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Figure 1. Expression and purification of GST-CA150 WW

A. System cartoon for air-bubbling cultivation. B. Time-dependent increase in the amount of GST-CA150 WW domain expression. Sizes of marker bands were 94, 67, 43, 30 and 20.1 from upper to bottom. The arrow indicates the band of the GST-CA150 WW domain. Lane 1 - 3, expression with shaking. Lane 4 - 8, expression with bubbling air. Lane 1 and 4, sampled culture at induction with 1 mM IPTG. Lane 2 and 5, 2 hours after the induction. Lane 3 and 6, 5 hours after the induction. Lane 7, 7.5 hours after the induction. Lane 8, 9 hours after the induction. C. Purity of WW domain of CA150. Sizes of marker bands were 94, 67, 43, 30, 20.1, 12.2 from upper to bottom. The arrow indicates the band of the CA150 WW domain.

IMPROVEMENT OF LIGATION-MEDIATED POLYMERASE CHAIN REACTION (LM-PCR) METHOD FOR GENOME WALKING WITH A SINGLE GENE-SPECIFIC PRIMER

Keina Kozono,1 Ayako Aoki,2 Masayoshi Tsukahara,1 and Haruhiko Tsumura1

'CMCR&D Laboratories, KIRINBrewery Co., Ltd., 100-1 Hagiwara, Takasaki, Gunma 3700013, Japan; 2Pharmaceutical Research Laboratories, KIRIN Brewery Co., Ltd., 3 Miyahara, Takasaki, Gunma 370-1295, Japan

Abstract: We have devised an improved ligation-mediated polymerase chain reaction (LM-PCR) for genome walking to obtain unknown genomic DNA sequences located adjacent to known sequences. Dozens of PCR-based methods have been reported for genome walking; however, these methods are limited to the amplification of a particular region due to their relatively high non-specific background. To increase the specificity and efficiency, we designed a unique oligo-cassette consisting of two different length oligo-nucleotides, a 3'-end amino group modified short one and a hairpin-shaped longer one with the arbitrary gene-specific sequence. Using these oligo-cassettes, we can amplify the 2.3 kb of genomic region coding the a-1, 6-fucosyltransferase (FUT8) gene from Chinese hamster ovary (CHO) cells by one-round PCR with a single gene-specific primer.

Key words: ligation-mediated polymerase chain reaction; a -1, 6-fucosyltransferase (FUT8); Chinese hamster ovary (CHO) cell

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