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Genomic walking (1)

To start, you need: the DNA sequence of a small region of the chromosome

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Genomic walking (1)

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  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 Genomic walking (1) Known region of the gene Adaptor Primers

  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. Genomic walking (2) Enzyme 1 cuts there: Enzyme 2 cuts there:

  3. So we get two test tubes, containing the same DNA, but this DNA has been cut in different places: Genomic walking (3) Enzyme 2 Enzyme 1

  4. Then we add lots of adaptors and a ligase enzyme which is going to bind the adaptor to the end bits of the DNA fragments Ligase Ligase Genomic walking (4) Enzyme 2 Enzyme 1

  5. Genomic walking (5) Enzyme 2 Enzyme 1

  6. PCR on the products of digestion by enzyme 1 Genomic walking (6) Enzyme 1

  7. PCR on the products of digestion by enzyme 1 Genomic walking (7) This fragment (100-1500bp) can be easily sequenced We have discovered more of the DNA sequence… Enzyme 1 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.

  8. So if you go back to the DNA sequence that we knew to start with: Genomic walking (8) Enzyme 2 Enzyme 1

  9. This shows the DNA sequence that we know now: Genomic walking (9) Enzyme 2 Enzyme 1

  10. This shows the DNA sequence that we know now: Genomic walking (10) Enzyme 2 Enzyme 1 This part of the new DNA sequence can be used to design new primers

  11. PCR on the products of digestion by enzyme 2 Genomic walking (11) New primer, it could have only been designed after getting new DNA sequence from the product of the first PCR Enzyme 2 This fragment can be easily sequenced, as a result, we obtain a small fragment of new sequence (100-1500bp)

  12. You can now go back to the products of the digestion by enzyme 1 and design new primers to walk further… Genomic walking (12) Enzyme 1 Extended know region of DNA sequence against Which new primers can be designed!!!!

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