Human Genome 1993 Program Report: Physical and Genetic Mapping
Date Published: March 1994
For a printed copy of this document, contact
Human Genome Management Information System
Oak Ridge National Laboratory
1060 Commerce Park, MS 6480
Oak Ridge, TN 37830
423-576-6669, Fax: 423-574-9888
Internet: bkq@ornl.gov
Projects New in FY 1993
A Method for Heterozygous Carrier Screening Using an E. coli Mismatch Binding Protein, MutS; Application to the Cystic Fibrosis Gene
Alla Lishanski and Jasper Rine
Human Genome Center; Lawrence Berkeley Laboratory; Berkeley, CA 94720
510/486-7332, Fax: -6818, Internet: allali@genome.lbl.gov
An experimental strategy for detecting heterozygosity in genomic DNA has been developed based on a preferential binding of Escherichia coli MutS protein to DNA molecules containing mismatched bases. The binding was detected by a gel mobility-shift assay. This approach was tested using the most commonly occurring mutations within the cystic fibrosis gene (CFTR) as a model. Genomic DNA samples were amplified using 5'-end-labeled primers that bracket the site of the DF508 3-bp deletion in exon 10 of the CFTR gene. The renatured polymerase chain reaction (PCR) products from homozygotes produced homoduplexes, and the PCR products from heterozygotes produced heteroduplexes and homoduplexes (1:1). MutS protein bound more strongly to heteroduplexes corresponding to heterozygous carriers of DF508 and containing a CTT or GAA loop in one of the strands than to homoduplexes corresponding to homozygotes. The ability of MutS to detect heteroduplexes in PCR-amplified DNA extended to fragments ~500 bp in length. The method was also able to detect carriers of the point mutations in exon 11 of the CFTR gene by a preferential binding of MutS to single-base mismatches in PCR-amplified DNA.
Vertically Integrated Analysis of Human DNA
Maynard Olson
University of Washington; Seattle, WA 98195
206/685-7346, Fax: -7344, Internet: mvo@u.washington.edu
A systematic approach will be developed for the vertically integrated analysis of human DNA segments ranging in size up to a few megabase pairs. Vertical integration denotes sequential analysis of a genomic region at all levels of detail from low-resolution physical mapping to finished sequence. This project will emphasize the steps preceding DNA sequencing, particularly the hierarchical analysis of genome segments in somatic cell lines, yeast artificial chromosomes, cosmids, lambdas, plasmids, and filamentous-phage clones. The goal will be precise, high-resolution physical maps on which the ends of the inserts of small-insert clones, suitable for use as sequencing templates, will be placed exactly.
The project will focus on simple, modular, experimental strategies that are good candidates for high-level automation. The raw data will be captured by electronically imaging electrophoretic gels that have banding patterns predictable enough for completely automated interpretation. Early steps will be taken toward automation of these procedures on a scale that would allow the analysis of millions of clones.
Construction of a Genetic Map Across Chromosome 21
Elaine A. Ostrander
Fred Hutchinson Cancer Research Center; Seattle, WA 98104
206/667-5000, Fax: -6124
The goal of our group is to develop and implement technologies aimed at high-resolution mapping of individual human chromosomes. Our initial efforts have focused on developing strategies for identifying and characterizing polymorphic microsatellite repeat sequences; these repeats are extremely abundant in all mammalian genomes and usually have multiple, highly informative alleles. We intend to construct a high-density genetic map of chromosome 21 with polymorphic repeat markers spaced every 0.5 to 1 cM, based on simple sequence microsatellite repeats.
We have fully sequenced 43 (CA)n repeat-based markers prepared from chromosome 21 cosmids. After eliminating 2 previously identified markers and 5 containing Alu sequences, we localized 14 of the remaining markers to their specific locations on chromosome 21 and identified yeast artificial chromosomes containing the markers.
With computer-analysis programs prepared by Lawrence Berkeley Laboratory's engineering group, we have adapted the automated laser fluorescent (called A.L.F.) sequencer for use in fluorescence-tagged genetic mapping studies. The markers identified and mapped to date are fairly well distributed and represent a strong foundation on which to continue our studies.
Advances in physical and genetic maps have set the stage for the next biological goals of genome research: obtaining genomic sequence from substantial regions of the genome and preparing the genetic infrastructure for genotyping populations. Toward these ends we are preparing to sequence a 3- to 4-Mb region from a medically significant portion of chromosome 21 and are working toward saturating the chromosome with genetic markers. Lawrence Berkeley Laboratory (LBL) has designed, implemented, and tested on an 80,000-bp segment of genomic DNA a directed sequencing approach that produced completed DNA sequences at a high rate of throughput and with high accuracy.
In the evaluation of potential DNA sources for sequencing templates, the yeast artificial chromosomes (YACs) used in chromosome 21 sequence tagged sites (STSs) failed to exhibit obvious deletions. Thus, YACs appear to be poor choices for sequencing studies. The rapid success of the directed genome-sequencing strategy at LBL has focused our attention on the human P1 library of Shepherd and Sternberg. P1 clones can carry 100,000 inserted bases and are the best source of material that can serve directly as sequencing templates for our strategy. We have constructed pools 1000 clones deep from the Shepherd and Sternberg library and have screened these with STSs from the 22.2 to 22.3 region of chromosome 21. These P1s are now in the pipeline for large-scale directed DNA sequencing. This process involves constructing physical maps in which the distance (d) and orientation (o) of each gene-sized sequencing template (g) is known and tagged by sequence from each end. This procedure, known as dog tagging, offers the best-known method for large-scale genome sequencing and avoids the sequence-assembly headaches of random strategies.
We have used marker-selection techniques to isolate a large number of simple sequence repeats from chromosome 21 as a source of genetic markers. STSs produced from about 40 of these repeats have been assigned to the map, with a resolution of a few hundred kilobases. We have approximately doubled the density of genetic markers on this chromosome, making it the most densely marked human chromosome.
Our physical mapping efforts have focused on the distal third of the q arm. In this region we have used fluorescence in situ hybridization to map about 280 YACs plus cosmids and have constructed contig maps. This mapping has allowed us to detect and correct errors in the recently published map (Chumakov et al., 1992), including gene misplacement of as much as 2 Mb. Corrected maps for these regions will be presented.
We have developed methods for physical selection of cDNAs corresponding to mapped YACs and cosmids. We have mapped 21 new cDNAs to their respective locations on chromosome 21. By sequence analysis, each of these defines new genes and pioneer proteins. The cDNA effort is now focused on saturating the multimegabase target of the genomic-sequencing effort.
All the programs involve close interaction among the center's biology, instrumentation and automation, and informatics groups. Major instrument development includes automation of steps in directed sequencing; a large-scale, extensible thermocycler; and a large-scale oligonucleotide developer. Software for sequence assembly and analysis is under development.
The capacity to produce genome information has outstripped the capacity of formal publication procedures to disseminate the information to the community. To help close the gap between producers and consumers, all of our unpublished cDNA sequences have been deposited in the cDNA Inform database (Los Alamos National Laboratory), all genetic markers in Genome Data Base, and the sequences from which they are derived in GenBank®. In addition, we are establishing a public database at LBL that will serve as an open notebook for chromosome 21 mapping data and sequence information from P1 clones. Our mapping data enter this database directly, and the sequence enters as each 3-kb dog unit is complete.
This project aims to develop a new, efficient approach for high-resolution chromosome mapping by in situ hybridization. The approach is based on the observation that the folding of DNA in the interphase nucleus can be described by a random walk model. This model provides the theoretical basis for using the average distance observed between hybridization sites in the interphase nucleus to estimate distance between two markers measured along the DNA strand. We have developed a rapid graphical method that makes possible the accumulation of thousands of distance measurements per day and have used this approach to confirm the published map of a 4-Mb region of chromosome 4. We plan to (1) further improve data collection methods and (2) develop software for calculating the most probable probe order and relative distance from a set of interphase distance measurements. We will develop similar graphical procedures for efficiently mapping probes to metaphase chromosomes and combine these techniques to build selected chromosome region maps with average density of 100 kb.
The main goal of this project is to contribute to contig mapping of human chromosome 13 with cosmids and YACs as elements. The starting point is production of microsatellite markers from cosmids already mapped to the 13q14 region. Sources of clones are an sCos cosmid chromosome 13 library from Los Alamos National Laboratory (LANL) and a similar Lorist-based library from the Imperial Cancer Research Fund (ICRF) in the United Kingdom. Restriction patterns for more than 150 cosmids have been established with one or two restriction enzymes, and a supporting database has been created to support contig assembly.
Chromosome 13-specific clones are found by hybridization with a nylon-gridded library of
all the cosmid clones. A total human genome YAC library has been obtained from ICRF; pools have been formed, and screening with chromosome 13-specific probes has begun. Some equipment for this project was supplied by the Russian Human Genome Organization board.
New Strategies for Closure of the Chromosome 19 Contig Map
DNA Sequence Mapping by Fluorescence In Situ Hybridization
Core Facility for Support of Chromosome 19 Physical Mapping
Mapping and Ordered Cloning of Human X Chromosome
Massive Isolation and Contig Building of Chromosome-Specific YAC Clones
Physical and Transcription Mapping of Human Chromosome 11
A Clone-Limited STS Strategy for Physical Mapping
Interdigitation of the Genetic and Physical/Cosmid Contig Maps of Human Chromosome 19
Physical Mapping of Human Chromosome 16
Assembly, Closure, and Characterization of a Chromosome 19 Contig Map
Developing a Physical Map of Human Chromosome 22
Physical Structure of Human Chromosome 21
Generating a Comparative Physical Map of Mouse Chromosome 7
Correlation of Physical and Genetic Maps of Human Chromosome 16
David F. Callen, Sinoula Apostolou, Elizabeth Baker, Liang Z. Chen, Helen Kozman, Sharon A. Lane, Julie Nancarrow, Hilary A. Phillips, Yang Shen, Andrew D. Thompson, Scott A. Whitmore, Norman A. Doggett,(1) Raymond L. Stallings,(1) C. Edgar Hildebrand,(1) John C. Mulley, Robert I. Richards, and Grant R. Sutherland
A Physical and Genetic Map of Human Chromosome 21--A Prelude to Sequence: Overview
Jasper Rine, R. Blajez, Jan-Fang Cheng, Jeffrey Gingrich, S. R. Lowry, Elaine Ostrander,(1) Stewart Scherer, Sylvia Spengler, D. Scott, F. Shadravan, T. Torok, K. M. Wilson, and Y. Zhu
Human Genome Center; Lawrence Berkeley Laboratory; Berkeley, CA 94720
510/643-5592, Fax: /642-6420
(1)Fred Hutchinson Cancer Research Center; Seattle, WA 98104Chromosome Mapping by Fluorescent In Situ Hybridization to Interphase Nuclei
Barbara Trask and Ger van den Engh
Department of Molecular Biotechnology; School of Medicine; University of Washington; Seattle, WA 98195
206/685-7347, Fax -7354, Internet: trask@fishnet.mbt.washington.edu*A Chromosome 13 Mapping Project Based on the Los Alamos Cosmid Library
Nick K. Yankovsky, B. I. Kapanadze, V. M. Brodjansky, and G. E. Sulimora
Laboratory of Genome Analysis; Institute of General Genetics; Russian Academy of Sciences; Moscow 117809, Russia
+7-095/135-4307, Fax: -1289, Internet: bion@glas.apc.orgProjects Continuing into FY 1993
Mark Batzer and Anthony V. Carrano
Human Genome Center; Biology and Biotechnology Research Program; Lawrence Livermore National Laboratory; Livermore, CA 94551
510/422-5721, Fax: /423-3608, Internet: batzer2@llnl.gov
Brigette Brandriff, Laurie Gordon, Anne Bergmann, Mari Christensen, Anne Fertitta, and Anthony Carrano
Human Genome Center; Biology and Biotechnology Research Program: Lawrence Livermore National Laboratory; Livermore, CA 94551
510/423-0758, Fax: -3608, Internet: brandriff@llnl.gov
Anthony V. Carrano, Anne Olsen, Mark Batzer, Jane Lamerdin, and Linda K. Ashworth
Human Genome Center; Biology and Biotechnology Research Program; Lawrence Livermore National Laboratory; Livermore, CA 94550
510/422-5698, Internet: carrano1@llnl.gov
C. Thomas Caskey and David L. Nelson
Institute for Molecular Genetics; Baylor College of Medicine; Houston, TX 77030-3498
Nelson: 713/798-3122, Fax: -5386, Internet: nelson@bcm.tmc.edu
Jan-Fang Cheng and Julia Nikolic
Human Genome Center; Cell and Molecular Biology Division; Lawrence Berkeley Laboratory; Berkeley, CA 94720
510/486-6549, Fax: -6816, Internet: jfcheng@lbl.gov
Glen A. Evans, David McElligott, Steven Clark, Suzanne Clancy, Licia Selleri, Michael Smith, Merl Hoekstra, and Gary Hermanson
Molecular Genetics Laboratory; Salk Institute for Biological Studies; San Diego, CA 92186-5800
619/453-4100 Ext. 279, Fax: /558-9513, Internet: gevans@salk-sd2.sdsc.edu
Christopher H. Martin, Carol A. Mayeda, and Michael J. Palazzolo
Human Genome Center; Cell and Molecular Biology Division; Lawrence Berkeley Laboratory; Berkeley, CA 94720
Martin and Palazzolo: 510/486-5909, Fax: -6816, Internet: chrism@genome.lbl.gov or mjpalazzolo@lbl.gov
Harvey W. Mohrenweiser, Elbert Branscomb, and Anthony V. Carrano
Human Genome Center; Biology and Biotechnology Research Program; Lawrence Livermore National Laboratory; Livermore, CA 94551
510/423-0534, Fax: /422-2282, Internet: harvey@cea.llnl.gov
N. A. Doggett, C. E. Hildebrand, M. K. McCormick,2 L. L. Deaven,(1) D. F. Callen,(3) G. R. Sutherland,(3) K. Okumura,(4) D. C. Ward,(5) and R. K. Moyzis(1)
Life Sciences Division and (1)Center for Human Genome Studies; Los Alamos National Laboratory; Los Alamos, NM 87545
Moyzis: 505/667-3912, Fax: /665-3024, Internet: moyzis@flovax.lanl.gov
(2)Massachusetts General Hospital; Charlestown, MA 02129
(3)Department of Cytogenetics and Molecular Genetics; Adelaide Children's Hospital; North Adelaide, South Australia 5006, Australia
(4)Juntendo University School of Medicine; Department of Immunology; Tokyo 113, Japan
(5)Department of Human Genetics; Yale University School of Medicine; New Haven, CT 06510
Anne S. Olsen, Emilio Garcia, Linda Ashworth, Alex Copeland, and Anthony V. Carrano
Human Genome Center; Biology and Biotechnology Research Program; Lawrence Livermore National Laboratory; Livermore, CA 94551
510/423-4927, Fax: -3608, Internet: olsen@ecor1.llnl.gov
Melvin I. Simon, Bruce Birren, and Hiroaki Shizuya
Biology Division; California Institute of Technology; Pasadena, CA 91125
818/356-3944, Fax: /796-7066
Cassandra L. Smith, Denan Wang,(1) Kaoru Yoshida,(1) Jesus Sainz,(2) Carita Fockler,(1) and Meire Bremer(1)
Center for Advanced Research in Biotechnology; Boston University; Boston, MA 02215
617/353-2800, Fax: -5929, Internet: cls@buenga.bu.edu
(1)Division of Chemical Biodynamics; Lawrence Berkeley Laboratory; Berkeley, CA 94720
(2)Cedars-Sinai Medical Center; Los Angeles, CA 90048
Lisa J. Stubbs, Eugene Rinchik,(1) and Estela Generoso
Biology Division; Oak Ridge National Laboratory; Oak Ridge, TN 37831-8077
615/574-0848 or -0864, Fax: -1283
(1)Sarah Lawrence College; Bronxville, NY 10708
Department of Cytogenetics and Molecular Genetics; Adelaide Children's Hospital; North Adelaide, South Australia 5006, Australia
Sutherland: + 61/8-204-7333 or -7284, Fax: -7384 or -7342
(1)Center for Human Genome Studies; Life Sciences Division; Los Alamos National Laboratory; Los Alamos, NM 87545