Analysis of Isolated YAC Clones

Divid D. Chaplin1, Bernard H. Brownstein1

1 Howard 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 λ vectors for higher‐resolution analysis is provided.

     
 
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Table of Contents

  • Basic Protocol 1: Propagation and Storage of YAC‐Containing Yeast Strains
  • Basic Protocol 2: Preparation of YAC‐Containing DNA from Yeast Clones for Analysis by Southern Blotting
  • Basic Protocol 3: Preparation of Yeast Chromosomes in Agarose Plugs for Pulsed‐Field Gel Electrophoresis
  • Basic Protocol 4: End‐Fragment Analysis Using PCR Amplification
  • Alternate Protocol 1: End‐Fragment Analysis by Subcloning into a Bacterial Plasmid Vector
  • Support Protocol 1: Design and Preparation of pUC19‐ES and pUC19‐HS Subcloning Vector
  • Basic Protocol 5: Preparation of High‐Molecular‐Weight YAC‐Containing Yeast DNA in Solution
  • Basic Protocol 6: Preparation and Analysis of a YAC‐Insert Sublibrary
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Propagation and Storage of YAC‐Containing Yeast Strains

  Materials
  • S. cerevisiae strain AB1380 containing pYAC4 with insert (from core facility; unit 6.9)
  • recipeAHC 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 2: 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 protocol 1basic protocol)
  • recipeAHC medium (ura−, trp−)
  • recipeSCE buffer
  • recipeSCEM 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 22)
  • 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 2.9 & 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 2.9), and pulsed‐field gel electrophoresis (unit 2.5)

Basic Protocol 3: Preparation of Yeast Chromosomes in Agarose Plugs for Pulsed‐Field Gel Electrophoresis

  Materials
  • recipeAHC medium (ura−, trp−)
  • Single colony of S. cerevisiae containing pYAC4 with insert (first protocol 1basic protocol)
  • 0.05 M EDTA, pH 8.0 ( appendix 22)
  • recipeSEM buffer
  • 10 mg/ml Lyticase (Sigma #L‐8137 or ICN Biomedicals #190123)
  • 2% InCert or SeaPlaque agarose (FMC Bioproducts), dissolved in recipeSEM buffer and equilibrated to 37°C
  • recipeSEMT buffer
  • recipeLithium lysis solution
  • recipe20% (v/v) NDS solution
  • 0.5× TBE ( appendix 22) or GTBE buffer (unit 2.5)
  • 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 2.5)

Basic Protocol 4: End‐Fragment Analysis Using PCR Amplification

  Materials
  • “Bubble‐top” and “bubble‐bottom” oligonucleotide primers (Fig. )
  • protocol 2YAC‐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. )
  • 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 1: 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. )
  • pUC19‐ES and pUC19‐HS plasmid vectors ( protocol 6 and Fig. )
  • Transformation‐competent Rec strain of E. coli (e.g., DH5; Table 97.80.4711)
  • 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 2.5), subcloning of DNA fragments (unit 3.16), transformation of E. coli (unit 1.8), Southern blotting and hybridization (unit 2.9), 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)

Support Protocol 1: Design and Preparation of pUC19‐ES and pUC19‐HS Subcloning Vector

  Materials
  • Single colony of S. cerevisiae containing pYAC4 with insert (first protocol 1basic protocol)
  • recipeAHC medium (ura−, trp−)
  • recipeSCEM buffer
  • recipeLysis buffer
  • Step‐gradient solutions: 50%, 20%, and 15% (w/v) sucrose
  • TE buffer, pH 8.0 ( appendix 22)
  • 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 2.5)
  • Additional reagents and equipment for size fractionation using a sucrose gradient (unit 5.3) and estimating DNA concentration (unit 2.6)

Basic Protocol 5: Preparation of High‐Molecular‐Weight YAC‐Containing Yeast DNA in Solution

  Materials
  • protocol 7High‐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 6.10basic protocol or 6.10)
  • 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

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Literature Cited

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