Methods for Korean UniGene Construction

1. mRNA Sources
   1. Stomach: cancer cell lines, Normal tissues, Primary cancer tissues
   2. Liver: cancer cell lines, Normal tissues, Primary cancer tissues
   3. Brain: Normal tissues, Primary cancer tissues
   4. Cervix: Cancer cell lines
   5. Thymus: Normal tissues
   * 각 library별로 사용된 mRNA source, vector, library type 등 보다 자세한 사항은 [Library Bank] 참조

2. Libray Types
   • Library Bank for distribution
   • [F] Full-length enriched cDNA library
   • [U] Universal cDNA library
   • [Fs] Subtracted full-length enriched cDNA library
   • [Un] Normalized universal cDNA library
   • [Us] Subtracted universal cDNA library

3. Vectors
   • pTZ18RP1
   • pT7T3-Pac
   • pCNS-D2: mammalian expression vector (CMV promoter)
   • pCNS (AF416744): mammalian expression vector (CMV promoter)
   • pME18S-FL3 (AB009864): mammalian expression vector (SV40 promoter)
     (*Kindly gifted by Dr. Sugano)
   • Other

4. Library Construction Methods
   • PCR based Oligo-capping method (EMM, 35 (6), 586-590, 2003) [PubMed]
   • Oligo-capping method (Gene 138: 171-174, 1994) [PubMed]
   • Capping method (Gene 150: 243-245, 1994) [PubMed]
   • 모든 clone은 그 제조 방법으로 미루어, 3'-untranslated region을 가지고 있을 것으로 예상됨.

5. Sequencing
   • Sequenced direction: 5'-end
   • Sequencing machines: ABI 3700, MegaBACE 1000, MegaBACE 4000
   • Sequencing primers

6. EST Analyses
   • Basecalling: phred
   • Vector-trimming: fasta
   • Repeat-masking: RepeatMasker
   • Annotation: BLASTN against human mRNA, human UniGene; BLASTX against NR proteins
   • Clustering: cap3
   • sequencing error나 overlap이 없는 경우 등, 다양한 이유로 인하여 동일 gene이 두개 이상의 cluster로 묶이거나, 비슷한 paralogous gene들이 하나의 cluster로 묶일 수 있음.

7. Clustering & Re-Arraying
   • Non-redundant clone의 유지 및 분양을 위하여 대표 clone을 모아 재배열함.
   • Contig generation: cap3
   • Annotation: BLASTN against human mRNA, human UniGene; BLASTX against NR proteins
   • Clone에 대한 정보는 annotation을 한 방법 및 시점에 따라 다소 차이가 있거나, 새로운 정보가 반영되지 못할 수도 있음.

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