Articles
CONSTRUCTION OF CHROMOSOME #1 SPECIFIC LIBRARY OF LILIUM TIGRINUM
Article number
855_19
Pages
143 – 148
Language
English
Abstract
Metaphase chromosome #1 of Lilium tigrinum was microdissected and microcloned.
DOP-PCR and linker-mediated PCR were used to amplify microdissected L. tigrinum chr. #1. DOP-PCR and linker-mediated PCR yielded products ranging in size from 100 to 3000 bp, with predominant fragments at 300 to 2000 bp.
When two methods were compared, linker-mediated PCR gave rise to a little bigger fragment.
Southern hybridization with DIG-labeled genomic DNA confirmed that the products from both methods were amplified from the L. tigrinum genome.
Cloning using size-fractionated DNA gave recombinant clones larger than 500 bp.
DOP-PCR gave larger recombinant clones than linker-mediated PCR. The insert size was approximately from 300 to 2000 bp.
DOP-PCR produced DNA fragments ranging in size from 400 to 2000 bp, and linker-mediated PCR from 300 to 1200 bp, with predominant fragment at 500 to 1000 bp.
When randomly selected 30 clones were sequenced, only two clones from DOP-PCR showed homology with Lilium henryi or L. philadelphicum and others were identified as unknown unique sequences.
On the other hand, 8 out of 15 clones from linker-mediated PCR showed homology with microsatellite region of other plant species.
Based on the results obtained in this experiment, DOP-PCR was more suitable for finding unknown unique sequences than linker-mediated PCR.
DOP-PCR and linker-mediated PCR were used to amplify microdissected L. tigrinum chr. #1. DOP-PCR and linker-mediated PCR yielded products ranging in size from 100 to 3000 bp, with predominant fragments at 300 to 2000 bp.
When two methods were compared, linker-mediated PCR gave rise to a little bigger fragment.
Southern hybridization with DIG-labeled genomic DNA confirmed that the products from both methods were amplified from the L. tigrinum genome.
Cloning using size-fractionated DNA gave recombinant clones larger than 500 bp.
DOP-PCR gave larger recombinant clones than linker-mediated PCR. The insert size was approximately from 300 to 2000 bp.
DOP-PCR produced DNA fragments ranging in size from 400 to 2000 bp, and linker-mediated PCR from 300 to 1200 bp, with predominant fragment at 500 to 1000 bp.
When randomly selected 30 clones were sequenced, only two clones from DOP-PCR showed homology with Lilium henryi or L. philadelphicum and others were identified as unknown unique sequences.
On the other hand, 8 out of 15 clones from linker-mediated PCR showed homology with microsatellite region of other plant species.
Based on the results obtained in this experiment, DOP-PCR was more suitable for finding unknown unique sequences than linker-mediated PCR.
Publication
Authors
Y.J. Hwang, I.S. Ahn, I.S. Park, K.B. Lim, S.Y. Kang
Keywords
chromosome isolation, DOP-PCR, linker-mediated PCR, microcloning, microdissection
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