Selective Isolation of Mammalian Genes by TAR Cloning

Natalay Kouprina1, Vladimir Larionov1

1 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.17
DOI:  10.1002/0471142905.hg0517s49
Online Posting Date:  May, 2006
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Abstract

Transformation‐associated recombination (TAR) cloning provides a unique tool for selective isolation of desired chromosome segments and full‐size genes from complex genomes in the form of a circular yeast artificial chromosome (YAC) up to 250 kb in size. The method has a broad application for structural and functional genomics, long‐range haplotyping, mutational analysis of gene families, characterization of chromosomal rearrangements, and evolutionary studies. This unit describes a procedure for gene isolation by TAR as well as a method for conversion of YAC‐TAR isolates into a bacterial artificial chromosome (BAC) form.

Keywords: transformation‐associated recombination cloning (TAR); yeast artificial chromosome (YAC); bacterial artificial chromosome (BAC); gene isolation; evolution; long‐range haplotyping

     
 
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Table of Contents

  • Basic Protocol 1: Gene Isolation by Recombination in Yeast
  • Support Protocol 1: Identification of Positive Pools by PCR
  • Support Protocol 2: PCR Identification of Individual Gene‐Positive Clones in Positive Pools
  • Support Protocol 3: Retrofitting of Circular YACs into BACs with a Mammalian Selectable Marker
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Gene Isolation by Recombination in Yeast

  Materials
  • Blood and cell culture DNA maxi kit (Qiagen 13362)
  • YPD medium
  • Saccharomyces cerevisiae strain VL6‐48N with deletions for HIS3, TRP1, and URA3, growing on YPD plate (see unit 5.5): available from Laboratory of Biosystems and Cancer, National Cancer Institute, NIH; or American Type Culture Collection (ATCC), #MBA‐212
  • 1 M sorbitol (Sigma; autoclave and store indefinitely at room temperature)
  • SPE solution (see recipe)
  • 10 mg/ml zymolyase 20 T (ICN Biomedicals) in 20% (w/v) glycerol (store in aliquots up to one year at −20°C)
  • 14 M 2‐mercaptoethanol (2‐ME)
  • 2% (w/v) SDS
  • STC solution (see recipe)
  • TAR cloning vector pVC604 (Laboratory of Biosystems and Cancer, National Cancer Institute, NIH; or ATCC, #MYA‐3666), with hooks specific for a gene of interest and linearized between the hooks (see )
  • PEG 8000 solution for spheroplast transformation (see recipe)
  • SOS solution (unit 5.2)
  • TOP agar–His (see recipe), equilibrated to 50°C
  • SORB–His plates with 1 M sorbitol (Teknova, http://www.Teknova.com or see recipe)
  • 250‐ml Erlenmeyer flasks
  • 30°C incubator and shaker
  • Spectrophotometer with visible‐light source
  • 15‐ and 50‐ml conical, screw‐cap centrifuge tubes (Falcon)
  • Beckman centrifuge with a JS‐5.2 swinging‐bucket rotor (or equivalent)
  • 1.5‐ml microcentrifuge tube
  • Refrigerated microcentrifuge (e.g., Eppendorf)
  • 15‐ml conical centrifuge tube (Falcon)

Support Protocol 1: Identification of Positive Pools by PCR

  Materials
  • Transformants
  • SD–His plates
  • 1 M sorbitol (Sigma; autoclave and store indefinitely at room temperature)
  • SPE solution (see recipe) containing 14 mM 2‐mercaptoethanol (2‐ME)
  • 10 mg/ml zymolyase 20T (ICN Biomedicals) in 20% (w/v) glycerol (store in aliquots up to one year at −20°C)
  • 50 mM EDTA containing 0.2% (w/v) SDS
  • Diethylpyrocarbonate (DEPC; Sigma)
  • 5 M potassium acetate
  • 100% ethanol
  • Isopropanol
  • Diagnostic PCR primers specific for the gene of interest
  • Toothpicks
  • 30°C incubator
  • 15‐ml conical centrifuge tubes (Falcon)
  • Beckman J‐6M centrifuge with JS‐5.2 swinging‐bucket rotor (or equivalent)
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge (e.g., Eppendorf)
  • 70°C water bath
  • Additional reagents and equipment for PCR (e.g., CPMBunit 15.1)

Support Protocol 2: PCR Identification of Individual Gene‐Positive Clones in Positive Pools

  Materials
  • Replica plates corresponding to the master plates analyzed in protocol 2
  • 10 mg/ml zymolyase 20T (ICN Biomedical) in 20% (w/v) glycerol (store in aliquots up to one year at −20°C)
  • 14 M 2‐mercaptoethanol (2‐ME)
  • 5 M potassium acetate
  • Isopropanol
  • Diagnostic PCR primers specific for the gene of interest
  • Sterile disposable pipet tips
  • 30°C and 70°C incubators
  • Microcentrifuge (Eppendorf or equivalent)
  • Additional reagent and equipment for PCR (e.g. CPMBunit 15.1)

Support Protocol 3: Retrofitting of Circular YACs into BACs with a Mammalian Selectable Marker

  Materials
  • SD–His synthetic liquid medium (see recipe)
  • Transformants ( protocol 3)
  • YPD medium (see recipe)
  • 0.1 M lithium acetate
  • Vector BRV1 (Laboratory of Biosystems and Cancer, National Cancer Institute, NIH) linearized in a BamHI site
  • 10 mg/ml carrier salmon sperm DNA (see recipe)
  • PEG solution for transformation of lithium acetate‐treated cells (see recipe)
  • 100‐mm SD–Ura plates (Teknova, http://www.Teknova.com or see recipe)
  • SD–His plates (Teknova, http://www.Teknova.com or see recipe)
  • EDTA mix (see recipe)
  • 10 mg/ml zymolyase 20T (ICN Biomedical) in 20% (w/v) glycerol (store in aliquots up to one year at −20°C)
  • 1% (w/v) low gelling/melting temperature agarose (LMP agarose; Life Technologies) prepared in 0.125 M EDTA, pH 7.5
  • LET solution: 0.5 M EDTA/0.01 M Tris·Cl, pH 7.5 (autoclave and store indefinitely at room temperature)
  • NDS cell lysis buffer (see recipe)
  • 1 U/µl β‐agarase (Boehringer Mannheim; store at −20°C)
  • DH10B E. coli competent cells (Life Technologies)
  • SOC solution (see recipe)
  • LB‐Cm plates: LB plates (supplemented with 12.5 µg/ml chloramphenicol)
  • 30°C incubator and shaker
  • 15‐ and 50‐ml conical centrifuge tubes
  • Beckman J‐6M centrifuge with JS‐5.2 swinging‐bucket rotor (or equivalent)
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge (e.g., Eppendorf)
  • 42°C, 45°C, 55°C, and 68°C heating blocks
  • Sterile toothpicks
  • 50°C water bath
  • Ultra micro pipet tips (Eppendorf)
  • 6‐cc syringe
  • Bio‐Rad Gene Pulser and cuvettes
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Figures

Videos

Literature Cited

Literature Cited
   Annab, L., Kouprina, N., Solomon, G., Cable, L., Hill, D., Barrett, J.C., Larionov, V., and Afshari, C. 2000. Isolation of functional copy of the human BRCA1 gene by TAR cloning in yeast. Gene 250:201‐208.
   Burke, D.T., Carle, G.F., and Olson, M.V. 1987. Cloning of large segments of DNA into yeast by means of artificial chromosome vectors. Science 236:806‐812.
   Cancilla, M., Tainton, K., Barry, A., Larionov, V., Kouprina, N., Resnick, M., Du Sart, D., and Choo, A. 1998. Direct cloning of human 10q25 neocentromere DNA transformation‐associated recombination (TAR) in yeast. Genomics 47:399‐404.
   Humble, M., Kouprina, N., Noskov, V., Graves, J., Garner, E., Tennant, R., Resnick, M.A., Larionov, V., and Cannon, R.E. 2000. Radial TAR cloning from the TgAC mouse. Genomics 70:292‐299.
   Kim, J.‐H., Leem, S.‐H., Sunwoo, Y., and Kouprina, N. 2003. Separation of long‐range human TERT gene haplotypes by transformation‐associated recombination cloning in yeast. Oncogene 22:2452‐2456.
   Kim, J., Noskov, V., Lu, X., Bergmann, A., Ren, X., Warth, T., Richardson, P., Kouprina, N., and Stubbs, L. 2000. Discovery of a novel, paternally expressed ubiquitin‐specific processing protease gene through comparative analysis of an imprinted region of mouse chromosome 7 and human chromosome 19q13.4. Genome Res. 10:1138‐1147.
   Kouprina, N. and Larionov, V. 2003. Exploiting the yeast Saccharomyces cerevisiae for the study of the organization of complex genomes. FEMS Microbiol. Rev. 27:629‐649.
   Kouprina, N., Graves, J., Resnick, M.A., and Larionov, V. 1997. Specific isolation of human rDNA genes by TAR cloning. Gene 197:269‐276.
   Kouprina, N., Annab, L., Graves, J., Afshari, C., Barrett, J.C., Resnick, M.A., and Larionov, V. 1998. Functional copies of a human gene can be directly isolated by TAR cloning with a small 3′ end target sequence. Proc. Natl. Acad. Sci. U.S.A. 95:4469‐4474.
   Kouprina, N., Leem, S.‐H., Solomon, G., Ly, A., Koriabine, M., Otstot, J., Pak, E., Dutra, A., Zhao, S., Barrett, J.C., and Larionov, V. 2003a. Segments missing from the draft human genome sequence can be isolated by TAR cloning in yeast. EMBO Rep. 4:257‐262.
   Kouprina, N., Ebersole, T., Koriabine, M., Pak, E., Rogozin, I.B., Katoh, M., Oshimura, M., Ogi, K., Peredelchuk, M., Solomon, G., Brown, W., Barrett, J.C., and Larionov, V. 2003b. Cloning of human centromeres by transformation‐associated recombination in yeast and generation of functional human artificial chromosomes. Nucl. Acids Res. 31:922‐934.
   Kouprina, N., Mullokandov, M., Rogozin, I., Collins, K., Solomon, G., Risinger, J., Koonin, E., Barrett, J.C., and Larionov, V. 2004a. The SPANX gene family of cancer‐testis specific antigens: Rapid evolution, an unusual case of positive selection and amplification in African great apes and hominids. Proc. Natl. Acad. Sci. U.S.A. 101:3077‐3082.
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   Kouprina, N., Pavlicek, A., Noskov, V.N., Solomon, G., Otstot, J., Isaacs, W., Carpten, J.D., Trent, J.M., Barrett, J.C., Jurka, J., and Larionov, V. 2005. Dynamic structure of the SPANX gene cluster mapped to the prostate cancer susceptibility locus HPCX at Xq27. Genome Res. 15:1477‐1486.
   Larionov, V., Kouprina, N., Graves, J., Chen, X.‐N., Korenberg, J., and Resnick, M.A. 1996a. Specific cloning of human DNA as YACs by transformation‐associated recombination. Proc. Natl. Acad. Sci. U.S.A. 93:491‐496.
   Larionov, V., Kouprina, N., Graves, J., and Resnick, M.A. 1996b. Highly selective isolation of human DNAs from rodent‐human hybrid cells as circular YACs by TAR cloning. Proc. Natl. Acad. Sci. U.S.A. 93:13925‐13930.
   Larionov, V., Kouprina, N., Solomon, G., Barrett, J.C., and Resnick, M.A. 1997. Direct isolation of human BRCA2 gene by transformation‐associated recombination in yeast. Proc. Natl. Acad. Sci. U.S.A. 94:7384‐7387.
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