The novel and abundant transcript gene, GS3582, in our established HCE cDNA library, corresponded to the daily updated dbEST and extended the expected sequence. 5′-Rapid amplification of cDNA ends (RACE)-PCR cloning was performed using a kit (Sure-RACE; Origene Technologies, Inc., Rockville, MD) that allows isolation of the 5′ sequence of the target transcript. The RACE panels in the kit consist of double-stranded cDNA from 24 individual human tissues arrayed in a multiwell plate and provides two contiguous adapter-specific primers at the 5′ end. Outer primer for first-round PCR, gene-specific primer (GSP)-1: 5′-TTTCCGCAACATTCTCCTTTT-3′) and inner primer for second-round (nested) PCR, GSP2: 5′-TTCTGTGTTTGGCTTGGT-3′) were designed from the expected sequence.
The RACE cDNAs were amplified with DNA polymerase (KOD-Plus; Toyobo). After incubation at 94°C for 3 minutes, the first round of PCR was performed with 5 cycles of 94°C for 3 seconds, 63°C for 30 seconds, and 72°C for 3 minutes and then 15 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 6 minutes, followed by 72°C for 6 minutes in a PCR thermal cycler (model MP; Takara, Kyoto, Japan). After incubation at 94°C for 3 minutes, the second round of PCR was performed with 35 cycles of 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 3 minutes, followed by 72°C for 6 minutes. Among human cDNAs in different tissues, PCR products 300 to 400 bp in length were circularized and subcloned into a vector (pBluescript; Stratagene, La Jolla, CA). Randomly selected plasmids were amplified and purified into DNA with a kit (Plasmid Midi; Qiagen, Hilden, Germany). The products were amplified with T7 promoter primer (T7 HT Primer; Toyobo) with 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes after incubation at 96°C for 30 seconds. Amplified products were sequenced with the autosequencer (ABI Prism 310 Genetic Analyzer; PE Biosystems). The resultant sequence was extended to the expected sequence, and the analysis of the total GS3582 sequence was concluded.