Purification and Characterization of YACs Containing Large Inserts

Robert M. Gemmill1, Richard Bolin2, William M. Strauss3, William Pavan4

1 Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado, 2 Nexagen, Boulder, Colorado, 3 Whitehead Institute, Cambridge, Massachusetts, 4 Princeton University, Princeton, New Jersey
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.7
DOI:  10.1002/0471142905.hg0507s00
Online Posting Date:  May, 2001
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Abstract

This unit provides protocols for characterizing DNA segments cloned in YACs and for purifying YACs from yeast chromosomes. The first basic protocol describes Southern blotting and partial‐digest restriction analysis of YACs. These methods are useful for determining the size and complexity of the cloned insert DNA, the presence and location of particular restriction sites or sequences, and even the species of origin of the insert DNA (indicated by hybridization to species‐specific repetitive elements such as Alu repeats). The second basic protocol describes gel purification of YACs for use in procedures requiring pure YAC DNA, such as mammalian‐cell transformation and subcloning into smaller insert vectors. The third basic protocol details characterizing and analyzing YACs: in vivo fragmentation via homologous recombination with specialized fragmentation vectors containing specific probe sequences or repetitive elements, followed by Southern blotting with YAC‐ and human‐derived probes.

     
 
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Table of Contents

  • Basic Protocol 1: Mapping YACS by Partial Restriction Digestion and Southern Blotting
  • Basic Protocol 2: Purification of YACs by Pulsed‐Field Gel Electrophoresis
  • Basic Protocol 3: Characterization of YACs by Recombination with Fragmentation Vectors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Mapping YACS by Partial Restriction Digestion and Southern Blotting

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix
  • Agarose blocks containing high‐molecular‐weight DNA (unit 5.1) prepared from single YAC‐bearing yeast colonies testing positive for a probe of interest
  • 1 mg/ml BSA, acetylated and nuclease‐free
  • Rare‐cutting restriction endonucleases (Table 97.80.4711) and 10×buffers
  • 100 mM spermidine
  • Stop/loading buffer: 0.1× gel loading buffer ( appendix 2D) in TEbuffer (store up to 6 months at room temperature)
  • pBR322 DNA
  • Control DNAs prepared in agarose blocks (unit 5.1): control λ‐concatemer molecular size standard, yeast chromosome molecular size standard (e.g.,YPHλ755; Gerring et al., ), and YAC strain negative for the internal marker
  • BamHI andPvuII restriction endonucleases and appropriate 10× buffers (for probe preparation)
  • NotI, SalI, SfiI, MluI, BssHII, and EagI restriction endonucleases and appropriate 10× buffers (for partial‐digest analysis)
  • Low melting/gelling temperature agarose (e.g., SeaPlaque GTG grade, FMC Bioproducts)
  • Charge‐modified nylon membrane of appropriate size
  • Pulsed‐field gel electrophoresis (PFGE) apparatus
  • Additional reagents and equipment for PFGE (unit 5.1), Southern blotting and hybridization (unit 2.7), radiolabeling DNA by oligonucleotide‐primed synthesis ( appendix 3E), and electroelution from agarose gel (CPMB UNIT )
CAUTION: Radiolabeled probes are hazardous; see appendix 2A for guidelines on handling,storage, and disposal.
Table 5.7.1   Materials   Interpretation of Recombination Events in His+ Transformants During Fragmentation (Fig. )   Interpretation of Recombination Events in His+ Transformants During Fragmentation

Lane Fragmenting vector Parental YAC (Ura+ Trp+) Fragmented YAC Event
 a pBP81 (Alu, HIS3, CEN) 360 kb His 250 kb His+ Trp+ Centric fragmentation with reduplication
 b pBP108 (Alu, HIS3) Absent 340 kb Ura+ His+ Acentric fragmentation
 c pBP108 (Alu, HIS3) Absent 340 kb; Ura+ His+ Acentric fragmentation
 d pBP108 (Alu, HIS3) Absent 580 kb; Ura+ Trp+ His+ Integration of vector and rearrangement of YAC
 e pBP81 (Alu, HIS3), CEN 360 kb; His 300 kb; His+ Trp+ Centric fragmentation with reduplication
 f pBP81 (Alu, HIS3), CEN 360 kb; His 75 kb His+ Trp+ Centric fragmentation with reduplication
 g pBP108 (Alu, HIS3) Absent 370 kb; Ura+ Trp+ His+ Integration of fragmenting vector into YAC
 h pBP81 (Alu, HIS3), CEN 360 kb; His 200 kb; His+ Trp+ Centric fragmentation with reduplication
 i pBP108 (Alu, HIS3) 360 kb; His 40 kb; Ura+ His+ Acentric fragmentation with reduplication
 j pBP81 (Alu, HIS3), CEN Absent 125 kb; His+ Trp+ Centric fragmentation

Basic Protocol 2: Purification of YACs by Pulsed‐Field Gel Electrophoresis

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D for suppliers, see suppliers appendix.
  • YAC‐bearing yeast strain
  • 0.5× TBE buffer( appendix 2D)
  • Low gelling/melting temperature agarose (e.g., SeaPlaque GTG grade, FMC Bioproducts)
  • High‐molecular‐weight size standards in agarose (unit 5.1)
  • Staining buffer: 0.1 mg/ml ethidium bromide in 0.5× TBE buffer ( appendix 2D)
  • recipeDialysis buffer (see recipe)
  • 10 mg/ml poly‐L‐lysine (Sigma)
  • Agarase: e.g., β‐agarase (New England Biolabs) or GELase (Epicentre Technologies)
  • Dialysis membrane
  • Customized gel casting plate (optional; Fig. )
  • Pulsed‐field gel electrophoresis (PFGE) apparatus, prechilled to 4°C
  • Short‐ or medium‐wave UV transilluminator with camera attachment
  • Rulers (preferably fluorescent)
  • 50‐ml conical centrifuge tube
  • Small conical tube
  • 65° and 40°C heating blocks or water baths
  • Additional reagents and equipment for preparation of high‐molecular‐weight DNA in agarose blocks (unit 5.1), agarose gel electrophoresis (unit 2.7), and PFGE (unit 5.1)
CAUTION: Ethidium bromide is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Basic Protocol 3: Characterization of YACs by Recombination with Fragmentation Vectors

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • YAC‐bearing yeast strain (unit 5.2 see and Table 5.7.2)
  • recipeSD dropout plates: SD −Ura −Ade; SD −His; SD −Ura; and SD −Trp (see recipe)
  • YPD medium (unit 5.5) containing 1× Ade and Trp amino acid supplements (see recipe)
  • recipeLithium acetate/Tris/EDTA solution (see recipe)
  • Fragmentation vector (see )
  • Appropriate restriction endonuclease and 10× buffer for linearizing fragmentation vector between target sequence and telomere
  • recipe40% PEG solution (see recipe)
  • recipeRadiolabeled probes made from HIS3, URA3, TRP1, and human Alu DNA (see recipe)
  • EcoRI orBamHI restriction endonuclease and appropriate buffer
  • 30°C shaking incubator
  • 15‐ml culture tube with loose‐fitting cap
  • 150‐ml Erlenmeyer flask with loose‐fitting cap
  • Beckman TJ‐6 centrifuge with TH4 rotor and buckets (or equivalent)
  • 42°C water bath
  • Nitrocellulose or nylon membranes
  • Additional reagents and equipment for preparation of agarose‐embedded high‐molecular‐weight DNA and PFGE (unit 5.1), agarose gel electrophoresis (unit 2.7), and Southern blotting (unit 2.7)
CAUTION: Radiolabeled probes are hazardous; see appendix 2A for guidelines on handling, storage, and disposal
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Figures

Videos

Literature Cited

Literature Cited
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