Both the PCR product containing the mammalian gene with the new restriction sites and the destination plasmid are subjected to restriction digestion, and the digest products are purified by gel electrophoresis.

Clones were analyzed by restriction digestion, and nucleotide sequencing was carried out on both strands by the dideoxy chain termination method.

The approach entails restriction digestion of the DNA sample then ligation of specially designed 'vectorette' linkers.

The construction of P51+6aa was carried out by removal of the 360 bp fragment from pKK-RT66+6aa by restriction digestion with SacI followed by re-ligation of the vector ends.

Genomic DNA was prepared from one Arg +Ura +segregant from each culture and subjected to restriction digestion and Southern analysis.

Srf I restriction digestion was then used to remove the Kan r cassette from the clones carrying in-frame insertions, thereby creating a sequence encoding a full-length fusion protein.

The technique is a variation of bisulfite sequencing, and combines bisulfite conversion based polymerase chain reaction with restriction digestion.

Sequence integrity of the cloned DNA was verified by cycle sequencing and restriction digestion.

Inactivation of the N25 or UV5 promoters was carried out by restriction digestion with Dra 1 or BstN 1 respectively, as previously described (12,15).

Candidates were screened with a combination of PCR analysis, restriction digestion, and DNA sequencing to identify those with successful allele replacement.