1

TERM
PAPER
OF
BIOTECHNOLO
GY
ON
“CHROMOSOME
WALKING”
 2

SUBMITTED TO : SUBMITTED BY:

MR. HARSH NAME :- ANKIT
KUMAR PANDEY
(LEC. LPU) SECTION :-B
ROLL NO:- R109X39
REG NO :-
1040070152

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

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
 4

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
 5

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 non-
repetitive 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
 6

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
 7

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 17-
33. 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:
 8

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 In this way chromosome Chromosome walking Chromosome walking

walking" walking among ... 632 x 400 - 57k - jpg 1650 x 1275 - 189k - jpg
410 x 376 - 50k - jpg 563 x 707 - 57k - gif homepages.strath.ac.uk web.nmsu.edu
www.entu.cas.cz 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 ... or chromosome

from un-10 toward ... walking)や DNA ... walking methods. walking.
561 x 261 - 3k - gif 570 x 255 - 5k - gif 808 x 648 - 167k - jpg 588 x 377 - 10k - gif
www.fgsc.net www.sc.fukuoka-u.ac.jp www.nature.com lifesci.ucsb.edu
[ More from [ More from
www.fgsc.net ] www.nature.com ]
 9

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.
 10

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.
 11

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.
 12

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
 13

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.
 14

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.