SHORT COMMUNICATION Chromosomal Assignment of 79 cDNAsfrom a Range of Human Tissues C AROLINE E VANS ,M ARK B OUZYK,S IMON C OX,DIANE WARNE , 1 S TEPHEN P. BRYANT, AND NIGEL K. S PURR 2 Human Genetic Resources Laboratory, ICRF Clare Hall Laboratories, Blanche Lane, South Mimms, Hertfordshire, EN6 3LD, United Kingdom Received July 24, 1995; accepted October 24, 1995 gene families. To make these genes more valuable, as- Single-pass DNA sequencing of cDNAs selected at signment to a specific chromosome would be the mini- random from a human mixed tissue cDNA library have mum information required. This has been carried out generated a series of more than 2000 expressed se- for over 4000 human gene transcripts (5, 8, 9). This quence tags. One hundred twenty-eight unique cDNA report describes the assignment of a further 70 genes fragments with little or no known protein or nucleic to specific chromosomes and the possible identification acid homologies have been selected for further analy- of 9 gene families assigned to two or more chromo- sis. Oligonucleotide primer pairs have been designed somes. from the cDNAs and used in PCR amplification in com- The expressed sequence tag (EST) sequences used in bination with genomic DNA from a panel of monochro- this work were generated by the UK Human Genome mosomal somatic cell hybrids. This has allowed us to Project Resource Centre (Hinxton Hall, Cambridge, assign 70 of these transcribed genes to a single chro- UK). The sequences had all been previously screened mosome, and a further 9 have been located on two or for homology to known genes or proteins using Blast three chromosomes. Additionally, 3 cDNAs contain and FastA. The sequences were obtained by file trans- short tandem repeats that may allow them to be fur- ther localized by linkage analysis.  1996 Academic Press, Inc. fer protocol (ftp) and downloaded as text files. Novel cDNA sequences from uncharacterized human tran- scripts were selected for assignment. The cDNA se- One of the current aims of the Human Genome Proj- quences were analyzed, for open reading frame and ect is a construction of a transcription map of all the intron – exon junctions, using the program INTRON (9). expressed sequences. The transcription map has the Those with a high likelihood of containing a transcribed potential to make a significant impact on human genet- gene and no introns were used to develop a set of oligo- ics in the next few years. It will speed the identification nucleotide primers specific for that gene. The program of genes involved in human genetic diseases and should PRIMER was used to select oligonucleotide primers lead to the elimination of one of the current bottlenecks specific for the fragments (PRIMER is available via in positional cloning, making most projects a search of anonymous ftp to genome.wi.mit.edu). This program candidate genes in a selected region. In the past 3 chose several alternative primers, and the final pair of years, several laboratories have sequenced partially se- primers used in the PCR amplification were selected lected clones from tissue-specific cDNA libraries. This based on their GC basepair content and their overall has been demonstrated to be a rapid and efficient nucleic acid composition and by limiting the annealing means of generating extensive sequence data (1 – 5, 7, temperature to approximately 55°C. 10, 11). The data generated from homology screening More than 350 cDNA sequences with little or no show that approximately 30% of the cDNAs sequenced known protein or nucleic acid homology were selected represent novel, previously uncharacterized human for intronic analysis, and from these, oligonucleotide transcripts. primer pairs were designed and synthesized for 128. Transcribed genes identified through cDNA sequenc- These cDNA sequences were the least likely to contain ing have limited value in the analysis of disease genes potential introns and were short fragments of 51 – 212 if no homology is found to already mapped genes or bp, but long enough to generate a PCR product that could be visualized. These relatively short fragments 1 Current address: Axis Genetics Limited, Babraham, Cambridge, were used under standard PCR conditions. Briefly, for CB2 4AZ, UK. each PCR reaction, 300 ng of each primer was used in 2 To whom correspondence should be addressed. Telephone: (44)171 269 3846. Fax: (44)171 269 3802. a mix containing 1.25 units Taq polymerase, 0.2 mM 130 GENOMICS 31, 130–134 (1996) Article No. 0021 0888-7543/96 $12.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. / m4752$3831 12-05-95 12:39:39 gnmas AP-Genomics