title: Human Unigene I, part 1
status: Public on May 31 2001
contact.city: Heidelberg
contact.first: Wolfgang
contact.last: Huber
contact.email: w.huber@dkfz.de
contact.address: Im Neuenheimer Feld 280
contact.department: Molecular Genome Analysis
contact.country: Germany
contact.web_link: http://www.dkfz-heidelberg.de/abt0840
contact.institute: German Cancer Research Center
contact.phone: +49-6221-424709
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 µm 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