Random Shear Custom BAC Libraries

NO GAPS, even in centromeres!

• Unbiased BAC libraries without gaps.
• Larger inserts, fewer empty clones.
• Generate more results, faster, for less money.

• BAC/Fosmid Libraries
  • Random Shear BAC
  • Novel (large insert) Fosmid
  • Partial Digestion BAC
• Sequencing
  • NextGen Sequencing
  • BAC end Sequencing
  • BAC Shotgun Sequencing
  • Sanger DNA Sequencing
• Other Libraries/Cloning
  • Difficult Genome
  • Large/Difficult Insert Cloning
  • NanoClone® & GapFree Shotgun
  • Metagenomic
• Library Screening & Tools
  • Pooling & Superpooling
  • Gridding & Colony Filters
  • Colony Preparation
  • Library Screening
• DNA Prep
  • BAC Miniprep / DNA Prep
  • HMW Genomic DNA Prep

Because of the very large insert sizes, constructing BAC (Bacterial Artificial Chromosome) libraries is far more challenging than making typical, small-insert plasmid libraries. Even if successful, conventional BAC libraries are inherently biased and incomplete, due to non-random methods of DNA preparation and problems with conventional cloning vectors. Lucigen has developed technologies that overcome many of these problems, producing much more even, complete, and higher quality genomic libraries.

• Download the Technical Application Note which describes Random Shear BAC Library technologies.
• True, unbiased BAC libraries. Lucigen’s exclusive Random Shear Cloning Technology and CloneSmart® transcription-free vectors eliminate biases in DNA preparation and cloning. Clone gaps and sequencing gaps are practically eliminated.
• More BAC recombinants per nanogram of input DNA. Vector preparation methods, high efficiency CopyRight® v2.0 BAC vectors, and the 10G BAC-Optimized Replicator™ v2.0 Competent Cells substantially increase recombinant yields.
• Large BAC insert size. Random Shear BAC Libraries have a guaranteed average insert size of 100 kb or larger.
• Easy production of BAC DNA. A simple, single-step copy number induction in the CopyRight v2.0 system ensures high yields of BAC DNA, resulting in a >95% success rate in BAC end sequencing.
• Dramatically reduced finishing costs. Lucigen’s custom Random Shear BAC Libraries offer the greatest value in what counts…results obtained per dollar spent.
• Custom fosmid library construction also available. Get the same unique advantages in a Lucigen Fosmid Library.
• Efficient screening. Lucigen offers colony picking, rearraying, and duplicating as well as creation of high-density, gridded colony filters, and generation of clone DNA pools and superpools to improve the efficiency of library screening.

Problems with conventional BAC libraries

Gaps due to partial restriction digestion
Standard BAC library construction methods are based on partial restriction digestion of high molecular weight (HMW) genomic DNA. Because restriction enzyme sites are not evenly distributed across a genome, the resulting libraries are substantially biased, with numerous gaps (see Figure 1 below). Generating up to 40X genomic coverage in a BAC library may not eliminate this inherent shortcoming.

Figure 1. BAC and fosmid libraries generated from genomic DNA cut with restriction enzymes are biased due to the non-random distribution of restriction sites for any given restriction enzyme. This results in over-representation, under-representation, and no representation (gaps) in the contig map. Lucigen’s Random Shearing technology avoids this bias.

Deleterious transcription from lacZ
Almost all BAC cloning vectors rely on indicator genes like lacZ for colony screening. Expression of the indicator gene often results in transcription/translation of the insert, which can lead to partial deletions, sequence rearrangements, or complete loss of inserts, particularly if the clone expresses products that are deleterious to the host cell.

Lucigen's solutions to problems with conventional BAC libraries:

Gaps eliminated by Random Shearing
Gaps due to restriction enzyme bias are eliminated by novel methods to randomly shear HMW genomic DNA into very large fragments up to ~250 kb (Figure 2, below). This Random Shear technology is coupled with Lucigen's DNATerminator® End Repair reagents to prepare inserts for efficient ligation.

Figure 2. Mouse genomic DNA was digested to completion by several restriction enzymes (Left panel) or fragmented by random shearing (Right panel). Lanes: 1, Uncut DNA; 2, Sau3A; 3, BamHI; 4, HindIII; 5, EcoRI. Only Sau3A digested the band at ~1 Mb. In contrast, all of the DNA was reduced to 100-400 kb as the degree of random shearing was increased. The arrow indicates undigestable megabase DNA.

Stable inserts in a transcription-free vector:
The absence of lacZ sequence and the presence of transcription terminators in the CopyRight® v2.0 BAC cloning vector (Figure 3) eliminates spurious transcription/translation of inserts and results in high insert stability. The lacZ-sacB stuffer region allows uncut vector to be detected by blue/white screening and selected against by plating on sucrose which completely eliminates uncut vector background and improves the efficiency of colony picking.

Figure 3. Diagram of CopyRight vector pSMART BAC. ori2, repE, IncC - origin of replication (single copy); oriV - inducible origin of replication; par A,B,C- partition genes; Cmr- chloramphenicol-resistance gene; cosN - lambda packaging signal; T – CloneSmart transcription terminators; sacB, sucrase gene; lacZ, alpha peptide portion of the beta galactosidase gene. Approximate positions of sequencing primers and transcription terminators are indicated.

Figure 4. Genomic DNA was isolated from potato tissue, randomly sheared, size-selected to >100 kb, and cloned into the pSMART® BAC vector. DNA from minipreps was digested with NotI to excise inserts. The vector band is visible at 7 kb.

The combination of Random Shear technology coupled with Lucigen's DNATerminator® End Repair reagents, high stability CopyRight® v2.0 BAC cloning vectors, and E. cloni® 10G BAC-Optimized ReplicatorT v2.0 Competent Cells results in truly unbiased BAC genomic libraries with consistently large inserts (Figure 4).

The power of the Random Shear BAC technology is illustrated in Figure 5, where clone coverage was uniform across all the probed regions, including the centromeric region, in the Random Shear Library (Figure 5, red horizontal bars). In contrast, these regions show vastly different representation (Figure 5, black vertical bars) in the Arabidopsis genome project (=1, 15, 75, or =1 clone per 0.1 Mb for each region probed, blue lines from left to right; 17X coverage overall).

Most importantly, the Random Shear BAC Library closed several existing centromeric gaps of this "finished" physical and sequence genomic map.

Figure 5. Closing gaps with a Random Shear Library. An Arabidopsis Random Shear BAC Library provides even coverage throughout the genome, including centromeric regions that were gaps in the “finished” sequence from partial-digestion libraries.

With these novel techniques and tools, Lucigen is the only source of custom BAC libraries that are truly representative of the input genomic DNA, without gaps or missing sequences. As a result, finishing costs (library screening, positional gene cloning, physical mapping, sequencing, etc.) are dramatically reduced, genomic analyses are completed faster, and critical genes are obtained with much less work and expense (Table 1, below).

Table 1. Comparison of finishing costs for an average BAC library

** Finishing cost includes BAC end sequencing, whole genome physical mapping, and integrating the physical map with about 1000 genetic markers.

Contact Lucigen for a free quote on any of our custom services.