Technique for cloning and sequencing the ends of bacterial artificial chromosome inserts.
Bacterial artificial chromosome (BAC) libraries are an important tool for positional cloning, gene analysis and physical mapping. During studies using BAC clones, it is often necessary to organize them into contiguous sequences (contigs). To finalize, join and extend the contigs, both cloning and sequencing of the ends of the inserts are required. Here, we describe a low-cost, accessible, fast and powerful method for the routine isolation of BAC ends. This method allows the isolation of 20 BAC clone ends in one day. The analysis of the ends reveals fragment sizes compatible with sequencing, and the structure of these clones allows the sequencing of both ends using the same plasmid. Moreover, long end fragments can be sequenced in both directions.
Ripoll
P J
PJ
University of Bristol, Long Ashton, UK. pierre-jean.ripoll@bbsrc.ac.uk
O'Sullivan
D M
DM
Edwards
K J
KJ
Rodgers
M
M
D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000737:chemistry / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D010957:Plasmids / Q000737:chemistry; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA / Q000379:methods*; D014169:Transformation, Bacterial; D003313:Zea mays / Q000235:genetics
D004269:DNA, Bacterial; D018744:DNA, Plant; D004274:DNA, Recombinant; D011933:Reagent Kits, Diagnostic