HUM-MOLGEN archive: BIOT: September 1997

New BIOTs! The BIOT section is open for requests and offers of information regarding molecular biology and molecular genetics (protocols, techniques, products, collaboration, etc.). You can reach over 4250 of your colleagues, and on average you may expect up to twenty replies to a single message. This service is absolutely FREE of charge. Help yourself by helping your colleagues! -Please send high quality messages only, including full name, address and purpose. -Please use the appropriate TOPIC subject heading in your message. -Please state non-trivial questions only. -Please reply by private E-mail only, unless your request is of general interest to the entire HUM-MOLGEN community Messages may be refused without further notification. Edward Wilcox Arthur Bergen (BIOT editors) This BIOT message contains: 1) Do you know of any evidence for a GAPDH pseudogene 2) A request for help in making a cDNA library from 100-5000 cells 3) How do aberrant RNA splicing errors or PCR artifacts come about 4) How can short tandem repeat loci from selected Philippine linguistic groups be used to compute genetic distances. 5) The latest international ImMunoGeneTics database is now available 6) A request for probes from the Retinoblastoma gene 7) A request for a clone expressing the human EPO (Erythropoietin) gene 8) By what mechanisms do pseudogenes arise ******************************************************************************** GAPDH pseudogene? 9/3/97 We are performing RT-PCR on some human tissue samples and have made primers to GAPDH as a positive control. These primers are from the first and fifth exons, therefore they should generate about a 320 BP band when amplifying fully processed mRNA (cDNA) or a 2.2 Kb band when amplifying genomic DNA. However, when we perform PCR on human genomic DNA we obtain a 320 BP band. We believe we have eliminated the possibility that the DNA is contaminated by RNA or the possibility that the PCR reaction (reagents) is contaminated. At this time we believe the only other explanation is that the human genome has a pseudogene for GAPDH. We have checked the Genbank databases and find no evidence for a GAPDH pseudogene. Has anyone else ever experienced this same (or similar) problem? Is there any evidence for a GAPDH pseudogene? Thank you for any comments/suggestions. Michael J. Kern, Ph.D. E-mail: kernmj@musc.edu ******************************************************************************** Hi! I am interested in making a cDNA library starting with material from 100-5000 cells only. Any help from people working with small number of cells about methods/Kits for RNA extraction, RT-PCR, and cDNA library construction is appreciated. Thanks, Mauricio Neira, Ph.D. Department of Medicine University of Wisconsin, Madison ******************************************************************************** Dear Sir/Madam, My project centers around developing the protein truncation test to screen the ATM gene in familial breast cancer patients that are analyzed by my department. I have noticed within my research the existence of aberrant RNA splicing and am wondering how such splicing errors are caused. Such splicing errors have been observed by other ATM researchers. I sequenced one such fragment around 900 BP long. I was trying to generate the entire ATM transcript of 9.2 kb but got this band instead. Upon sequencing, the band contained 350 BP 5' end, 323 BP 3' end with the middle of this product being intronic regions. Splicing did not occur at exon intron sites. Any comments as to the nature of such aberrant splicing or PCR artifacts would be greatly appreciated. Yours faithfully, Aaron Simpson Clinical Chemistry PMH, Western Australia Australia 61 08 93408482 ph 08 9450 2197 fax 089 6455 225 asimpson@central.murdoch.edu.au ******************************************************************************** Hi! I intend to study genetic variation at short tandem repeat loci among selected Philippine linguistic groups and compute for genetic distances between them for my dissertation. This study shall be contributory to our national effort of constructing a population database primarily for forensic purposes and hopefully for other population studies. My questions (among hundreds) include the following: 1. What factors should I consider in choosing a "sample size" that would give me the best estimate of actual population allele frequencies? 2. At the minimum, how many loci/alleles do I need to study to allow for computation of genetic distance between the linguistic groups (assuming high frequency of heterozygotes for these loci)? 3. What are the criteria for choosing a measure of genetic distance e.g. Nei's, and method of construction of a dendrogram e.g. Neighbor-joining or UPGMA to best examine the phylogenetic relationships of these linguistic groups? As I've said, I have more to ask but I do not want to take so much of your precious time (for now ;-)). Any input shall be greatly appreciated. Thank you. Jiji Miranda Molecular Biology and Biotechnology Program College of Science University of the Philippines Diliman, Quezon City PHILIPPINES jmiranda@SKYINET.NET ******************************************************************************** Dear Colleague, I am pleased to send you the latest IMGT news for information and diffusion. With many thanks. Best regards. Professor Marie-Paule LEFRANC _____________________ IMGT NEWS-August 1997 "Alleles and mutations" IMGT, the international ImMunoGeneTics database, announces a STANDARDIZED description of allele polymorphisms and mutations for all immunoglobulin and T cell receptor V-REGIONs of all species, based on the IMGT unique numbering (IMGT NEWS - March 1997). Allele alignments and tables for the human IGH, IGK and IGL V-REGIONs are freely available at IMGT http://imgt.cnusc.fr:8104 IMGT initiator and coordinator : Prof. Marie-Paule Lefranc Laboratoire d'ImmunoGenetique Moleculaire, LIGM UMR 5535 (CNRS - Universite Montpellier II) 1919 route de Mende 34293 Montpellier Cedex 5 - France Tel: +33 (0)4 67 61 36 34 - Fax: +33 (0)4 67 04 02 31 lefranc@ligm.crbm.cnrs-mop.fr IMGT reference : Giudicelli et al., Nucleic Acids Research, 25, 206-211(1997) ******************************************************************************** I am working on the Retinoblastoma gene and we are in need of probes for this gene. Probes must be from within the gene. Sequences are all we need, for we can make them ourselves. Thank you very much. Helizna Kilian Department of Haematology UOFS South Africa email: GNHMHSK@med.UOVS.ac.za ******************************************************************************** Dear colleagues, I am looking for someone from whom I could obtain a pro- or eukaryotic clone expressing human EPO (Erythropoietin) for in vitro experiments. Thank you in advance, sincerely Stefan Schmidt stefan@biozentrum.uni-wuerzburg.de ******************************************************************************** We have cloned and sequenced a "gene" of interest from a human liver library. On analysis of the sequence, we believe that the "gene" is, in fact, a pseudogene of a class of genes that are of interest to us. Can anyone direct me to a recent review or book that deals with pseudogenes? For example, we'd like to know the different mechanism by which pseudogenes arise, their predicted abundance, importance, etc. Thanks for any help. Bill McIntire wsm@itsa.ucsf.edu ******************************************************************************** ************************************************************************ Dr. Arthur A.B. Bergen Department of Ophthalmogenetics The Netherlands Ophthalmic Research Institute (IOI) Royal Academy of Sciences of the Netherlands (KNAW) ** Snail-mail: ** ** FAX: ** ** E-mail: ** P.O.Box 12141 (+31)206916521 A.Bergen@IOI.KNAW.NL 1100 AC Amsterdam The Netherlands ************************************************************************