PCR Primer: A Laboratory Manual Edited by Carl Dieffenbach, National Institute of Al- lergy and Infectious Diseases, Gabriela Dveksler, Uniformed Services University of the Health Sciences

From its first-published account in 1985, the polymerase chain reaction has become a standard re- search tool in a wide range of laboratories. Its impact has been felt in basic molecular biological research, clinical research, forensics, evolutionary studies, and the Human Genome Project. The PCR technique originally conceived by Nobel laureate Kary Mullis has proven to be exceptionally adaptable and has been transformed into a myriad array of methods, each with different applications.

PCR Primer: A Laboratory Manual introduces the complex world of PCR by beginning at an accessible level and then moving to more advanced levels of ap- plication. First, the practical requirements for perform- ing PCR and other amplification techniques in the lab are introduced and then the basic aspects of the techni- que are explained by exploring important issues such as sample preparation, primer design, efficiency, detec- tion of products, and quantitation. Protocols for a wide range of PCR and amplification techniques-each writ- ten by an expert investigator-are presented for cloning, sequencing, mutagenesis, library construction and screening, exon trapping, differential display, and ex- pression, and these include RT-PCR, R N A PCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expres- sion PCR, 3 ' and 5 ' RACE, in situ PCR, and ligation- mediated PCR. Each protocol is augmented by analysis and troubleshooting sections and complete references.

C O N T E N T S Introduction to PCR Setting Up a PCR Laboratory (C.W. Dieffenbach et al.); A Standard PCR Protocol: Rapid Isolation of DNA and PCR Assay for 15-Globin (M.T. Vahey et al.); Enzymatic Control of Carryover Contamination in PCR (J.L. Hartley, A. Rashtchian); Ultraviolet Irradiation of Sur- faces to Reduce PCR Contamination (R.W. Cone, M.R. Fairfax); Specificity, Efficiency, and Fidelity of the PCR (R.S. Cha, W.G. Thilly); Optimization and Troubleshooting in PCR (K.H. Roux); Long-Distance PCR (O.S. Foord, E.A. Rose) Sample Preparation Rapid Pr.~e[~aration of DNA for PCR Amplification with Gene Releaser am (E.P. Dawson et al.); PCR Amplification from Paraffin-

embedded Tissues: Sample Preparation and the Effects of Fixation (C.E. Greer et al.); RNA Purification (J.L Adamovicz, W.C. Gause) Primer Design General Concepts for PCR Primer Design (C.W. Dieffenbach et al.); Design and Use of Mismatched and Degenerate Primers (S. Kwok et al.); Multiplex PCR (M.C. Edwards, R.A. Gibbs) Detection of PCR Products: Quantitation and Analysis Immunological Detection of PCR Products (J.G. Lazar); Quantitative PCR Using the AmpliSensor Assay (C.N. Wang); DNA Fingerprint- ing Using Arbitrarily Primed PCR (M. McClelland, J. Welsh); RNA Fingerprinting Using Arbitrarily Primed PCR (M. McClelland, J. Welsh); In Situ PCR (G.J. Nuovo); Single-strand Conformational Polymorphism (K. Fujita, J. Silver); Genetic Subtyping of Human lmmunodeficiency Virus Using a Heteroduplex Mobility Assay (E.L. Delwart et al.); Sensitive and Fast Mutation Detection by Solid-phase Chemical Cleavage (L.L. Hansen et al.) PCR Starting from RNA Use of the PCR to Ouantitate Relative Differences in Gene Expres- sion (W.C. Gause, J.J. Adamovicz); Quantitative Liquid Hybridiza- tion PCR Method Employing Storage Phosphor Technology (M.T. Vahey, M.T. Wong); Use of the SNuPE Assay to Quantitate Allele- specific Sequences Differing by a Single Nucleotide (J. Singer-Sam); Trapping Internal and 3'-Terminal Exons (P.E. Nisson et ai.); Expression-PCR (D.E. Lanar, K.C. Kain) PCR-mediated Cloning Rapid Amplification of eDNA Ends (M.A. Frohman); Panhandle PCR (D.H. Jones); Detection and Identification of Expressed Genes by Differential Display (P. Warthoe et al.); Construction of Subtrac- tive eDNA Library Using Magnetic Beads and PCR (A. Lonneborg); PCR-based Method for Screening DNA Libraries (D.I. Israel); Screening of YAC Libraries with Robotic Support (M.M. Blanchard, V. Nowotny); Phagemid Display Libraries Derived from PCR- immortalized Rearranged Immunoglobulin Genes (H.H. Hogrefe, B. Shopes) PCR Sequencing Direct Sequencing of PCR-amplified DNA (V.B. Rao); Cycle Se- quencing (K. Kretz et al.) Cloning of PCR Products Strategies for Cloning PCR Products (R. Levis); Cloning and Analy- sis of PCR-generated Fragments (G.L. Costa, M.P. Weiner) Mutagenesis by PCR Mutagenic PCR (R.C. Cadwell, G.F. Joyce); PCR Mutagenesis and Recombination In Vivo (D.H. Jones); Mutagenesis and Synthesis of Novel Recombinant Genes Using PCR (A.N. Vallejo et al.); Rapid PCR Site-directed Mutagenesis (M.P. Weiner, G.L. Costa) Alternative Amplification Technologies Ligase Chain Reaction (M. Weidmann et al.); Optimization and Characterization of 3SR-based Assays (T.R. Gingeras et al.); One- tube Quantitative HIV-1 RNA NASBA (B. van Gemen et al.) Appendices Computer Software for Selecting Primers; Reagents and Equipment

1995, 625 pp. (approx.), illus., appendices, index Cloth $160 ISBN 0-87969-447-5 Plastic comb binding $80 ISBN 0-87969-448-3

Reader Service No. 424

V O L U M E 9

GENES

DEVELOPMENT NUMBER 18 PAGES 2203-2324 SEPTEMBER 15, 1995

EDITORIAL BOARD

J. Adams (Melbourne, Australia) J. Beckwith (Boston, USA) A. Berns (Amsterdam, The Netherlands) E. Blackburn (San Francisco, USA) T. Cech (Boulder, USA) P. Chambon (Strasbourg, France) N.-H. Chua (New York, USA) E. Coen (Norwich, UK) S. Courtneidge (Heidelberg, FRG) R. Evans (La Jolla, USA) G. Fink (Cambridge, USA) P. Goodfellow (Cambridge, UK) S. Gottesman (Bethesda, USA) T. Graf (Heidelberg, FRG) C. Gross (San Francisco, USA) R. Grosschedl (San Francisco, USA) F. Grosveld (Rotterdam, The Netherlands) M. Groudine (Seattle, USA) L. Guarente (Cambridge, USA) R. Harland (Berkeley, USA) E. Harlow (Charlestown, USA) W. Herr (Cold Spring Harbor, USA) J. Hodgkin (Cambridge, UK) R. Horvitz (Cambridge, USA) P. Ingham (London, UK) T. Jessell (New York, USA) N. Jones (London, UK)

Editorial~Production N. Dumser, Technical Editor V. Nicolette, Production Editor L. Olsewski, Editorial Secretary

J. Kadonaga (La Jolla, USA) R. Lehmann (Cambridge, USA) M. Levine (San Diego, USA) D. Livingston (Boston, USA) R. Losick (Cambridge, USA) J. Manley (New York, USA) W. McGinnis (New Haven, USA) S. McKnight (South San Francisco USA) R. McKay (Bethesda, USA) A. McMahon (Cambridge, USA) P. Nurse (London, UK) R. Palmiter (Seattle, USA) L. Parada (Dallas, USA) C. Prives (New York, USA) G. Rubin (Berkeley, USA) U. Schibler (Geneva, Switzerland P. Sharp (Cambridge, USA) C. Sherr (Memphis, USA) D. Sherratt (Oxford, UK) D. Solter (Freiburg, FRG) P. Soriano (Seattle, USA) J. Steitz (New Haven, USA) M. Takeichi (Kyoto, Japan) T. Taniguchi (Tokyo, Japan) S. Tilghman (Princeton, USA) R. Tjian (Berkeley, USA) M. Wigler (Cold Spring Harbor, USA)

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GENES & DEWLOPMENT (ISSN 0890-9369) is published semimonthly for $485 (institutional), $120 (individual making personal payment), $167 (Genet- ical Society of Great Britain members) by Cold Spring Harbor Laboratory Press, Bungtown Road, Cold Spring Harbor, New York 11724, in associ- ation with the Genetical Society of Great Britain. Second-class postage is paid at Cold Spring Harbor and additional mailing offices. POSTMAS- TER: Send address changes to Cold Spring Harbor Laboratory Press, POB 100, 1 Bungtown Rd., Cold Spring Harbor, New York 11724-2203. Subscription Price Orders may be sent to Cold Spring Harbor Labora- tory Press, Fulfillment Department, 10 Skyline Drive, Plainview, NY 11803-9729. Telephone: Continental US except NY State, 1-800-843- 4388; all other locations, 516-349-1930/1931/1932. FAX 516-349-1946. Volume 9, 1995, $485, U.S. institutional; $580, R.O.W. institutional. Personal subscription rate: $120, U.S.; $215, R.O.W. Price includes sur- face postage for U.S. and airlift for R.O.W. Genetical Society members, $167.00. All subscriptions are entered for the calendar year and must be prepaid. Personal subscriptions must be prepaid by personal check, credit card, or money order. All checks must be for US dollars and drawn on a US bank. Genetical Society members may also subscribe by check, payable to the Genetical Society, for $167 (includes airlift). Send to: Dr.

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Claims for missing issues must be received within 4 months of issue date. Change of address Please enclose recent mailing label with address change; allow 4 weeks. Advertising To advertise in Genes & Development, contact Deborah Dufton, Advertising Manager, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 11724-2203; telephone 516-367-8351. Photo Copy Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Gold Spring Harbor Laboratory Press for libraries and other users registered with the Copyright Clearance Center (CCC) Transactional Reporting Service, provided that the base fee of $5.00 per copy is paid directly to CCC, 21 Congress St., Salem, MA 01970 (0890-9369/95 $5.00 + 0.). This consent does not extend to other kinds of copying, such as copying for general distribution for advertising or promotional purposes, for creating new collective works, or for resale. Copyright �9 1995 by Cold Spring Harbor Laboratory Press

Contents GENES & DEVELOPMENT September 15, 1995

Research papers Targeted deletion of 5'HS2 of the murine [3-globin LCR reveals that it is not essential for proper regulation of the 13-globin locus Steven Fiering, Elliot Epner, Kirsten Robinson, Yuan Zhuang, Agnes Telling, Ming Hu, David I.K. Martin, Tariq Enver, Timothy J. Ley, and Mark Groudine

Altering specific telomerase RNA template residues affects active site function David Gilley, Margaret S. Lee, and Elizabeth H. Blackburn

Boundary elements of the Tetrahymena telomerase RNA template and alignment domains Chantal Autexier and Carol W. Greider

2203

2214

2227

The basis for a heat-induced developmental defect: defining crucial lesions Michael A. Welte, Ian Duncan, and Susan Lindquist

sur-2, a novel gene, functions late in the let-60-ras-mediated signaling pathway during Caenorhabditis elegans vulval induction Natasha Singh and Min Han

Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities John P. Lydon, Francesco J. DeMayo, Cindee R. Funk, Shiala K. Mani, Angela R. Hughes, Charles A. Montgomery Jr., Gopalan Shyamala, Orla M. Conneely, and Bert W. O'Malley

fyn tyrosine kinase is involved in keratinocyte differentiation control Enzo Calautti, Caterina Missero, Paul L. Stein, Robert M. Ezzell, and G. Paolo Dotto

Ectopic expression of the knox homeo box gene rough sheathl alters cell fate in the maize leaf Richard G. Schneeberger, Philip W. Becraft, Sarah Hake, and Michael Freeling

A novel bacterial transcription cycle involving r Yin Tintut, Jonathan T. Wang, and Jay D. Gralla

54

A U6 snRNA:pre-mRNA interaction can be rate-limiting for Ul-independent splicing John D. Crispino and Phillip A. Sharp

Erratum

2240

2251

2266

2279

2292

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2314

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Cover A progesterone-deficient mouse model. (Background) Ductal structure in PR mutant mammary gland. (Inset) Terminal end of wild-type (left) and PR mutant (right) duct. (For details, see Lydon et al., p. 2266.)