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Analysis of Isolated YAC Clones

Divid D. Chaplin1,  Bernard H. Brownstein1

1Howard Hughes Medical Institute and Washington, University School of Medicine, St. Louis, Missouri

Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 6.10
DOI: 
10.1002/0471142727.mb0610s20
Online Posting Date: 
May, 2001
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Abstract

This unit provides a series of protocols describing the analysis and manipulation of an isolated YAC clone. The procedures are based upon the use of the YAC vector pYAC4. Once an isolated YAC clone has been obtained from a core laboratory, the clone can be analyzed as described herein. Methods for analysis involve growing and storing YAC-containing yeast strains and purifying YAC DNA in a form suitable for assessing the size of the artificial chromosome and for conventional Southern blotting. Preparation of yeast chromosomes in agarose plugs for subsequent analysis by pulsed-field gel electrophoresis is also described. Additional protocols are provided for recovering DNA fragments from the ends of a YAC genomic insert to be used as probes for detecting chimerism and for chromosome walking. Finally, preparation of high-molecular-weight YAC DNA is described and a general method for subcloning YAC inserts into cosmid or vectors for higher-resolution analysis is provided.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Propagation and Storage of YAC-Containing Yeast Strains
  • Basic Protocol: Preparation of YAC-Containing DNA from Yeast Clones for Analysis by Southern Blotting
  • Basic Protocol: Preparation of Yeast Chromosomes in Agarose Plugs for Pulsed-Field Gel Electrophoresis
  • Basic Protocol: End-Fragment Analysis Using PCR Amplification
  • Alternate Protocol: End-Fragment Analysis by Subcloning into a Bacterial Plasmid Vector
  • Support Protocol: Design and Preparation of pUC19-ES and pUC19-HS Subcloning Vector
  • Basic Protocol: Preparation of High-Molecular-Weight YAC-Containing Yeast DNA in Solution
  • Basic Protocol: Preparation and Analysis of a YAC-Insert Sublibrary
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol: Propagation and Storage of YAC-Containing Yeast Strains

 Materials
  • S. cerevisiae strain AB1380 containing pYAC4 with insert (from core facility; UNIT 6.9)
  • AHC plates (ura–, trp–)
  • YPD medium (UNIT 13.1)
  • 80% (v/v) glycerol in YPD medium
  • 30°C orbital shaking incubator (e.g., New Brunswick Scientific #G-24)
  • Cryovials
  • Additional reagents for preparation of yeast media (UNIT 13.1) and growth and manipulation of yeast (UNIT 13.2)

Basic Protocol: Preparation of YAC-Containing DNA from Yeast Clones for Analysis by Southern Blotting

 Materials
  • Single colony of S. cerevisiae AB1380 containing pYAC4 with insert (first basic protocol)
  • AHC medium (ura–, trp–)
  • SCE buffer
  • SCEM buffer
  • 50 mM Tris×Cl (pH 7.6)/20 mM EDTA (Tris/EDTA lysis buffer)
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • 5 M potassium acetate, pH 4.8, ice-cold (UNIT 1.6)
  • 95% ethanol, room temperature
  • TE buffer, pH 8.0 (APPENDIX 2)
  • 1 mg/ml DNase-free RNase A (UNIT 3.13)
  • Isopropanol, room temperature
  • 5 M NaCl
  • Total genomic DNA of the species or individual from which the library was made (e.g., UNITS 2.2 2.3 & 5.3)
  • Appropriate single-copy probe designed to hybridize with the YAC insert (see UNITS 2.9A & 6.9)
  • Orbital shaker (e.g., New Brunswick Scientific #G-24)
  • 50-ml conical plastic centrifuge tubes
  • Beckman JS-4.2 rotor or equivalent
  • Additional reagents and equipment for digestion of DNA with restriction endonucleases (UNIT 3.1), Southern blotting and hybridization (UNIT 2.9A), and pulsed-field gel electrophoresis (UNIT 2.5B)

Basic Protocol: Preparation of Yeast Chromosomes in Agarose Plugs for Pulsed-Field Gel Electrophoresis

 Materials
  • AHC medium (ura–, trp–)
  • Single colony of S. cerevisiae containing pYAC4 with insert (first basic protocol)
  • 0.05 M EDTA, pH 8.0 (APPENDIX 2)
  • SEM buffer
  • 10 mg/ml Lyticase (Sigma #L-8137 or ICN Biomedicals #190123)
  • 2% InCert or SeaPlaque agarose (FMC Bioproducts), dissolved in SEM buffer and equilibrated to 37°C
  • SEMT buffer
  • Lithium lysis solution
  • 20% (v/v) NDS solution
  • 0.5× TBE (APPENDIX 2) or GTBE buffer (UNIT 2.5B)
  • 30°C rotary platform shaking incubator
  • Beckman JS-4.2 rotor or equivalent
  • Gel sample molds (e.g., CHEF gel molds, Bio-Rad #1703622)
  • 60-mm tissue culture plate
  • Additional reagents and equipment for pulsed-field gel electrophoresis (UNIT 2.5B)

Basic Protocol: End-Fragment Analysis Using PCR Amplification

 Materials
  • “Bubble-top” and “bubble-bottom” oligonucleotide primers (Fig. 6.10.2)
  • YAC-containing DNA (second basic protocol)
  • RsaI and HinfI restriction endonucleases and appropriate buffers (UNIT 3.1)
  • 10× T4 DNA ligase buffer and 1 U/µl T4 DNA ligase (UNITS 3.4 & 3.14)
  • PCR reaction mix
  • PCR amplification primers HYAC-C, HYAC-D, 224, and RA-2, 4 µM each (Fig. 6.10.2)
  • Thermal cycling apparatus
  • 65° and 68°C water baths
  • Additional reagents and equipment for phosphorylating synthetic oligonucleotides (UNIT 3.10), restriction endonuclease digestion (UNIT 3.1), PCR (UNIT 15.1), nondenaturing PAGE (UNIT 2.7), preparing radiolabeled oligonucleotide probes (UNITS 3.10, 4.6 & 5.2), and blunt-end ligation (UNIT 3.6)

Alternate Protocol: End-Fragment Analysis by Subcloning into a Bacterial Plasmid Vector

 Additional Materials
  • ClaI, SalI, and other appropriate restriction endonucleases and digestion buffers (UNIT 3.1)
  • Left- and right-vector-arm probes (Fig. 6.10.4)
  • pUC19-ES and pUC19-HS plasmid vectors (Support Protocol and Fig. 6.10.5)
  • Transformation-competent Rec– strain of E. coli (e.g., DH5; Table 1.4.5)
  • 2× TY or LB agar plates (UNIT 1.1) containing 50 to 100 µg/ml ampicillin
  • Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.5A), subcloning of DNA fragments (UNIT 3.16), transformation of E. coli (UNIT 1.8), Southern blotting and hybridization (UNIT 2.9A), labeling by random-primed synthesis (UNIT 3.5), isolation and purification of DNA fragments from agarose gels (UNIT 2.6), replica plating (UNIT 1.3), and purification of plasmid DNA (UNITS 1.6 & 1.7)

Basic Protocol: Preparation of High-Molecular-Weight YAC-Containing Yeast DNA in Solution

 Materials
  • Single colony of S. cerevisiae containing pYAC4 with insert (first basic protocol)
  • AHC medium (ura–, trp–)
  • SCEM buffer
  • Lysis buffer
  • Step-gradient solutions: 50%, 20%, and 15% (w/v) sucrose
  • TE buffer, pH 8.0 (APPENDIX 2)
  • Dry granular sucrose
  • 30°C orbital shaking incubator (e.g., New Brunswick Scientific #G-24)
  • 250-ml conical centrifuge bottles (e.g., Corning #25350)
  • 65°C water bath
  • 25 × 89–mm tube (e.g., Beckman #344058)
  • Beckman JS-4.2 and SW-27 rotors (or equivalents)
  • Dialysis tubing (APPENDIX 3A)
  • Pyrex baking dish
  • CHEF pulsed-field gel apparatus or equivalent (UNIT 2.5A)
  • Additional reagents and equipment for size fractionation using a sucrose gradient (UNIT 5.3) and estimating DNA concentration (UNIT 2.6)

Basic Protocol: Preparation and Analysis of a YAC-Insert Sublibrary

 Materials
  • High-molecular-weight YAC-containing DNA (fifth basic protocol)
  • Vector DNA (e.g., SuperCos 1, Stratagene #251301)
  • 32P-labeled (UNIT 3.10) probes: total genomic DNA of the individual or species from which the library was made (e.g., UNITS 2.2 2.3 & 5.3), end-specific DNA (UNIT 3.10) or RNA (UNIT 3.8), and end fragment from YAC (fourth basic protocol or Alternate Protocol)
  • Additional reagents and equipment for restriction endonuclease digestion (UNIT 3.1), genomic DNA library production (UNIT 5.7), plating and transferring a cosmid library (UNIT 6.2), and hybridization with radioactive probes (UNITS 6.3 & 6.4)
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Figures

  • Figure 6.10.1
    Structure of a representative pYAC4 clone at the vector/insert junction. Open boxes represent portions of the pYAC4 vector derived from yeast sequences (CEN4, the two halves of the SUP4 element, and URA3). Thin lines represent sequences derived from pBR322 and the bold line represents the YAC genomic insert fragment. Sites of annealing of the HYAC-C, LS-2, RA-2 and HYAC-D oligonucleotides are indicated by arrows 1, 2, 3, and 4, respectively. TheEcoRI cloning site, the ClaI and SalI sites (used for the end-fragment subcloning Alternate Protocol), and theHinfI sites that are utilized in the bubble linker end-fragment isolation protocol are indicated.

    View Image
  • Figure 6.10.2
    Oligonucleotides for amplification and sequencing of YAC insert end-fragments. (A) Annealing of the 53-mer universal “bubble-bottom” oligonucleotide to the 53-mer RsaI bubble oligonucleotide yields a blunt-ended DNA duplex in which 12-bp complementary sequences flank a 29-nucleotide “bubble” of noncomplementary sequence. This bubble linker can be ligated to any blunt-ended fragment (e.g., one generated by digestion with RsaI). The 224 primer does not anneal to either strand of the bubble, but is fully complementary to any DNA strand that is generated during PCR using the universal bubble-bottom strand as a template (see Fig. 6.10.3). (B) Annealing of the 53-mer universal bubble-bottom oligonucleotide to the 56-mer HinfI bubble-top oligonucleotide yields a DNA duplex with one blunt end and one cohesive end with the degenerateHinfI site. A mixture of all four nucleotides at a specific position is indicated by (N). (C) The HYAC-C, HYAC-D, RA-2, and LS-2 primers anneal to sequences in the pYAC4 vector (see Fig. 6.10.1). The bubble sequencing primer anneals to the RsaI andHinfI bubble-top sequences near their 5¢ ends, permitting DNA sequencing from the bubble linker back into the YAC insert end-fragment.

    View Image
  • Figure 6.10.3
    Selective PCR amplification from the YAC insert end-fragment. (A) Result of ligation of the bubble linker to a random fragment from the internal portion of the YAC insert. Because this fragment is not derived from the end of the YAC genomic insert, it contains no sequences from the YAC vector and has no site for annealing any of the HYAC-C, HYAC-D, RA-2, or LS-2 primers or for annealing of the 224 primer. Consequently, no fragment is amplified by PCR. (B) Result of ligation of the bubble linker to a fragment derived from the end of the YAC genomic insert and containing its associated YAC vector sequences. During the first cycle of PCR, extension from the YAC vector priming site produces sequences complementary to the universal bubble-bottom primer. This extended fragment provides a template for annealing of the 224 primer, thus permitting successful amplification of the insert end-fragment.

    View Image
  • Figure 6.10.4
    Generation of left- and right-vector-arm probes. The 351-bp ClaI-BamHI and 276-bpBamHI-SalI fragments of pBR322, which hybridize to sequences immediately flanking the sup4 sequences of the YAC vector, are appropriate probes for the YAC left and right vector arms. These probes can be obtained by restriction digestion and gel fractionation of pBR322 plasmid DNA or generated by PCR using 10 ng pBR322 as template for the primers illustrated here. Perform PCR using 25 cycles of 1 min at 92°C, 1 min at 50°C, and 2 min at 72°C. Extract the amplified material once with phenol and once with chloroform, then precipitate with ethanol (UNIT 2.1A). Label directly by random priming (UNIT 3.5) without further purification.

    View Image
  • Figure 6.10.5
    Structure of the pUC19-ES and pUC19-HS plasmids.

    View Image
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