chromosome walking


Summary: A technique with which an unknown region of a chromosome can be explored. It is generally used to isolate a locus of interest for which no probe is available but that is known to be linked to a gene which has been identified and cloned. A fragment containing a known gene is selected and used as a probe to identify other overlapping fragments which contain the same gene. The nucleotide sequences of these fragments can then be characterized. This process continues for the length of the chromosome.

Top Publications

  1. Trinh Q, Xu W, Shi H, Luo Y, Huang K. An A-T linker adapter polymerase chain reaction method for chromosome walking without restriction site cloning bias. Anal Biochem. 2012;425:62-7 pubmed publisher
    ..amplification efficiency, greater flexibility, and easier manipulation compared with other PCR methods for chromosome walking. Experimental results from 143 Arabidopsis mutants illustrate that this method is reliable and efficient in ..
  2. Zhao G, Zhang Z, Sun H, Li H, Dai H. Isolation of Ty1-copia-like retrotransposon sequences from the apple genome by chromosome walking based on modified SiteFinding-polymerase chain reaction. Acta Biochim Biophys Sin (Shanghai). 2007;39:675-83 pubmed
    ..retrotransposon (named Tcrm1), including RT-ribonuclease H-LTR domain sequences, was achieved by chromosome walking based on modified SiteFinding-polymerase chain reaction...
  3. Rosenthal A, MacKinnon R, Jones D. PCR walking from microdissection clone M54 identifies three exons from the human gene for the neural cell adhesion molecule L1 (CAM-L1). Nucleic Acids Res. 1991;19:5395-401 pubmed
    ..This is the first description of extension of a human derived microclone by PCR mediated walking within total human genomic DNA. These results show that anonymous DNA sequences may be extended into coding or any sequence. ..
  4. Wang Z, Ye S, Li J, Zheng B, Bao M, Ning G. Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning. BMC Biotechnol. 2011;11:109 pubmed publisher
    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...
  5. Yuanxin Y, Chengcai A, Li L, Jiayu G, Guihong T, Zhangliang C. T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends. Nucleic Acids Res. 2003;31:e68 pubmed
    Dozens of PCR-based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation-mediated PCR and randomly primed PCR...
  6. Budiman M, Chang S, Lee S, Yang T, Zhang H, de Jong H, et al. Localization of jointless-2 gene in the centromeric region of tomato chromosome 12 based on high resolution genetic and physical mapping. Theor Appl Genet. 2004;108:190-6 pubmed
    ..Based on high resolution genetic and physical mapping, we conclude that the jointless-2 gene is located within or near the chromosome 12 centromere where 1 cM is approximately 25 Mb in length. ..
  7. Bae J, Sohn J. Template-blocking PCR: an advanced PCR technique for genome walking. Anal Biochem. 2010;398:112-6 pubmed publisher
    ..The method was successfully applied for the cloning of the PGK1 promoter from Pichia ciferrii and two novel cellulase genes from Penicillium sp. ..
  8. Liu F, Ma B, Zhao Y, Zhang Y, Wu Y, Liu X, et al. Characterization of the gene encoding glycoprotein C of duck enteritis virus. Virus Genes. 2008;37:328-32 pubmed publisher
    ..Phylogenetic analysis of the DEV gC gene revealed that the gC gene had a close evolutionary relationship with the subfamily of Alphaherpesvirinae...
  9. Dong Q, Wilkerson M, Brendel V. Tracembler--software for in-silico chromosome walking in unassembled genomes. BMC Bioinformatics. 2007;8:151 pubmed
    ..We have developed software, Tracembler, that facilitates in silico chromosome walking by recursively assembling reads of a selected species from the NCBI Trace Archive starting with reads that ..

More Information


  1. Wang Y, Tsukamoto T, Yi K, Wang X, Huang S, McCubbin A, et al. Chromosome walking in the Petunia inflata self-incompatibility (S-) locus and gene identification in an 881-kb contig containing S2-RNase. Plant Mol Biol. 2004;54:727-42 pubmed
    ..We first conducted chromosome walking at the S2 locus using BAC clones that contained either S2-RNase or one of the nine markers tightly linked to ..
  2. Uchiyama T, Watanabe K. Improved inverse PCR scheme for metagenome walking. Biotechniques. 2006;41:183-8 pubmed
    ..Since rare sequences are a critical element of natural genetic diversity, IAN-PCR enables access to undiscovered diverse genes in the environment. ..
  3. Reddy P, Mahanty S, Kaul T, Nair S, Sopory S, Reddy M. A high-throughput genome-walking method and its use for cloning unknown flanking sequences. Anal Biochem. 2008;381:248-53 pubmed publisher
    ..The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5' flanking regions/promoters of selected plant genes. ..
  4. Ji J, Braam J. Restriction site extension PCR: a novel method for high-throughput characterization of tagged DNA fragments and genome walking. PLoS ONE. 2010;5:e10577 pubmed publisher
    ..RSE-PCR has high potential application in identifying tagged (T-DNA or transposon) sequence or walking from known DNA toward unknown regions in large-genome plants, with likely application in other organisms as well. ..
  5. Liu Y, Whittier R. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics. 1995;25:674-81 pubmed
    ..of this method to expedite amplification and sequencing of insert end segments from P1 and YAC clones for chromosome walking. In this study we present protocols that are amenable to automation of amplification and sequencing of ..
  6. Tan G, Gao Y, Shi M, Zhang X, He S, Chen Z, et al. SiteFinding-PCR: a simple and efficient PCR method for chromosome walking. Nucleic Acids Res. 2005;33:e122 pubmed
    In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific ..
  7. Myrick K, Gelbart W. Universal Fast Walking for direct and versatile determination of flanking sequence. Gene. 2002;284:125-31 pubmed
    ..Despite the complexity of banding patterns in these fingerprints, we observed that the reaction products were directly sequenceable. In view of its speed, reliability and generality, we term the described method Universal Fast Walking. ..
  8. Chibana H, Heinecke E, Beckerman J, Magee P. A system of rapid isolation of end-DNA from a small amount of fosmid DNA, with vector-based PCR for chromosome walking. Genome. 2001;44:305-8 pubmed
    ..However, the low copy number requires laborious end-DNA preparation for end sequencing and chromosome walking. Here we describe efficient methods for end-DNA isolation...
  9. Le Cunff L, Garsmeur O, Raboin L, Pauquet J, Telismart H, Selvi A, et al. Diploid/polyploid syntenic shuttle mapping and haplotype-specific chromosome walking toward a rust resistance gene (Bru1) in highly polyploid sugarcane (2n approximately 12x approximately 115). Genetics. 2008;180:649-60 pubmed publisher
    ..syntenic shuttle mapping with two model diploid species (sorghum and rice) and haplotype-specific chromosome walking. Their applications allowed us (i) to develop a high-resolution map including markers at 0.28 and 0...
  10. Satyanarayana K, Chandrashekar A, Ravishankar G. Evaluation of PCR-based methods for isolating flanking regions of genes. Mol Biotechnol. 2006;32:111-6 pubmed
    ..The methods adopted in this study differed in the sizes of the 5'-flanking regions obtained. The efficiencies of various methods used reflect the inherent limitations of the PCR-based methods for isolation of unknown flanking regions. ..
  11. Tonooka Y, Fujishima M. Comparison and critical evaluation of PCR-mediated methods to walk along the sequence of genomic DNA. Appl Microbiol Biotechnol. 2009;85:37-43 pubmed publisher
    ..The cassette PCR seems to be a standard method, but suitable method should be selected in consideration of the characteristics of the material. ..
  12. Wang S, He J, Cui Z, Li S. Self-formed adaptor PCR: a simple and efficient method for chromosome walking. Appl Environ Microbiol. 2007;73:5048-51 pubmed
    We developed a self-formed adaptor PCR (termed SEFA PCR) which can be used for chromosome walking. Most of the amplified flanking sequences were longer than 2.0 kb, and some were as long as 6.0 kb...
  13. Garcia Fernandez J, Holland P. Archetypal organization of the amphioxus Hox gene cluster. Nature. 1994;370:563-6 pubmed
    ..Remarkably, this organization is compatible with that inferred for a direct ancestor of the vertebrates; we conclude that amphioxus is a living representative of a critical intermediate stage in Hox cluster evolution. ..
  14. Milan S, D Ari L, Chamberlin M. Structural analysis of ternary complexes of Escherichia coli RNA polymerase: ribonuclease footprinting of the nascent RNA in complexes. Biochemistry. 1999;38:218-25 pubmed
    ..Our results rule out the existence of a stable RNA-DNA hybrid in these ternary complexes of greater than 3 base pairs in length. ..
  15. Hou J, Parrish J, Ludecke H, Sapru M, Wang Y, Chen W, et al. A 4-megabase YAC contig that spans the Langer-Giedion syndrome region on human chromosome 8q24.1: use in refining the location of the trichorhinophalangeal syndrome and multiple exostoses genes (TRPS1 and EXT1). Genomics. 1995;29:87-97 pubmed
  16. Carattoli A, Aschbacher R, March A, Larcher C, Livermore D, Woodford N. Complete nucleotide sequence of the IncN plasmid pKOX105 encoding VIM-1, QnrS1 and SHV-12 proteins in Enterobacteriaceae from Bolzano, Italy compared with IncN plasmids encoding KPC enzymes in the USA. J Antimicrob Chemother. 2010;65:2070-5 pubmed publisher
    ..This plasmid family is now playing a crucial role in the global spread of diverse carbapenemase genes in Klebsiella spp. ..
  17. Maier T, Förster H, Asperger O, Hahn U. Molecular characterization of the 56-kDa CYP153 from Acinetobacter sp. EB104. Biochem Biophys Res Commun. 2001;286:652-8 pubmed
    ..The relevance of the remarkably long N-terminus and of other sequence motives like the hydrophobic F-G loop is discussed with respect to substrate binding and recognition. ..
  18. He J, Magarvey N, Piraee M, Vining L. The gene cluster for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway homologues and a monomodular non-ribosomal peptide synthetase gene. Microbiology. 2001;147:2817-29 pubmed
    ..As has been shown for ORF2, disrupting ORF3, ORF7, ORF8 or ORF9 blocked Cm production. ..
  19. Wisotzkey R, Johnson A, Takaesu N, Newfeld S. Alpha/beta hydrolase2, a predicated gene adjacent to mad in Drosophila melanogaster, belongs to a new global multigene family and is associated with obesity. J Mol Evol. 2003;56:351-61 pubmed
    ..Overall, the data suggests that CG3488 functions as a lipase and that analyses of its homologs will provide unique insights into lipid metabolism in many species. ..
  20. Tahlan K, Park H, Jensen S. Three unlinked gene clusters are involved in clavam metabolite biosynthesis in Streptomyces clavuligerus. Can J Microbiol. 2004;50:803-10 pubmed
    ..Since antibiotic biosynthetic genes are invariably clustered on the chromosome in prokaryotes, chromosome walking was undertaken in an attempt to show that the three groups of clavam genes would resolve into a single super-..
  21. Jo Y, Ha Y, Lee J, Park M, Bergsma A, Choi H, et al. Fine mapping of Restorer-of-fertility in pepper (Capsicum annuum L.) identified a candidate gene encoding a pentatricopeptide repeat (PPR)-containing protein. Theor Appl Genet. 2016;129:2003-17 pubmed publisher
    ..Through six cycles of chromosome walking, the co-segregating region harboring the Rf locus was delimited to be within 821 kb of sequence...
  22. Chaplin D, Brownstein B. Analysis of isolated YAC clones. Curr Protoc Mol Biol. 2001;Chapter 6:Unit6.10 pubmed publisher
    ..DNA fragments from the ends of a YAC genomic insert to be used as probes for detecting chimerism and for chromosome walking. Finally, preparation of high-molecular-weight YAC DNA is described and a general method for subcloning YAC ..
  23. Watanabe T, Kankel D. Molecular cloning and analysis of l(1)ogre, a locus of Drosophila melanogaster with prominent effects on the postembryonic development of the central nervous system. Genetics. 1990;126:1033-44 pubmed
    ..An open reading frame (ORF) of 1,086 base paris was found, and a protein sequence of 362 amino acids with one highly hydrophobic segment was deduced from conceptual translation of this ORF. ..
  24. Sim B, Grosveld F, Hui K. Inactivation of the H-2Klk gene could involve the substitutions of methylated CpGs. J Immunogenet. 1990;17:133-50 pubmed
    By the isolation of overlapping cosmid clones and 'chromosome walking' studies from the H-2Kk gene, we have obtained cosmid clones encoding the H-2Klk gene from two separate cosmid libraries...
  25. Schweifer N, Barlow D. The mouse plasminogen locus maps to the recombination breakpoints of the tLub2 and TtOrl partial t haplotypes but is not at the tw73 locus. Mamm Genome. 1992;2:260-8 pubmed
    ..However, the mapping of Plg relative to the tLub2 deletion and mRNA analysis of plasminogen in tw73 heterozygotes suggests that Plg does not lie at the tw73 locus. ..
  26. Trinh Q, Shi H, Xu W, Hao J, Luo Y, Huang K. Loop-linker PCR: an advanced PCR technique for genome walking. IUBMB Life. 2012;64:841-5 pubmed publisher
    ..The experimental results demonstrated that loop-linker PCR is an efficient, reliable, and cost-effective method for identifying flanking sequences in transgenic crops and could be applied for other genome walking applications. ..
  27. Feng G, Deak P, Kasbekar D, Gil D, Hall L. Cytogenetic and molecular localization of tipE: a gene affecting sodium channels in Drosophila melanogaster. Genetics. 1995;139:1679-88 pubmed
    ..Sequencing these transcripts will elucidate the role of the tipE gene product in sodium channel functional regulation. ..
  28. Tripoulas N, Hersperger E, La Jeunesse D, Shearn A. Molecular genetic analysis of the Drosophila melanogaster gene absent, small or homeotic discs1 (ash1). Genetics. 1994;137:1027-38 pubmed
    ..During the larval stages the transcript accumulates primarily in imaginal discs. During oogenesis the transcript accumulates in the nurse cells of developing egg chambers. ..
  29. Cox R, Shedlovsky A, Hamvas R, Goldsworthy M, Whittington J, Connelly C, et al. A 1.2-Mb YAC contig spans the quaking region. Genomics. 1994;21:77-84 pubmed
    ..This contig will form the basis for identification and mapping of expressed sequences and for an investigation of genome organization...
  30. Trofatter J, MacCollin M, Rutter J, Murrell J, Duyao M, Parry D, et al. A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor. Cell. 1993;72:791-800 pubmed
    ..The NF2 gene may therefore constitute a novel class of tumor suppressor gene. ..
  31. Acevedo Garcia J, Collins N, Ahmadinejad N, Ma L, Houben A, Bednarek P, et al. Fine mapping and chromosome walking towards the Ror1 locus in barley (Hordeum vulgare L.). Theor Appl Genet. 2013;126:2969-82 pubmed publisher
    ..These factors have prevented the molecular identification of the Ror1 gene for the time being. ..
  32. Zhou S, Yang Y, Scott M, Pannuti A, Fehr K, Eisen A, et al. Male-specific lethal 2, a dosage compensation gene of Drosophila, undergoes sex-specific regulation and encodes a protein with a RING finger and a metallothionein-like cysteine cluster. EMBO J. 1995;14:2884-95 pubmed
  33. Shippy T, Brown S, Denell R. Molecular characterization of the Tribolium abdominal-A ortholog and implications for the products of the Drosophila gene. Dev Genes Evol. 1998;207:446-52 pubmed
    ..In addition, we describe the distribution of Abdominal transcripts during embryogenesis. The Tribolium expression pattern closely resembles that of Drosophila, and does not account for the differences in mutant phenotypes. ..
  34. Sanangelantoni A, Bocchetta M, Cammarano P, Tiboni O. Phylogenetic depth of S10 and spc operons: cloning and sequencing of a ribosomal protein gene cluster from the extremely thermophilic bacterium Thermotoga maritima. J Bacteriol. 1994;176:7703-10 pubmed
    ..DNA spanning 6,613 bp downstream from the gene tuf for elongation factor Tu was sequenced by use of a chromosome walking strategy...
  35. Jagla K, Jagla T, Heitzler P, Dretzen G, Bellard F, Bellard M. ladybird, a tandem of homeobox genes that maintain late wingless expression in terminal and dorsal epidermis of the Drosophila embryo. Development. 1997;124:91-100 pubmed
    ..Lack of late wingless expression and anal plate formation can be rescued with the use of a heat-shock-ladybird transgene. ..
  36. Tonooka Y, Mizukami Y, Fujishima M. One-base excess adaptor ligation method for walking uncloned genomic DNA. Appl Microbiol Biotechnol. 2008;78:173-80 pubmed
    ..Using this technique, the 3'- and 5'-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined. ..
  37. Aitchison K, Gonzalez F, Quattrochi L, Sapone A, Zhao J, Zaher H, et al. Identification of novel polymorphisms in the 5' flanking region of CYP1A2, characterization of interethnic variability, and investigation of their functional significance. Pharmacogenetics. 2000;10:695-704 pubmed
    ..In addition, we report 532 bases of 5' flanking sequence further upstream than that reported to date, and four sequence discrepancies compared to the original published sequence (G-3649C, deltaT-3650, deltaA-4072, and C-4093 ins). ..
  38. Chen S, Li X. Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes. BMC Evol Biol. 2007;7:46 pubmed
    ..These results indicate that TEs are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes. ..
  39. Wang J, Radford D, Holt M, Helms C, Goate A, Brandt W, et al. Sequence-ready contig for the 1.4-cM ductal carcinoma in situ loss of heterozygosity region on chromosome 8p22-p23. Genomics. 1999;60:1-11 pubmed
    ..In addition, four unique cDNA clones from the region were isolated and fully sequenced. This integrated YAC/BAC resource provides the starting point for transcription mapping, genomic sequencing, and positional cloning of this region. ..
  40. Schmitt Wrede H, Koewius H, Tschuschke S, Greven H, Wunderlich F. Genomic organization of the cadmium-inducible tandem repeat 25-kDa metallothionein of the oligochaete worm Enchytraeus buchholzi. Biochim Biophys Acta. 2004;1680:24-33 pubmed
    ..The TATA box-, MREc- and the AP-2, -3-containing region are required for high crp promoter activity. Our data support the view that the crp gene is a unique MT-gene and has evolved by exon duplications from a MT-like ancestral gene. ..
  41. Yamato T, Orikasa K, Fukushige S, Orikasa S, Horii A. Isolation and characterization of the novel gene, TU3A, in a commonly deleted region on 3p14.3-->p14.2 in renal cell carcinoma. Cytogenet Cell Genet. 1999;87:291-5 pubmed
    ..Considering the fact that we found no genetic alterations in TU3A, it is possible that some epigenetic alteration may have suppressed its expression. ..
  42. Marculescu R, Le T, Böcskör S, Mitterbauer G, Chott A, Mannhalter C, et al. Alternative end-joining in follicular lymphomas' t(14;18) translocation. Leukemia. 2002;16:120-6 pubmed
    ..This unusual and restricted window of differentiation opens intriguing questions concerning the etiology of the translocation. ..
  43. Miwa T, Sudo K, Nakamura Y, Imai T. Fifty sequenced-tagged sites on human chromosome 11. Genomics. 1993;17:211-4 pubmed
    ..These STSs can serve as new reagents for investigating human DNA in somatic cell hybrids and for isolating yeast artificial chromosomes to anchor large DNA contigs and fine-scale physical maps of chromosome 11. ..
  44. Szebenyi G, Rotwein P. The mouse insulin-like growth factor II/cation-independent mannose 6-phosphate (IGF-II/MPR) receptor gene: molecular cloning and genomic organization. Genomics. 1994;19:120-9 pubmed
    ..abstract truncated at 250 words) ..
  45. Davidow L, Breen M, Duke S, Samollow P, McCarrey J, Lee J. The search for a marsupial XIC reveals a break with vertebrate synteny. Chromosome Res. 2007;15:137-46 pubmed
    ..The latter argues that marsupial XCI does not require XIST and opens the search for alternative mechanisms of dosage compensation...
  46. Hagemann T, Surosky R, Monaco A, Lehrach H, Rosen F, Kwan S. Physical mapping in a YAC contig of 11 markers on the human X chromosome in Xp11.23. Genomics. 1994;21:262-5 pubmed
    ..2 and ELK1 are less than 50 kb apart. Together these data reveal the following order for the markers in this region: Xpter-UBE1-ARAF1-TIMP-SYN1.2-PFC-ELK1++ +-ZNF81-ZNF21-OATL1-(SYP,TFE3)-Xcen. ..
  47. Kudo F, Numakura M, Tamegai H, Yamamoto H, Eguchi T, Kakinuma K. Extended sequence and functional analysis of the butirosin biosynthetic gene cluster in Bacillus circulans SANK 72073. J Antibiot (Tokyo). 2005;58:373-9 pubmed
    ..In addition, we constructed disruption mutants of btrR1 and btrP-V, and found that the btr genes (ca. 24Kb) between btrR1 and btrP-V are at least required for the butirosin biosynthesis. ..
  48. Gawin B, Niederfuhr A, Schumacher N, Hummerich H, Little P, Gessler M. A 7.5 Mb sequence-ready PAC contig and gene expression map of human chromosome 11p13-p14.1. Genome Res. 1999;9:1074-86 pubmed
    ..Genomic sequencing of clones along a minimal tiling path through the contig is currently under way and will facilitate these expression studies by in silico gene identification approaches. ..
  49. Smoller D, Petrov D, Hartl D. Characterization of bacteriophage P1 library containing inserts of Drosophila DNA of 75-100 kilobase pairs. Chromosoma. 1991;100:487-94 pubmed
    ..The P1 cloning system has general utility in molecular genetics and may provide an important intermediate level of resolution in physical mapping of the Drosophila genome. ..
  50. Liu B, Su Q, Tang M, Yuan X, An L. [Progress of the PCR amplification techniques for chromosome walking]. Yi Chuan. 2006;28:587-95 pubmed
    Various PCR-based methods are available for chromosome walking from a known sequence to an unknown region...
  51. Shirvan A, Srivastava M, Wang M, Cultraro C, Magendzo K, McBride O, et al. Divergent structure of the human synexin (annexin VII) gene and assignment to chromosome 10. Biochemistry. 1994;33:6888-901 pubmed
    ..We conclude that the annexin VII gene may have arisen by a divergence from the evolutionary pathway taken by both annexins I and II. ..
  52. van Heerden H, Collins N, Brayton K, Rademeyer C, Allsopp B. Characterization of a major outer membrane protein multigene family in Ehrlichia ruminantium. Gene. 2004;330:159-68 pubmed
    ..All 16 map1 paralogs were transcriptionally active in E. ruminantium grown in endothelial cells and paralogs with short intergenic spaces were co-transcribed with their adjacent genes...
  53. Clark R, Marker P, Kingsley D. A novel candidate gene for mouse and human preaxial polydactyly with altered expression in limbs of Hemimelic extra-toes mutant mice. Genomics. 2000;67:19-27 pubmed
    ..The human ortholog of Lmbr1 maps within the recently described interval for human PPD, strengthening the possibility that both mouse and human limb abnormalities are due to defects in the same highly conserved gene. ..