A High
Efficient Approach Used for BAC-contig Extension of Oryza sativa with
PCR Screening the BAC Clone Pools
HU Hao,LI Tao,MU Jie,HAN Bin*,HONG
Guo-Fan*
( National Center for Gene Research,Shanghai Institutes for Biological
Sciences,the Chinese Academy of Sciences,Shanghai 200233,China )
Abstract To extend 8
BAC contigs, which were previously located in the 56.1―68 cM region of the
chromosome 4 of the Oryza sativa indica GuangLuAi4, 14 pairs of primers
were designed according to the terminal sequences of the existing seed BACs and
were deliberately divided into 3 groups. With the 3 groups of primer mixtures,
233 pools of BAC DNA that represent 22 368 BAC clones from O.sativa indica
GuangLuAi4 genomic library were screened. 65 positive clones corresponding
to the 8 contigs were isolated and 29 clones of them were confirmed to be
extended to the seed BACs by end-sequencing and fingerprinting.The protocol
greatly enhanced the efficiency of the contig extension and was also superior
for its specificity,sensitivity and reusability to the colony in situ
hybridization which is a conventional method employed in contig extension and
physical map construction.
Key words Oryza sativa; BAC; PCR; contig
*Corresponding authors: HAN
Bin: Tel, 86-21-64845260;Fax,86-21-64825775; e-mail, [email protected]; HONG
Guo-Fan: Tel, 86-21-64516371; Fax, 86-21-64825775; e-mail, [email protected]