"crossref","unstructured":"Roux KH: Optimization and troubleshooting in PCR. PCR Methods Appl. 1995, 185-194. Suppl 4","DOI":"10.1101\/gr.4.5.S185"},{"key":"169_CR39","doi-asserted-by":"crossref","first-page":"478","DOI":"10.2144\/19962003478","volume":"20","author":"KH Hecker","year":"1996","unstructured":"Hecker KH, Roux KH: High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. Biotechniques. 1996, 20: 478-485.","journal-title":"Biotechniques"},{"key":"169_CR40","doi-asserted-by":"publisher","first-page":"351","DOI":"10.1016\/S0014-5793(99)00661-4","volume":"452","author":"S Yazynin","year":"1999","unstructured":"Yazynin S, Lange H, Mokros T, Deyev S, Lemke H: A new phagemid vector for positive selection of recombinants based on a conditionally lethal barnase gene. FEBS Lett. 1999, 452: 351-354.","journal-title":"FEBS Lett"},{"key":"169_CR41","doi-asserted-by":"publisher","first-page":"411","DOI":"10.1016\/0965-1748(95)00070-4","volume":"26","author":"DJ Leisy","year":"1996","unstructured":"Leisy DJ, Mattson JD, Quistad GB, Kramer SJ, Van Beek N, Tsai LW, Enderlin FE, Woodworth AR, Digan ME: Molecular cloning and sequencing of cDNAs encoding insecticidal peptides from the primitive hunting spider, Plectreurys tristis (Simon). Insect Biochem Mol Biol. 1996, 26: 411-417.","journal-title":"Insect Biochem Mol Biol"},{"key":"169_CR42","doi-asserted-by":"publisher","first-page":"1651","DOI":"10.1126\/science.2047873","volume":"252","author":"MD Adams","year":"1991","unstructured":"Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF, Kerlavage AR, McCombie WR, Venter JC: Complementary DNA sequencing: expressed sequence tags and human genome project. Science. 1991, 252: 1651-1656.","journal-title":"Science"},{"key":"169_CR43","first-page":"186","volume":"31","author":"PA Zhulidov","year":"2005","unstructured":"Zhulidov PA, Bogdanova EA, Shcheglov AS, Shagina IA, Wagner LL, Khazpekov GL, Kozhemyako VV, Lukyanov SA, Shagin DA: A method for the preparation of normalized cDNA libraries enriched with full-length sequences. Bioorg Khim. 2005, 31: 186-194.","journal-title":"Bioorg Khim"},{"key":"169_CR44","doi-asserted-by":"publisher","first-page":"32","DOI":"10.1186\/1471-2199-7-32","volume":"7","author":"G Spinsanti","year":"2006","unstructured":"Spinsanti G, Panti C, Lazzeri E, Marsili L, Casini S, Frati F, Fossi CM: Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies. BMC Mol Biol. 2006, 7: 32-","journal-title":"BMC Mol Biol"}],"container-title":["BMC Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/1471-2199-8-32.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,9,1]],"date-time":"2021-09-01T10:56:08Z","timestamp":1630493768000},"score":1,"resource":{"primary":{"URL":"https:\/\/bmcmolbiol.biomedcentral.com\/articles\/10.1186\/1471-2199-8-32"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2007,5,11]]},"references-count":44,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2007,12]]}},"alternative-id":["169"],"URL":"https:\/\/doi.org\/10.1186\/1471-2199-8-32","relation":{},"ISSN":["1471-2199"],"issn-type":[{"value":"1471-2199","type":"electronic"}],"subject":[],"published":{"date-parts":[[2007,5,11]]},"assertion":[{"value":"30 October 2006","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"11 May 2007","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"11 May 2007","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}}],"article-number":"32"}}