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A molecular genetic technique used to clone a gene of interest from its

closest known piece of DNA, as the starting probe.

What is “Chromosome Walking”?

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The known piece of DNA that links to the gene of interest is used to probe a genomic library. Most distal part of the most distal clone is used as probe in

order to sequence the gene. This process is repeated several times to

walk across the chromosome and reach the gene of interest.

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Purpose of Chromosome Walking

To obtain the gene of interest

To obtain the remainder of a gene when you have isolated a

part of it.

“Chromosome Walking" is used instead when we know the sequence but don't have

a clone of a gene.

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Chromosome walking

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1. Firstly, create genomic libraries by cutting the DNA into fragments (for example, by partial digestion with a restriction enzyme).

2. Next, hybridizing a primer of known sequence to a clone from an unordered genomic library and synthesizing a short complementary strand.

3. The complementary strand of clone 2 is then sequenced and its end is used as next primer for further walking to find an overlapping clone 3 in genomic library.

4. Clone 4, in turn, be used as a probe to identify clone 5, and so on.

5. It is, therefore, possible to walk along the chromosome until target gene (gene 6) is reached and cloned.

6. In this way, the adjacent of the region, which is previously unknown, is finally identified and sequenced. 

1. Firstly, create genomic libraries by cutting the DNA into fragments (for example, by partial digestion with a restriction enzyme).

2. Next, hybridizing a primer of known sequence to a clone from an unordered genomic library and synthesizing a short complementary strand.

3. The complementary strand of clone 2 is then sequenced and its end is used as next primer for further walking to find an overlapping clone 3 in genomic library.

4. Clone 4, in turn, be used as a probe to identify clone 5, and so on.

5. It is, therefore, possible to walk along the chromosome until target gene (gene 6) is reached and cloned.

6. In this way, the adjacent of the region, which is previously unknown, is finally identified and sequenced. 

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Chromosome Walking

ADVANTAGES DISADVANTAGES

No need the construction of DNA libraries

Requires only little information for screening

Sequence the entire chromosome

Difficult to sequence the sequence at the end of the chromosome

Difficult to sequence the sequence at the end of the chromosome

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Why Chromosome Walking?

Identify, isolate and clone gene whose product is unknown

Identify the molecular lesion in the gene of interest that can cause diseases – cystic fibrosis

Discovery CTFR, BRCA1 & BRCA2 gene