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Platform GPL9 Query DataSets for GPL9
Status Public on May 31, 2001
Title Human Unigene I, part 1
Technology type spotted DNA/cDNA
Distribution non-commercial
Organism Homo sapiens
 
Description Human cDNA filter from the Resource Centre of the German Human Genome Project. Each of the two parts (GPL9 and GPL10) contains half of 31,488 PCR amplificates of human cDNAs selected as representatives of UniGene clusters (Build #17, NCBI)

Selection of a Global Human Clone Set:

In order to generate a non-redundant human clone set, we postprocessed the UniGene clusters (Build 17, NCBI), which represent a large number of human genes. All processing steps are part of the GeneNest software (Haas et al., 2000), that additionally provides an interactive graphical interface to the postprocessed UniGene database (http://www.dkfz.de/tbi/services/GeneNest/index). To identify the most reliable, representative clone from each cluster, we analyzed the following criteria (sorted according to their importance): (1) Availability of clones at the RZPD. (2) Quality of cDNA library of origin. (3) Presence of more than one read of the same clone in a cluster, ensuring a higher confidence in the sequence - clone relationship. (4) Calculated insert size, selecting for larger inserts. (5) Presence of a polyA signal. We used a fuzzy logic based rule system to combine all clone selection criteria in order to obtain a quality measure of each clone in the entire UniGene set. For each cluster we selected the clone with the highest quality as representative. To estimate the redundancy of the global clone set, we re-sequenced over 2,700 clones of the set and found 12.8% wrongly assigned clones. All of these belonged to clusters that were already represented by another clone. Therefore, the overall redundancy of the clone set was estimated to be 1.44-fold, and the 31,500 clones on the cDNA array represent an estimated 21,875 different transcripts.

31,500 Clone Human cDNA Array:

From the Human UniGene 1 clone set, cDNA inserts of the clones from the 82 first 384-well microtiter plates were amplified by PCR in a 384-well format (MJ Research, Boston, Massachusetts) using M13 forward (5'-CGTTGTAAAACGACGGCCAGT-3') and reverse primers (5'-TTTCACACAGGAAACAGCTATGAC-3'). The 31,488 PCR products were transferred in a 4x4 pattern onto a set of two 22x22 cm Hybond N+ nylon membranes (Amersham Pharmacia Biotech, Uppsala, Sweden) soaked in 0.4 M NaOH using a Picking-Spotting-Robot (Linear Drives LDT, Essex, UK) with 400 ┬Ám pins (Genetix, Hampshire, UK). After spotting the arrays were carefully floated for 2 minutes on 0.4 M NaOH and 5 x SSC (pH 7.5) successively, air dried and cross-linked by UV. Every 4x4 block contained one spot with PCR product from the bacterial kanamycin resistance gene to serve as a guide spot in automated image analysis, one empty spot to serve as background measurement and DNA from seven clones spotted in duplicate. On each filter, a control plate containing putative housekeeping genes and positive and negative controls was spotted. To assure even quality between subsequent rounds of hybridization, cDNA arrays were pre-stripped before their first use (Hauser et al., 1998). To check for filter quality, M13 forward oligonucleotide hybridizations were carried out.
Keywords = membrane, filter
 
Contributor(s) Huber W, Boer J, Sueltmann H, Wilmer F, von Heydebreck A, Haas S, Korn B, Vente A, Vingron M, Poustka A
Citation(s) 12199289
Submission date May 30, 2001
Last update date Oct 28, 2005
Contact name Wolfgang Huber
E-mail(s) w.huber@dkfz.de
Phone +49-6221-424709
Fax +49-6221-423470
URL http://www.dkfz-heidelberg.de/abt0840
Organization name German Cancer Research Center
Department Molecular Genome Analysis
Street address Im Neuenheimer Feld 280
City Heidelberg
ZIP/Postal code 69120
Country Germany
 
Samples (218) GSM81, GSM82, GSM83, GSM84, GSM85, GSM86 
Series (2)
GSE3 Renal Cell Carcinoma Differential Expression
GSE112 Distinctive gene expression patterns in gastrointestinal stromal tumors identified with cDNA arrays

Data table header descriptions
ID
X X coordinate
Y Y coordinate
CLONE_ID Visit http://www.rzpd.de
RZPD_ID Visit http://www.rzpd.de
SPOT_ID Spot identifier
GB_LIST
DESCRIPTION description

Data table
ID X Y CLONE_ID RZPD_ID SPOT_ID GB_LIST DESCRIPTION
1 1 1 IMAGE:256538 IMAGp950P2439 h95144, h95180 ests, weakly similar to no definition line found [c.elegans]
2 2 1 IMAGE:67006 IMAGp950P243 t69711, t70368
3 3 1 IMAGE:25807 IMAGp950P249 r12124, r36886 glutamate receptor 3 precursor
4 4 1 IMAGE:42546 IMAGp950P2415 r61114, r61836
5 1 2 IMAGE:154233 IMAGp950P2421 r51964, r53631 homo sapiens cell cycle-regulated factor p78 mrna, complete cds,homo sapiens cell cycle-regulated factor p78 mrna, complete cds,mcrs1
6 2 2 heterologous
7 3 2 IMAGE:234760 IMAGp950P2433 h77659, h77660 human gamma-tubulin mrna, complete cds,tubg1,tubulin, gamma polypeptide : tubg
8 4 2 IMAGE:183462 IMAGp950P2427 h45455, h45547 homo sapiens alpha mannosidase 6a8b (6a8b) mrna, complete cds,homo sapiens alpha-mannosidase (6a8) mrna, complete cds,man2c1
9 1 3 IMAGE:183462 IMAGp950P2427 h45455, h45547 homo sapiens alpha mannosidase 6a8b (6a8b) mrna, complete cds,homo sapiens alpha-mannosidase (6a8) mrna, complete cds,man2c1
10 2 3 IMAGE:25807 IMAGp950P249 r12124, r36886 glutamate receptor 3 precursor
11 3 3 IMAGE:154233 IMAGp950P2421 r51964, r53631 homo sapiens cell cycle-regulated factor p78 mrna, complete cds,homo sapiens cell cycle-regulated factor p78 mrna, complete cds,mcrs1
12 4 3 IMAGE:256538 IMAGp950P2439 h95144, h95180 ests, weakly similar to no definition line found [c.elegans]
13 1 4 IMAGE:234760 IMAGp950P2433 h77659, h77660 human gamma-tubulin mrna, complete cds,tubg1,tubulin, gamma polypeptide : tubg
14 2 4 IMAGE:67006 IMAGp950P243 t69711, t70368
15 3 4 IMAGE:42546 IMAGp950P2415 r61114, r61836
16 4 4 empty
17 5 1 IMAGE:254308 IMAGp950P2339 n22261, n81157 ests, weakly similar to pot. orf i [h.sapiens]
18 6 1 IMAGE:24643 IMAGp950P233 r38718, t80233 homo sapiens mrna for kiaa0652 protein, complete cds,kiaa0652
19 7 1 IMAGE:129876 IMAGp950P239 r11406, r19156
20 8 1 IMAGE:42008 IMAGp950P2315 r60710, r60711 ests, weakly similar to coded for by c. elegans cdna yk86e5.5 [c.elegans],kiaa1091

Total number of rows: 36864

Table truncated, full table size 3254 Kbytes.






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