How would you design a primer for sequencing a DNA fragment of Unknown sequence?

If you are given a tissue sample and asking you to sequence DNA.. you don't have any idea about the sequence..And sanger's method is the simplest method for sequencing DNA.. but this method requires a Primer. since you don't have any idea about the given DNA , how would you design primer for it?

How would you design a primer for sequencing a DNA fragment of Unknown sequence?
I like that you post this to Yahoo answers. Makes me laugh.
Reply:You digest the DNA with restriction enzymes, insert the fragments into a plasmid or a phage reacted with the same restriction enzyme(these are called vectors), and transform a bacteria with the vector. This creates a library. You know where the fragments are inserted because the the vector only has one restriction site for the enzyme you choose. The sequence of the the vector is already known, so you can prime on the vector DNA upstream from the restriction site.
Reply:If you have the money and resources, you shotgun sequence it. Use random primers.





Afterward use some assembly software to figure out a tentative sequence for you; then go back and sequence the rest in a less random manner.





If you're specifically trying to sequence a gene, that's easier. You might not know what the sequence is, but you can guess based on homologous sequences from the same organism and other related species.
Reply:Is this from a tissue sample: in other words, from all the genomic DNA? Or is it from a pre-isolated fragment (generated by restriction fragment digest).





In the first case, I would isolate the mRNA and perform rtPCR to get a cDNA library (you can use a poly-T probe to target the poly-A tail of mRNA, IIRC).





In the second case, if you know the restriction enzyme used to digest the DNA, then you know the sequence where that enzyme cuts, and you can design a primer to target that.





Good luck.