Genomic walking (1)To start, you need:-the DNA sequence of a small region of the chromosome

-An adaptor: a small piece of DNA, 10-15 nucleotides long and double stranded of which you also know the sequence. The adaptor can be easily ordered from a lab. This will be ligated to fragments of the chromosomes after digestion.

- To order PCR primers against the known region of the gene and your small piece of DNA

Known regionof the gene

Adaptor

Primers

Genomic walking (2)Preparation of the DNA

A few digestion reactions need to be set, each using a restriction enzyme which will cut DNA at different place (i.e. different group of 6 nucleotide). For simplicity, we will show only 2 in here.

Enzyme 1 cuts there:

Enzyme 2 cuts there:

Genomic walking (3)So we get two test tubes, containing the same DNA, but this DNA has been cut in different places:

Enzyme1

Enzyme2

Genomic walking (4)Then we add lots of adaptors and a ligase enzyme which is going to bind the adaptorto the end bits of the DNA fragments

Enzyme1

Enzyme2

LigaseLigase

Genomic walking (5)

Enzyme1

Enzyme2

Genomic walking (6)

Enzyme1

PCR on the products of digestion by enzyme 1

Genomic walking (7)

Enzyme1

PCR on the products of digestion by enzyme 1

This fragment (100-1500bp)can be easily sequenced We have discovered more of the DNA sequence…

However, we cannot use the products of this digestion immediately again because we have no other known sequence against which we can order primers. Doing a PCR using a pair of primers against the adaptor would yield too many products which could not be isolated individually.

Genomic walking (8)

Enzyme1

Enzyme2

So if you go back to the DNA sequence that we knew to start with:

Genomic walking (9)

Enzyme1

Enzyme2

This shows the DNA sequence that we know now:

Genomic walking (10)

Enzyme1

Enzyme2

This shows the DNA sequence that we know now:

This part of thenew DNA sequence can be used to design new primers

Genomic walking (11)PCR on the products of digestion by enzyme 2

Enzyme2

New primer, it could have only been designed after getting new DNA sequence from the product of the first PCR

This fragment can be easily sequenced, as a result, we obtain a small fragment ofnew sequence (100-1500bp)

Genomic walking (12)You can now go back to the products of the digestion by enzyme 1 and design new primers towalk further…

Extended know region of DNA sequence againstWhich new primers can be designed!!!!

Enzyme1