Lucigen Corporation 2120 W. Greenview Dr., Ste 9 Middleton, WI 53562 USA www.lucigen.com

David A. Mead1, Nikolai Ravin2, Sarah Vande Zande1, Rebecca Hochstein1, Karen Usdin3, Attila Karsi4, Cheng-Cang Wu1, Ronald Godiska1

1 Lucigen Corporation, Middleton, WI 53562 USA 2 Centre BioEngineering RAS, Moscow, Russia 3 NIH, Bethesda, MD 20892 USA 4 Mississippi State Univiversity

Stable Cloning of Repetitive, GC-rich and AT-rich DNAs in a Linear Plasmid

AbstractA linear cloning vector for transformation of E. coli has been developed from the coliphage N15. The linear “pJAZZ” vector provides exceptional ability to clone otherwise unclonable DNAs of any size up to ~30 kb. The ends of the molecule are free to rotate during replication, so the vector and inserted DNA fragments are not subject to the torsional stress caused by supercoiling. In addition, transcriptional terminators flank the cloning site, preventing transcriptional interference between the vector and insert. Regions of potential secondary structure appear to be quite stable in this relaxed, non-transcribed state. For example, up to 300 copies (~ 1 kb) of the GCC repeat of the Fragile X locus were stable in the pJAZZ vector, but could not be maintained in a circular vector. AT-rich fragments of >20 kb and other highly repetitive DNAs also have been easily and reproducibly cloned and maintained through numerous rounds of sub-culturing. These regions led to frequent deletions of both the vector and insert when cloned into a supercoiled plasmid. The pJAZZ vector is particularly well suited to library construction and genomic sequencing, as its lack of cloning bias greatly simplifies sequence assembly and finishing. In addition, modifications of the vector allow for simplified cloning of PCR fragments of up to 20 kb, which is useful for closing sequence and clone gaps from genomic libraries.

SummaryLinear, transcription-free pJAZZ vector allows cloning of large, AT- or GC-rich DNAs and repetitive DNA. Libraries of 10-20 kb clones of AT-rich genomic DNA were routinely created with the linear vector. Likewise, repetitive cDNAs were maintained without rearrangement.

Efficient Genomic Cloning and Sequencing. Powerful, unbiased cloning greatly reduced the need for manual finishing of genomic libraries. A 3-Mb microbial genome was assembled without the use of fosmid clones.

Rapid and simple protocol. No vector preparation or special techniques are needed to generate high quality, large-insert libraries of otherwise “unclonable” DNAs.

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A)

Cloning Repetitive DNARepetitive DNA sequences can be extremely difficult to clone in circular plasmids. For example, a library of mollusk cDNA inserts of 0.3-2 kb was unclonable in all circular plasmids tested. However, the pJAZZ linear vector produced a complex library of full length clones. Sequence traces from two of the clones show the repetitive regions (Figures A & B).

Cloning large GC-rich genomic DNAThe GC-rich genome of B. animalis (67% GC) was sheared to 6-20 kb, end-repaired, and cloned into the pJAZZ vector. Clones from this library were similar in size and number to those from the AT-rich L. helveticus library. Uncut DNA from transformants was 18-40 kb, corresponding to inserts of 4-26 kb.

97 kb

48 kb33 kb

15 kb

L. helveticus (65% AT) B. animalis (67% GC)

Replication of the pJAZZ Linear Vector

The pJAZZ vector is derived from the linear phage N15 of E. coli. The vector contains the repA and telN genes of N15, encoding replicase and pro-telomerase. Bi-directional replication is initiated by RepA and carried out by host DNA polymerase. TelN cleaves the replicated telomeres and covalently joins the free ends to each other, creating terminal hairpins (telL and telR).

Insert

Insert

Insert

Insert

Insert

Insert

telL telR

RepA

TelN

TelN

TelN

B) L. helveticus DNA in the pJAZZ vector

9.4 kb

6.6 kb

23 kb

Left Arm (12 kb)

Right Arm (2 kb)

[ran off gel]

A) AT-rich inserts are unstable in pUC19M V

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Intact clones

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Deleted clones

(GCC)220

Right arm

Left arm

pJAZZ-OC pSMART-LCKan pUC19

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0.25

The human Fragile X locus consists of over 200 copies of the triplet GCC in affected individuals. The transcription-free, linear pJAZZ vector was able to stably maintain fragments containing 220-300 copies of the GCC repeat. The fragment could be cloned in the circular, transcription-free vector pSMART-LCKan, but it was unstable upon subculturing. No full-length clones were recovered in the conventional plasmid pUC19, suggesting that transcription of this fragment interferes with cloning.

Number of contigs vs. genomic sequence coverage of Flavobacterium columnare.

Efficient Library Construction and Assembly The pJAZZ vector was used to construct genomic libraries of Flavobacterium columnare (75% AT, 3.2 Mb). Clones of 2-10 kb were readily obtained, and gave sequence reads of ~800 bp.

Automated assembly closely followed the theoretical Lander Waterman curve, yielding 21 major contigs. Further analysis revealed just 10 sequence gaps. Therefore, this AT-rich genome was completely cloned and nearly completely sequenced without the use of fosmid or BAC libraries.

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Coverage

Contigs

Minimal Size BiasSheared E. coli genomic DNA was cloned into the pJAZZ vector or pUC19 without size selection. As expected, the size distibution of the pUC19 inserts was clearly skewed toward smaller inserts. In contrast, the distribution of the pJAZZ inserts closely matched the input DNA.

DNAs were sheared to 6-20 kb, end-repaired, size-selected, and cloned into the pJAZZ vector. Plasmid DNA from transformants was cut with NotI to excise the insert. Vector bands are 12 kb and 2 kb.

Libraries have been constructed from numerous other AT-rich genomes as well, including Piromyces sp. (>85% AT) and Flavobacterium columnare (68% AT) (data not shown).

Cloning“Unclonable” AT-rich genomic DNAThe AT content of Lactobacillus helveticus is >65%. Attempts to clone 2 kb inserts into pUC19 yielded a small library, containing very few intact clones (Figure A). Such results are commonly observed when AT-rich genomes are cloned into supercoiled circular vectors. In contrast, inserts of > 20 kb were routinely cloned into the pJAZZ vector (Figure B).

pJAZZ™ Linear Vectors for High Stability Cloning

Multiple Cloning Site

lacZ Camr

Camr

pJAZZ-OC

pJAZZ-OKKanr

pJAZZ-OC

pJAZZ-OK

Insert DNA

Kanr

cBtelN repA

cBtelN repA

PAG 2008

The (GCC)220 fragment was cloned into the pJAZZ, PSMART, or pUC19 vectors. Plasmid DNA was isolated and the inserts were excized by digestion. No intact inserts were obtained in pUC19.

The pJAZZ vector is derived from the linear phage N15 of E. coli. The linear pJAZZ vectors lack the structural and lysis genes of N15 but retain the repA and telN genes, which encode replicase and protelomerase. Bi-directional replication is initiated by RepA and is carried out by the host DNA polymerase. TelN cleaves the replicated telomeres and covalently joins the 5’ and 3’ strands of each free end, re-creating the terminal hairpin loops.

Dual selection ensures that recombinant clones contain both arms of the vector. The origin of replication is used to select for the left arm vectors; antibiotic resistant selects for the right arm. The ends of the vectors are free to rotate during replication, so cloned inserts are not subject to torsional stress caused by supercoiling. Transcriptional terminators at the cloning site minimize transcriptional interference between the

insert and the vector, increasing insert stability. The pJAZZ vectors are low-copy (~5/cell) to further promote stable propagation of inserts, and their copy number can be induced 5-20X for DNA preparation. Linear plasmid DNA is electroporated into BigEasy™ TSA E. coli cells, which contain N15 genes needed for partitioning and copy number regulation. Linear plasmid DNA is efficiently isolated by standard alkaline lysis preparation.

Stable Cloning PAG 08.indd 1 12/21/2007 5:11:10 PM