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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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Construction of genomic libraries from unclonable dna
Construction of Genomic Libraries from “Unclonable” DNA

Ronald Godiska, Melodee Reuter,

Tom Schoenfeld, David A. Mead

Lucigen Corporation

Middleton, WI

www.lucigen.com


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 390:249 (1997).


What is unclonable dna
What is “Unclonable” DNA ? are mentioned only in the Methods section of publications. For example:

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 (>10 kb)

  • Trace amounts


  • Drawbacks of current vectors

    P are mentioned only in the Methods section of publications. For example:

    lac

    Cloned fragment

    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.


    Psmart vectors
    pSMART are mentioned only in the Methods section of publications. For example:™ 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.


    Psmart vectors1
    pSMART are mentioned only in the Methods section of publications. For example:™ Vectors

    pSMART™ -HC pSMART™ -LC


    Cloning a t rich dna
    Cloning A/T Rich DNA are mentioned only in the Methods section of publications. For example:

    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).


    Cloning a t rich dna1
    Cloning A/T Rich DNA are mentioned only in the Methods section of publications. For example:

    Lucigen’s approach:

    • HydroshearTM fragment L. helveticus DNA

    • Clone into:

      • pUC19 (control)

      • pSMARTTM–HC

      • pSMARTTM–LC


    Cloning a t rich dna2
    Cloning A/T Rich DNA are mentioned only in the Methods section of publications. For example:

    Increased number of stable cloneswith pSMARTTM


    Cloning a t rich dna3
    Cloning A/T Rich DNA are mentioned only in the Methods section of publications. For example:

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


    Cloning a t rich dna4

    Maltose ABC transport system: are mentioned only in the Methods section of publications. For example:

    transmembrane

    permeases

    maltose-

    binding

    ATP-

    binding

    b-phosphogluco

    mutase

    maltose

    phosphorlase

    maltose

    amylase

    pJDC9/Sau3A library

    pSMART-LC library

    Cloning A/T Rich DNA

    Sequence analysis of the L. helveticus libraries confirms the reduced stacking with pSMARTTM-LC.


    Cloning strong promoters
    Cloning Strong Promoters are mentioned only in the Methods section of publications. For example:

    The phage lambda PRpromoter (400 bp; 60 pg) was easily cloned in pSMART, but difficult to clone into pUC19:


    Cloning toxic genes
    Cloning Toxic Genes are mentioned only in the Methods section of publications. For example:

    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:


    Cloning large dna 10 kb

    kb are mentioned only in the Methods section of publications. For example:

    16

    12

    8

    6

    4

    2

    Cloning large DNA (>10 kb)

    Large-insert libraries are routinely made at Lucigen. Shown are 8-14 kb clones from a Shigella genomic library in pSMARTTM-LC.

    • T. Whittam, Michigan State University


    Cloning modified genomic dna
    Cloning Modified Genomic DNA are mentioned only in the Methods section of publications. For example:

    A Streptococcus thermophilus genomic library presented unexpected difficulties for cloning.

    • P.Richardson, JGI

      Lucigen’s approach:

      Hydroshear TM fragmented DNA to 2-3 kb.

      End-repaired

      Cloned into pSMART TM-LC:

      • Directly

        – or –

      • With Linker Amplification


    Cloning modified genomic dna1

    16 are mentioned only in the Methods section of publications. For example:

    8

    6

    16

    4

    8

    3

    6

    2

    4

    3

    Linker

    Ampl’d.

    Cloning

    (1000x cfu)

    Cloning Modified Genomic DNA

    kb

    Direct

    Cloning


    Cloning trace amounts of dna
    Cloning trace amounts of DNA are mentioned only in the Methods section of publications. For example:

    The low background and high efficiency of Lucigen’s 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


    Duplex cloning cloneplex
    Duplex Cloning (ClonePlex are mentioned only in the Methods section of publications. For example:™)

    Two inserts per vector doubles throughput

    pLEXXTM-AK

    2.9 kb


    Duplex cloning cloneplex1
    Duplex Cloning (ClonePlex are mentioned only in the Methods section of publications. For example:™)

    • Lambda DNA cloned into pLEXX-AK


    Paired end duplex cloning

    KanL1 are mentioned only in the Methods section of publications. For example:

    KanR1

    pLEXXTM -PE

    2.9 kb

    AmpL1

    AmpR1

    Paired-End Duplex Cloning

    • Vector components are in fixed orientation

      • Sequencing primers are “paired”

    • One insert per site (no chimeras)

    • Increased efficiency


    Paired end duplex cloning1

    Ligate to Linker ‘C’ are mentioned only in the Methods section of publications. For example:

    Ligate to Linker ‘T’

    • Remove linkers (Gel)

    Paired-End Duplex Cloning

    • Shear and End-repair DNA

    • Ligate to pLEXX- PE

    • Transform

    • Select for Amp + Kan


    Paired end duplex cloning2
    Paired-End Duplex Cloning are mentioned only in the Methods section of publications. For example:

    A LacZ fragment+linker C (300 bp) and a GentR fragment+linker T (700 bp) were ligated with pLEXX-PE.

    Over 99% of the clones containing the GentR insert also contained the LacZ insert.


    Summary
    Summary are mentioned only in the Methods section of publications. For example:

    • 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


    Acknowledgements
    Acknowledgements are mentioned only in the Methods section of publications. For example:

    • 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.


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