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TERM PAPER OF
BIOTECHNOLO GY ON
“CHROMOSOME WALKING”

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SUBMITTED TO : MR. HARSH KUMAR (LEC. LPU)

SUBMITTED BY: NAME :- ANKIT PANDEY SECTION :-B ROLL NO:- R109X39 REG NO :1040070152

INDEX
S. NO. 1. 2. 3. 4. 5. 6. 7. 8. 9. TOPIC ACKNOWLEGEMENT INTRODUCTION HISTORY OF CHROMOSOME WALKING WHAT IS CHROMOSOME WALKING METHODS OF CHROMOSOME WALKING APPLICATION OF CHROMOSOME WALKING SUMMARY LATEST RESEARCH REFRENCES PAGE NO.

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ACKNOWLEDGEMENT

I gratefully acknowledge my indebtedness to my respected teacher MR . HARSH SIR who is always a source of inspiration for me, for providing this opportunity. Under his able guidance, I have learned how to overcome the odds and trying circumstances. I am also very thankful to my colleagues with whom I

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could discuss and give the final shape to this term paper. At the same time, I am also thankful to my class incharge Dr. PRABHOJOT JASSAL who gave us an immense help in the completion of this project. Any constructive comments, suggestions, criticism from students and colleagues will be highly appreciated and gratefully acknowledged.

INTRODUCTION
CHROMOSOME WALKING
chromosome walking involves localization of gene families whiten a chromosome domain through the use of overlapping clones. Chromosome walking techniques

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and the mapped marker permit identification of specific loci and isolation of gene for the study of their expression. Large region sf genome can thus be cloned through the isolation of series of overlapping recombinants in this method a segment of nonrepetitive DNA isolated from the end of recombinant. Can be used as a probe for identification of CLONES with adjacent sequence because of slowness process the use cosmid rather than bacteriophase is preferred . In cosmid the foreign DNA of approx 45 kb can be inserted . Chromosome walking involves in the localization of gene families within a chromosome domain through the use . The principle of chromosome walking It initially involves in the selection an identified gene from the library/ and subcloning of its end segment The sub cloned segment is hybridized with other cones and on the basic of hybridisation of the overlapping end sequence the adjunct clone is chosen the end segment of the second cloned is hybridised again with clones fro the library and the third adjacent sequence is chosen on the basis of hybridization of the overlapping sequences . the repetition of this process utilising overlapping restriction sites ultimately leads to mapping all the adjacent genes along the length of the chromosome . The primary seep in cloning genes through chromosome walking in to secure probe within a few hundred kilo bases of the desired locus in order to

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achieve this objective meiotic segregation of the mutants is correlated with restriction fragments length polymorphism. After identification of linked RFLPs, cloning of desired genes entails isolation of cloned DNA fragments bridging the gene chromosome walking through small steps through cosmid or bacteriophage is time consuming and laborious . the latter development of the technique for cloning and maintenance of lon DNA fragments as insert in yeast artificial chromosome vector in yeast cell has emerged as a powerful too for chromosome walking it enables DNA fragments of several hundred kilo bases to be coloned

HISTORY OF CHROMOSOME

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Discovery To answer your question stricktly, no one discovered the process of chromosome walking, as the word discovery suggests that the thing that was discovered had been present but unnoticed. But I quibble. The technique known as chromosome walking (or a chromosomal walk) was developed by Welcome Bender, Pierre Spierer, and David S. Hogness in the Early 1980's. The paper in which they first describe the technique is: Bender W, Spierer P, and Hogness DS. (1983) Chromosomal Walking and Jumping to Isolate DNA from the ACE and rosy Loci and the Bithorax Complex in Drosophila melanogaster. J Mol Biol 168 1733. Reading the abstract of that paper, I find that the term "chromosomal walk" is used rather casually, as if everyone already knows what it is (which may have been the case, see below). But in the Introduction, the authors state, "The strategy we have used is called chromosomal walking and jumping; it is shown diagrammatically in Figure 1.", after which they explain how chromosomal walking works, followed by an explanation of chromosomal jumping. Interestingly though, this paper is not the first paper to present genetic mapping research that relied on chromosmal walking. In 1981, C. Weldon Jones and Fortis C. Kafatos published the following two papers, which describe the use of chromosomal walks to map Chorion genes in Silkmoths:

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Jones CW, Kafatos FC. (1981) Linkage and evolutionary diversification of developmentally regulated multigene families: tandem arrays of the 401/18 chorion gene pair in silkmoths. Mol Cell Biol. (9):814-28.

"Chromosome walking" 410 x 376 - 50k - jpg www.entu.cas.cz

In this way chromosome Chromosome walking Chromosome walking walking among ... 632 x 400 - 57k - jpg 1650 x 1275 - 189k - jpg 563 x 707 - 57k - gif homepages.strath.ac.uk web.nmsu.edu biology200.gsu.edu

... time that ... Gene by ... Gene by Chromosomal Walking chromosome walking Chromosome Walking. Chromosome Walking. to Clone the ... was ... 455 x 423 - 8k - gif 373 x 344 - 14k - gif 342 x 442 - 6k - gif 769 x 549 - 96k - jpg www.mindfully.org www.ornl.gov www.bio.davidson.edu www.shigen.nig.ac.jp [ More from www.bio.davidson.edu ]

Chromosome walking 染色体歩行(chromosome ... chromosome from un-10 toward ... walking methods. walking)や DNA ... 561 x 261 - 3k - gif 808 x 648 - 167k - jpg 570 x 255 - 5k - gif www.fgsc.net www.nature.com www.sc.fukuoka-u.ac.jp [ More from [ More from www.fgsc.net ] www.nature.com ]

... or chromosome walking. 588 x 377 - 10k - gif lifesci.ucsb.edu

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METHOD OF CHROMOSOME WORKING Chromosome walking is a method in genetics for identifying and sequencing long parts of a DNA strand, e.g., a chromosome. As long DNA strands cannot be sequenced, this method works by dividing the long sequence into several short ones. Basically, chromosome walking works as follows: 1)A primer that matches the beginning of the DNA to sequence is used to synthesize a short DNA strand complementray to the unknown sequence, starting with the primer (see PCR). 2)The new short DNA strand is sequenced. 3)The end of the sequenced strand is used as a primer for the next part of the long DNA sequence. That way, the short part of the long DNA that is sequenced keeps "walking" along the sequence. The method can be used to sequence entire chromosomes (thus, chromosome walking). A different method with the same purpose which becomes more popular for large-scale sequencing (e.g., the Human Genome Project) is shotgun sequencing.

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APPLICATION OF CHROMOSOME WALKING A more general approach is to clone genes by chromosome walking. This strategy is general in the sense that the cloning of the gene is based solely on the mutant phenotype and genetic map position. Therefore, chromosome walking can be used to clone any gene which can be genetically identified. The first step toward cloning the gene is to identify DNA probes residing within one to several cM of the locus of interest. Typically this is achieved by analyzing the meiotic segregation of restriction fragment length polymorphisms (RFLPs). Once a linked RFLP(s) has been identified, it can be used as the starting point to initiate a chromosome walk. Briefly, chromosome walking entails the progressive isolation and characterization of overlapping sets of genomic clones. The overlapping clones are selected by hybridization using end-specific probes (probes generated from the extremities of the clone/contig). The walk is continued in this manner until the region spanning the intervening gap has been bridged by an overlapping set of clones. While chromosome walking is technically straight forward, in practice the procedure is extremely labor intensive and ill-suited for large projects where more than a few steps are required.

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SUMMARY
Chromosome walking is a technique for cloning everything in the genome around a known piece of DNA (the starting probe). You screen a genomic library for all clones hybridizing with the probe, and then figure out which one extends furthest into the surrounding DNA. The most distal piece of this most distal clone is then used as a probe, so that ever more distal regions can be cloned. This has been used to move as much as 200 kb away from a given starting point (an immense undertaking). Typically used to "walk" from a starting point towards some nearby gene in order to clone that gene. Also used to obtain the remainder of a gene when you have isolated a part of it.

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LATEST RESEARCH
Genet Res. 2005 Apr ;85 (2):93-100 16174327 (P,S,G,E,B) [Cited?]Map-based isolation of disease resistance genes from bread wheat: cloning in a supersize genome.

[My paper] Beat Keller, Catherine Feuillet, Nabila Yahiaoui Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland. bkeller@botinst.unizh.ch The genome of bread wheat is hexaploid and contains 1.6 x 10 10 bp of DNA, of which more than 80% is repetitive sequences. Its size and complexity represent a challenge for the isolation of agronomically important genes, for which we frequently know only their position on the genetic map. Recently, new genomic resources and databases from genome projects have simplified the molecular analysis of the wheat genome. The first genes to be isolated from wheat by map-based cloning include three resistance genes against the fungal diseases powdery mildew and leaf rust. In this review, we will describe the approaches and resources that

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have contributed to this progress, and discuss genomic strategies that will simplify positional cloning in wheat in the near future. Mesh-terms: Chromosome Walking; Chromosome Mapping :: methods; Cloning, Molecular :: methods; Genes, Plant; Genome, Plant :: genetics; Immunity, Natural :: genetics; Mycoses; Oryza sativa :: genetics; Research Support, Non-U.S. Gov't; Triticum :: genetics; Triticum :: microbiology;

REFRENCES
Berg, D. E. and Howe, M. M. (1989). Mobile DNA. (American Society for Microbiology, Washington, D.C.). Burke, D. T., Carle, G. F., and Olson, M. V. (1987). Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science, 236, 806-812. Chang, C., Bowman, J. L., DeJohn, A. W., Lander, E. S. and Meyerowitz, E. M. (1988). Restriction fragment length polymorphism linkage map for Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA, 85, 6856-6860. Coulson, A., Sulston, J., Brenner, S., and Karn, J. (1986). Toward a physical map of the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA, 83, 7821-7825. Coulson, A., Waterston, R., Kiff, J., Sulston, J., and Kohara, Y. (1988). Genome linking with yeast artificial chromosomes, Nature, 335, 184-186. Feldman, K. A., Marks, M. D., Christianson, M. L., and Quatrano, R. S. (1989). A dwarf mutant of Arabidopsis generated by T-DNA insertion mutagenisis. Science, 243, 1351-1354. Grill, E. and Somerville, C. (1991). Construction and characterization of a yeast artificial chromosome library of Arabidopsis which is suitable for chromosome walking. Molec. Gen. Genet., in press. Hauge, B. M. and Goodman, H. M. (1991). Physical Mapping by Random Clone Fingerprint Analysis. In Plant Genomes: Methods for Genetic and Physical Mapping, T. Osborn and J.S. Beckmann eds. (Kluwer). In press.

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Hauge, B. M., Hanley, S., Giraudat, J., and Goodman, H. M. (1991). Mapping the Arabidopsis Genome. In Molecular Biology of Plant Development, G. Jenkins and W. Schurch eds. In press. Hwang, I., Kohchi, T., Hauge, B. M., Goodman, H. M., Schmidt, R., Cnops, G., Dean, C., Gibson, S., Iba, K., Lemieux, B. L., Danhoff, L., and Somerville, C. (1991). Identification and map position of YAC Clones comprising one third of the Arabidopsis genome. (submitted). Kohara, Y., Akiyama, K., and Isono, K. (1987). The physical map of the whole E. coli chromosome: Application of a new strategy for rapid analysis and sorting of a large genomic library. Cell, 50, 495-508. Kuspa, A., Vollrath, D., Cheng, Y., and Kaiser, D. (1989). Physical mapping of the Myxococcus xanthus genome by random cloning in yeast artificial chromosomes. Proc. Natl. Acad. Sci. USA, 86, 8917-8921.

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