Restriction endonuclease (e) cut specific palindromic sequence ex. (aagctt) Cs-9 can cut dna at any specific sequence. It pairs up with hydrogen bonds, in order to increase the strength, you put them together with a condensation reaction, this creates a phosphodiester bond. 4 million base pairs in a chromosome and 1 in 4096 chance of getting a palindrome. So there will be about 1000 cuts in a chromosome for electrophoresis. To avoid this, we use a probe that is complementary to the dna of interest. Most common probes are radioactive, luciferase, or colored enzyme. The tag is always placed on the 5" end to leave the 3" end open to start replicating dna. You then put a mismatch in the original probe so you know where it began. This also causes one of its daughter cells to be a mutant with that particular sequence. This is referred to as oligonucleotide site-directed mutagenesis.