Construction of Genomic Libraries from Unclonable DNA

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Construction of Genomic Libraries from
Unclonable DNA

• Ronald Godiska, Melodee Reuter,
• Tom Schoenfeld, David A. Mead

Lucigen Corporation Middleton, WI www.lucigen.com
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• Difficulties in library construction are common,
  but often are mentioned only in the Methods
  section of publications. For example
• A major technical difficulty was the inability
  to construct in E. coli gene banks representative
  of the entire B. subtilis chromosome....
• Kunst et al. The complete genome sequence of the
  Gram-positive bacterium Bacillus subtilis. Nature
  390249 (1997).

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What is Unclonable DNA ?

• Difficult cloning targets include several
  different types of sequences, such as
• Toxic coding sequences
• Promoters
• True Promoters
• Random A/T Rich DNA
• Modified bases
• Large fragments (gt10 kb)
• Trace amounts

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Drawbacks of Current Vectors

• Vector driven transcription and translation into
  the insert induce expression of the cloned
  sequence.
• Fortuitous transcription out of the insert can
  interfere with vector maintenance.
• False positives and false negatives arise from
  inappropriate transcription.
• High copy number can cause plasmid instability.

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pSMART Vectors

• Lucigen has developed the transcription-free
  pSMART vectors to overcome many common cloning
  problems
• Vector-driven expression of the cloned insert is
  eliminated.
• Transcripts initiated within the insert are
  terminated.
• The vectors are provided pre-cut and
  dephosphorylated to produce near zero background.
  Therefore, no colony screening is needed.
• Low-copy and high-copy versions are available.

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pSMART Vectors

• pSMART -HC pSMART -LC

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Cloning A/T Rich DNA

• The pSMART vectors are very useful for cloning
  A/T rich DNA, e.g. Lactobacillus helveticus
  genomic DNA (65 A/T 2.3 Mb genome)
• J.Steele (U.Wisc.) J. Broadbent (Utah St.
  Univ.)
• Libraries with conventional vectors were highly
  biased
• Sau3A L.h. genomic DNA in pJDC9
• 3.7 X depth of sequence (8.7 Mb) resulted in only
  63 coverage of the genome (98 was expected for
  an unbiased library).

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Cloning A/T Rich DNA

• Lucigens approach
• Hydroshear TM fragment L. helveticus DNA
• Clone into
• pUC19 (control)
• pSMART TMHC
• pSMART TMLC

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Cloning A/T Rich DNA
Increased number of stable clones with pSMART TM
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Cloning A/T Rich DNA

• pSMART TM-LC yielded random coverage of the L.
  helveticus genome, whereas pJDC9 did not.

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Cloning A/T Rich DNA

• Sequence analysis of the L. helveticus libraries
  confirms the reduced stacking with pSMART TM-LC.

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Cloning Strong Promoters

• The phage lambda PR promoter (400 bp 60 pg) was
  easily cloned in pSMART, but difficult to clone
  into pUC19

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Cloning Toxic Genes

• A lethal RNase gene (350 bp no promoter) was
  also easily cloned in both orientations in
  pSMART, but could only be recovered in the
  reverse orientation in pUC19

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Cloning large DNA (gt10 kb)

• Large-insert libraries are routinely made at
  Lucigen. Shown are 8-14 kb clones from a Shigella
  genomic library in pSMART TM-LC.
• T. Whittam, Michigan State University

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Cloning Modified Genomic DNA

• A Streptococcus thermophilus genomic library
  presented unexpected difficulties for cloning.
• P.Richardson, JGI
• Lucigens approach
• Hydroshear TM fragmented DNA to 2-3 kb.
• End-repaired
• Cloned into pSMART TM-LC
• Directly
• or
• With Linker Amplification

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Cloning Modified Genomic DNA
kb
Direct Cloning
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Cloning trace amounts of DNA

• The low background and high efficiency of
  Lucigens custom cloning system allows
  construction of libraries from trace amounts of
  DNA, such as
• Phage genomic libraries from 10 ng DNA
• Bacterial genomic libraries from 100 ng DNA
• Libraries from 10 ng of DNA isolated from the
  environment

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Duplex Cloning (ClonePlex )

• Two inserts per vector doubles throughput

pLEXX TM-AK 2.9 kb
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Duplex Cloning (ClonePlex )

• Lambda DNA cloned into pLEXX-AK

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Paired-End Duplex Cloning

• Vector components are in fixed orientation
• Sequencing primers are paired
• One insert per site (no chimeras)
• Increased efficiency

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Paired-End Duplex Cloning

• Shear and End-repair DNA

• Ligate to pLEXX- PE

• Transform

• Select for Amp Kan

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Paired-End Duplex Cloning

• A LacZ fragmentlinker C (300 bp) and a GentR
  fragmentlinker T (700 bp) were ligated with
  pLEXX-PE.
• Over 99 of the clones containing the GentR
  insert also contained the LacZ insert.

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Summary

• Transcription-free vectors allow cloning of
• Toxic coding sequences
• Strong promoters
• A/T-rich DNA
• Fixed linker amplification allows cloning of
  modified or trace amounts of DNA
• Low copy vector reduces plasmid instability
• Dual insert cloning is feasible with low
  background vector and efficient transformation

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Acknowledgements

• P. Richardson, C. Detter- JGI
• J. Steele J. Broadbent- U. Wisc. and Utah St.
• K. Montgomery/ R. Kucherlapati lab- Harvard
  Partners Genome Center
• F. Rohwer- San Diego St.
• T. Whittam- Michigan St.
• Supported by NIH SBIR grants.