End Rescue from YACs Using the Vectorette
互联网
628
The vectorette unit (1 ) consists of a pair of annealed oligonucleotides that contain two regions of complementary nucleotide sequence flanking a 29-bp noncomplementary segment (Fig. 1 ). The 5′ terminus of one of these complementary regions is phosphorylated and displays a restriction enzyme-specific sticky (or blunt) end that permits ligation of vectorette units to both ends of a restriction fragment. A nested pair of polymerase chain reaction (PCR) primers, VP1 and VP2, directed toward the phosphorylated end have most of their sequence, including their 3′ termini, in the noncomplementary region of the vectorette. These oligonucleotides cannot function as PCR primers on vectorette units without synthesis of a complementary strand from a primer located in the ligated DNA. Thus, although vectorette units will ligate to themselves and to all restriction fragments with matching ends, only DNA flanked by a specific primer (from known sequence) and a vectorette will be amplified in PCR.
Fig. 1. Oligonucleotides used in vectorette end rescue from YACs: (A) The vectorette unit and associated PCR and sequencing primers. In the blunt-ended vectorette residue X = 5′ Phospho-T; Y = A. In the GATC sticky-ended vectorette (NNNN)X = 5′ Phospho-GATCG; Y = C. (B) PCR and sequencing primers for the r(ight) and l(eft) arms of the YAC vector pYAC4 (based on sequence in ref. 3 ).
