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INDEX1.Terminology
2.Introduction of chromosome walking
3.Procedure of chromosome walking
4.Introduction of chromosome jumping
5.Procedure of chromosome jumping
6.Application of chromosome walking &
jumping.
7 .References
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Chromosome walking
It is technique used to isolate contiguous cloned DNA fragments by using
each fragment as a probe to isolate adjacent
cloned regions.
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Chromosome jumping
A technique used to isolate non-
contiguous regions of DNA by jumping across gaps that may appear as a consequence of
uncloned regions of DNA in gene library.
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Probes: Probes are small (15-30 bases long) nucleotides
(RNA & DNA) sequences used to detect the presence of completely nucleotide sequence used for disease diagnosis.
Contig:Clones containing neighboring DNA fragments
having an overlapping region.
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Genomic library: A collection of clones made from a set of
randomly generated overlapping DNA fragments representing the entire genome of an organism.
Hybridization:
The process of joining two complementary strands of DNA or one each of DNA and RNA to form a double-stranded molecule. One strand is often labeled and used as a probe to detect the presence of the second strand.
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Jumping library: A clone such a library contains sequences on one
side each of two recognition sites of rare cutting enzyme .
e.g-Not I for human genome.
Linking library:
A clone of such a library has sequences on either side of a single recognition site of a rare cutting enzyme like Not I for human genome.
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Introduction of chromosome walking
Chromosome walking is DNA base sequencing method in which the chromosome is analysed by extending along the chromosome from known location to reach an unknown location .
Chromosome walking technique is applicable for much smaller DNA fragment.
These technique is used as a means of
finding adjacent gene or parts of a gene which are missing in the original clone and
analyse long streachs of eukaryotic DNA.
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If chromosome walking is done in conjaction
with DNA sequencing would take several
years to cover 1 mu distance.
For this reason, researchers are agar to locate starting point in a exp. That are
close as possible to the gene of interest.
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Procedure of chromosome walking
1.The first step in ‘walking’ a chromosome
consist of the isolation of a DNA fragment
containing a known gene or marker located near some region of interest in the
given chromosome this fragment provide
starting point .
2.A ristriction map of this fragment is
prepared .A small segment representing
one end of this original fragment 1 is
isolated and cloned ; this is called subcloning. This subclone now used as a prob for the identification of such clone
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in the genomic library that overlap fragment
1.
3.The clone(s) identified in this way will contain such a DNA insert that overlaps
fragment 1 prefareby at one end; the new
genomic fragment may be referred to as
fragment 2.
A ristriction map of fragment 2 is prepared
, and the sequence at the other end of this
fragment is now used as a probe to identify
clones having DNA inserts overlapping with fragment 2.
4.The DNA inserts obtained from such clones will be overlapping fragment 2 prefereby at
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on end ; this new genomic fragment may be
called fragment 3.
The process of step 3 is repeated till we reach one end of chromosome.
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Chromosome
walking
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Introduction of chromosome jumping
The technique of chromosome jumping is used for the creation of contigs and for construction of restriction map of genomes.
Chromosome jumping is used for rather
large DNA fragments.
A simple approch to chromosome jumping uses ‘jumping’ and ‘linking’ libraries generated by a rare cutting enzyme, e.g. NotI for humans.
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each clone in jumping library contains DNA sequences on one side each e.g. sequences 2 and 3 , of two neighboring cutting sites of enzyme Not I ,while a clone in linking library has the DNA sequences located on either side of a single Not I , site e.g. regions 1 and 2 in clone 1 and regions 3 and 4 in clone 2.
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Procedure of chromosome jumping• In construction of Not I jumping library• Genomic DNA is completely digested with NotI and
the resulting fragments are digested with NotI, and resulting fragments are circularized in presence of SupF marker.
• The circularized fragments are digested with a frequent cutting enzyme like BamH1 to delete bulk of DNA leaving a small sequence of DNA on either side of suppressor molecule(SupF marker) .
• These restricuion fargnents are ligated in a suitable λ vector and cloned
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• Similarly, the linking library clones are prepared. The genomic DNA is partially digested with a frequent cutting enzyme like a Sau 3A and the fragments are circularized in presence of sup F marker. The circularized fragments are cut open with enzyme Not I and linear fragments are integrated in a suitable λ vector.
• Only those circles that have Not I site will become linear and, hence, integrated in the λ vector. This is important to not that the regions surrounding a single Not I sites are present in one clone.
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Chromosome
jumping
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• SupF is a tRNA gene that has a mutated anticodon , which recognizes a polypeptide chain termination codon generated by a suppressor –sensitive mutation within a gene
• Therefore, SupF serves as selectable marker when it is introduced in an E coli strain that carries the concerned suppressor- sensitive muatation with in aessential gene.
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• Physical mapping of a chromosomes begins with the preparations of YAC clones from the target chromosome, using Not I to digest the chromosomal DNA .
• In order to create a contig of these YAC clones. Linking and jumping library are made from DNA of the same chromosome using Not I and Bam H1 restriction enzymes as described above. Each clone of linking library hybridizes with two different clones of the jumping library and vice versa.
• But each linking library clone hybridize with only one YAC clone , while each clone of jumping library will hybridizes with two different YAC clones.
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Application of chromosome walking and jumping
• The gene responsible for the disease cystic
fibrosis (CF) has been identified by chromosome walking and jumping.
The gene of protein responsible for CF is
called cystic fibrosis transmembrane
conductance regulator(CFTR) and located
on the long arm of chromosome 7.
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• for many human disorders, which are each controlled by a single gene (e.g. Huntington disease, cystic fibrosis, duchenne muscular dystrophy, etc.), the gene product is not known despite intensive investigations. Such genes are cloned by a process, in which the gene is identified primarily by its map position.
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Cystic fibrosis Procedure to identify the gene responsible for this
disease.
Isolate the DNA of the patient
Go For genetic mapping
Go For physical mapping or Restriction
Candidate region
Chromosome Walking
Chromosome jumping
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References:
• Genetics by B.D sing.• Genomes.• Principle of genetics.• Biotechnology by U.Satyanarayan.• Genetics engineering sec. edition by Desmond
S.T. Nicholl.• www.studentguide .com
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