Construction of Genomic Libraries from “Unclonable” DNA

1. Construction of Genomic Libraries from “Unclonable” DNA Ronald Godiska, Melodee Reuter, Tom Schoenfeld, David A. Mead Lucigen Corporation Middleton, WI www.lucigen.com

2. 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).

3. 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 (>10 kb) • Trace amounts

4. P 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.

5. 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.

6. pSMART™ Vectors pSMART™ -HC pSMART™ -LC

7. 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).

8. Cloning A/T Rich DNA Lucigen’s approach: • HydroshearTM fragment L. helveticus DNA • Clone into: • pUC19 (control) • pSMARTTM–HC • pSMARTTM–LC

9. Cloning A/T Rich DNA Increased number of stable cloneswith pSMARTTM

10. Cloning A/T Rich DNA pSMARTTM-LC yielded random coverage of the L. helveticus genome, whereas pJDC9 did not.

11. Maltose ABC transport system: 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.

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

13. 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:

14. kb 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

15. Cloning Modified Genomic DNA 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

16. 16 8 6 16 4 8 3 6 2 4 3 Linker Ampl’d. Cloning (1000x cfu) Cloning Modified Genomic DNA kb Direct Cloning

17. Cloning trace amounts of DNA 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

18. Duplex Cloning (ClonePlex™) Two inserts per vector doubles throughput pLEXXTM-AK 2.9 kb

19. Duplex Cloning (ClonePlex™) • Lambda DNA cloned into pLEXX-AK

20. KanL1 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

21. Ligate to Linker ‘C’ 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

22. Paired-End Duplex Cloning 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.

23. 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

24. 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.