Construction of YAC Libraries with Large Inserts

Simon Foote1, Christopher Denny2

1 Whitehead Institute, Cambridge, Massachusetts, 2 University of California at Los Angeles, School of Medicine, Los Angeles, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.2
DOI:  10.1002/0471142905.hg0502s31
Online Posting Date:  February, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The yeast artificial chromosome (YAC) cloning system makes it possible to clone large pieces of genomic DNA into yeast. Libraries have been made containing clones with inserts in the megabase‐pair range. The basic protocol in this unit describes preparation of YAC vectors and transformation of ligated DNA into yeast spheroplasts. A support protocol describes titration of Lyticase to make spheroplasts. Additional support protocols detail two methods for partial digestion of genomic DNA: EcoRI restriction endonuclease‐EcoRI methylase competition and the partial digestion of genomic DNA by use of limiting amounts of Mg2+, respectively.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Construction of a Yeast Artificial Chromosome Library
  • Support Protocol 1: Titration of Lyticase
  • Support Protocol 2: Partial Digestion of Chromosomal DNA Using Restriction Enzyme–Methylase Competition
  • Support Protocol 3: Partial Restriction Enzyme Digestion by Mg2+ Limitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Construction of a Yeast Artificial Chromosome Library

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2D ; for suppliers, see suppliers appendix.
  • E. coli strain carrying a pYAC4 vector (Figs. and ; ATCC #67379)
  • 20 U/µl HindIII, BamHI, and EcoRI restriction endonucleases and appropriate buffers
  • DNA molecular size markers
  • 1 U/µl calf intestine phosphatase (CIP; Boehringer Mannheim)
  • TE buffer
  • Size‐selected, partially digested insert DNA in agarose block (second or third support protocol)
  • 1× ligase buffer: 50 mM Tris⋅Cl (pH 7.6)/10 mM MgCl 2 (store at room temperature) 100 mM ATP ( appendix 2)
  • 1 M DTT
  • 400,000 U/ml T4 DNA ligase (in cohesive‐end units; New England Biolabs)
  • SeaPlaque GTG agarose (FMC Bioproducts)
  • 0.5× TBE buffer ( appendix 2)
  • 0.5 µg/ml ethidium bromide solution ( appendix 2)
  • recipeT 50E buffer (see recipe)
  • recipeAgarase buffer (see recipe)
  • 100 mM spermidine (store at −20°C)
  • 1000 U/ml β‐agarase (New England Biolabs)
  • Yeast strain AB1380 (ATCC #20843)
  • YPD medium (unit 5.5)
  • 1 M sorbitol
  • SCE buffer (unit 5.1)
  • 2‐mercaptoethanol (2‐ME)
  • 10,000 U/ml Lyticase (see first support protocol; Sigma)
  • recipeSTC buffer (see recipe)
  • recipeCalf thymus DNA, purified (see recipe)
  • 20% (w/v) polyethylene glycol (PEG) 8000 solution
  • recipeSOS medium (see recipe)
  • recipe−Ura top agar, prewarmed to 50°C (see recipe)
  • 150‐mm recipe−Ura SORB dropout plates, 30°C (see recipe)
  • AHC plates (unit 5.5)
  • Glycerol, sterile
  • 50‐ml polypropylene centrifuge tubes (Falcon)
  • 4‐ and 12‐ml polypropylene tubes (Falcon), sterile
  • 50° and 68°C water baths
  • Pulsed‐field gel electrophoresis (PFGE) apparatus (e.g., Bio‐Rad CHEF)
  • 30°C humidified incubator
  • Beckman G6‐6R centrifuge with GH‐3.8 rotor (or equivalent)
  • 1000‐µl wide‐bore pipet tips
  • Dissecting microscope
  • Sterile toothpicks or plastic inoculating loops (VWR Scientific)
  • 96‐well tissue culture plates (Costar)
  • Transtar device with elevator (Costar)
  • Adhesive sealing membrane (Costar)
  • 96‐pin replicator (Dan Kar Scientific)
  • Additional reagents and equipment for CsCl plasmid preps (unit 5.3 and CPMB UNIT ), agarose gel electrophoresis (unit 2.7), phenol extraction, ethanol precipitation, and quantitation of DNA ( appendix 3C & ), and PFGE (unit 5.1)
CAUTION: Ethidium bromide and 2‐mercaptoethanol are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 1: Titration of Lyticase

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Yeast strain containing control YAC
  • 0.03 ng/µl YCp50 plasmid DNA in recipeagarase buffer (see recipe)Additional reagents and equipment for preparing YAC DNA in agarose blocks (unit 5.1)

Support Protocol 2: Partial Digestion of Chromosomal DNA Using Restriction Enzyme–Methylase Competition

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Mammalian cells or tissue
  • recipeEcoRI compromise buffer (prepare fresh; see recipe)
  • 40 U/µl EcoRI methylase (New England Biolabs)
  • Yeast chromosomal DNA size standards in agarose blocks (unit 5.1)
  • 100 mM S‐adenosylmethionine (SAM; Sigma)Additional reagents and equipment for preparation of high‐molecular‐weight mammalian genomic DNA in agarose blocks(unit 5.1)CAUTION: Human cells and tissue are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 3: Partial Restriction Enzyme Digestion by Mg2+ Limitation

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Mammalian cells or tissue
  • recipeEcoRI(−Mg2+) buffer (see recipe)
  • 10 and 100 mM MgCl 2 (store at −20°C)
  • 100 mM EDTA, ice‐cold (store at 4°C)
  • 15‐ml conical tubes, sterile
  • 2‐ml flat‐bottom microcentrifuge tubes, sterile
  • Additional reagents and equipment for preparation of high‐molecular‐weight DNA in agarose blocks (unit 5.1)
CAUTION: Human cells and tissue can be hazardous; see appendix 2A for guidelines on handling, storage, and disposal.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Albertsen, H.M., Le Paslier, D., Abderrahim, H., Dausset, J., Cann, H., and Cohen, D. 1989. Improved control of DNA restriction digestion in agarose using limited concentrations of Mg++. Nucl. Acids Res. 17:808.
   Albertsen, H.M., Abderrahim, H., Cann, H.M., Dausset, J., Le Paslier, D., and Cohen, D. 1990. Construction and characterization of a yeast artificial chromosome library containing seven haploid equivalents. Proc. Natl. Acad. Sci. U.S.A. 87:4256‐4260.
   Burgers, P.M.J. and Percival, K.J. 1987. Transformation of yeast spheroplasts without cell fusion. Anal. Biochem. 163:391‐397.
   Burke, D.T., Carle, G.F., and Olson, M.N. 1987. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806‐812.
   Cooke, H. and Cross, S. 1988. pYAC‐4 Neo, a yeast artificial chromosome vector which codes for G418 resistance in mammalian cells. Nucl. Acids Res. 16:11817.
   Shero, J.H., McCormick, M.K., Antonarakis, S.E., and Hieter, P. 1991. Yeast artificial chromosome vectors for efficient clone manipulation and mapping. Genomics 10:505‐508.
   Smith, D.R., Smyth, A.P., and Moir, D.T. 1990. Amplification of large artificial chromosomes. Proc. Natl. Acad. Sci. U.S.A. 87:8242‐8246.
Key References
   Albertsen et al., 1989. See above.
  Mg2+ limitation for genomic partial digestion.
   Albertsen et al., 1990. See above.
  The first description of use of PFGE to size select DNA for ligation.
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.). 1993. Current Protocols in Molecular Biology. Greene Publishing Associates and John Wiley & Sons, New York.
  Good reference for elementary yeast‐associated techniques.
   Burgers and Percival, 1987. See above.
  A detailed description of the factors that influence spheroplast transformation.
   Burke et al., 1987. See above.
  Original description of YACs and their construction, still of interest even though the method of making YACs has changed.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library