Getting to Megabase

Large Fragment Cloning Products and Services Gone are the days when all you had to do to get out of grad school was identify and clone a new gene. Besides, with the information rapidly gathered through the Human Genome Project, there will soon no longer be a "new gene." Enormous projects such as sequencing entire genomes have created a need to play with bigger pieces of the puzzle, and a new universe of technologies adapted to large DNA fragments has appeared. Assignment of a new gene to a particular chromosome is an established sport in elite journals. This has become possible through our increasing ability to manipulate entire chromosomes. We can now isolate and sort chromosomes, fractionate fragments, and clone and sequence these large pieces of genetic material. Clones obtained from microdissected DNA can be used to develop "contig" maps, a series of clones representing overlapping genomic regions. First, to characterize disease-related chromosomal loci, one creates a library restricted to DNA sequences from that region. Landmarks are then established at 100,000 base pair (bp) intervals using molecular tags of 200-500 bp (sequence-tagged sites, or STSs). When clones within one megabase of the disease locus are isolated, chromosome walking or jumping technology can lead to the gene. Custom services provided by Genome Systems Inc. Another use for large DNA fragments is in the functional characterization of gene products. Transfecting cells with large, continuous DNA stretches from microdissected chromosome fragments allows a gene to be studied in the context of native promoters, enhancers, or introns that can affect expression and regulation. Finally, the creation of ordered libraries of clones containing large DNA inserts has provided the starting material for large-scale sequencing projects. YACs The ability to clone DNA fragments >100 kb is crucial to physical mapping, positional cloning, and many types of molecular analysis of complex eukaryotic genomes. Conventional cosmid vectors hold 40-50 kb of DNA and are not usually sufficient for analysis of very large genomic regions. Scientists have developed chromosome-like vectors that can accommodate longer DNA inserts.1 These artificial chromosome (AC) vectors first consisted of sequences from Saccharomyces cerevisiae, common baker's yeast. Circular yeast plasmids replicate through cis-acting elements known as autonomously replicating sequences (ARSs). Addition of a centromere (CEN) and a telomere (TEL) sequence to an ARS results in a plasmid that acts like a normal yeast chromosome. These constructs are known as yeast artificial chromosomes, or YACs. In principle, YACs can incorporate inserts as large as 1 Mb. Partially overlapping YACs can be subjected to homologous recombination to obtain larger YAC clones. Contig maps created with YACs can span 4 Mb or more and are better suited for large fragment library construction than cosmids. For instance, 500 YACs could cover 150 Mb of a human chromosome; 3,500 cosmids would be needed for the same task. For individual cloning or library construction, YAC vectors such as pYAC4 are available from Sigma. If you don't want to construct your own YAC library, commercial libraries as well as library construction services are available. Research Genetics Inc. has a very complete collection of YAC libraries from mice (WI, WI/MIT-820, and Princeton), rat (WI or Harvard/EC/HGF), Zebrafish, and human (CEPH A and B, Washington University, or Y chromosome). Genome Systems offers YAC libraries from Zebrafish and mice. However, YAC technology has some nagging problems intrinsic to the use of yeast. Spontaneous deletions may occur in YACs, owing to the instability of long, repetitive elements in yeast. Also, coligation of multiple, noncontiguous DNA fragments is common. The prevalence of chimeric and deleted inserts in YAC libraries (40-50 percent of clones) complicates chromosome walking experiments. Another more pedestrian problem with YACs is that it can be difficult to separate YAC DNA from yeast chromosomal DNA. Luckily, many kits are now available to simplify isolation of YAC DNA. Many kits used for the isolation of genomic DNA have been adapted for work with YACs. Kits specifically designed for YAC isolation typically eliminate the use of enzymes such as proteinase K, the addition of phenol/chloroform, and shearing forces that can break fragile YAC DNA. Isolation of DNA from YAC clones can be carried out with CPG Inc.'s nonenzymatic, phenol-free DNA-Pure™ Yeast Genomic Kit. DNA-Pure kits extract 10-20 µg of DNA from 1.5-3 ml of culture. Clontech's NucleoBond® Plasmid Kits can also be used to isolate YAC DNA with high efficiency. BAC/PACs Since YACs are unstable and can be difficult to purify, other artificial chromosome systems have been developed. Bacterial artificial chromosomes (BACs) are based on the Escherichia coli fertility plasmid or F factor, and bacteriophage P1-derived artificial chromosomes (PACs) combine P1 and F-factor features. BACs allow cloning of DNA fragments up to 350 kb (average insert ~120 kb) while PAC inserts are typically ~150 kb. BAC/PACs are replacing YACs for the construction of contig and physical maps, since it is easier to insert DNA into these vectors than into YAC vectors. BAC libraries also typically contain fewer chimeric inserts. As a result of these advantages, BAC/PAC systems are typically used for long-range physical mapping, positional cloning, and whole genome sequencing projects. A number of BAC cloning vectors are available. Addition of the lacZ gene to the original pBAC108L vector has created pBeloBAC11 and its derivative pECBAC1. EcoR I fragments are easily cloned into pECBAC1, and pBeloBAC11 accepts Hind III-BamH I fragments. The second generation of pBeloBAC11 vectors includes pEBAC1 and pEBAC140. The Roswell Park Cancer Institute (RPCI) offers BAC vector pBACe3.6 on a cost-recovery basis (bacpac.med.buffalo.edu). Typical PAC vectors such as pCYPAC2 and pPAC4 are also available from RPCI. Genome Systems offers several services and supplies for the preparation and analysis of P1 (pAd10SacBII vector), PAC (pCYPAC2 vector), or BAC (pBeloBAC11 vector) clones. Typical BAC vector available through the Roswell Park Cancer Institute Commercially available BAC/PAC products include classic mouse, human, and rat libraries, and high-density filter kits for library screening. Genome Systems offers a large collection of BAC/PAC/P1 products including mammalian, insect, plant, and microbial libraries and screening/sequencing services. Research Genetics has mammalian BAC libraries (mouse and human), plant BAC libraries (Arabidopsis, tomato), a Trypanosoma brucei library, plus various mammalian PAC libraries (pPAC4 vector). RPCI has an extensive collection of libraries including mammalian BAC libraries (male and female human, chimpanzee, baboon, porcine, canine), and those made from lower organisms such as Drosophila melanogaster or T. brucei. They also have female human and mouse PAC libraries. These libraries are distributed in 384-well microtiter dishes or 22 x 22 cm nylon high-density filters for hybridization screening. The Texas A&M BAC Center (hbz.tamu.edu) has over 40 BAC libraries available for screening, and a variety of tools and protocols to help in the process. The drawbacks of using BAC/PAC vectors are that they are present in only one or two copies per cell, complicating isolation and screening. Large culture volumes are required to obtain sufficient quantities of BAC DNA for analysis, leading to a high degree of RNA contamination. However, several columns and kits have been specifically tailored for BAC/PAC/P1 isolation. Genome Systems offers Magnum columns for P1/PAC (KB-100 for 100 ml volume) and BAC (KB-500 for 500 ml culture) isolation. Princeton Separations Inc. has the PSI * CLONE BAC Kit, specifically developed for the isolation of high-quality BAC DNA. Yields from 3-5 ml of culture are typically sufficient for at least one sequencing reaction. Life Technologies Inc. has also developed BAC-specific protocols for its Concert High Purity Plasmid Purification Systems. These gravity-flow, anion-exchange columns isolate 0.3-1 µg DNA per ml of culture and come in mini- and maxi-prep scales. Product purity is touted as equivalent to that obtained by double banding on CsCl gradients. In addition to these systems, there is a new QIAGEN Large-Construct Kit to isolate BAC/PAC/P1 DNA from up to 500 ml culture without contamination with genomic DNA. This kit uses a gravity-flow, anion-exchange column and an ATP-dependent exonuclease to break down genomic DNA. Clontech's NucleoBond Plasmid Kits can also be used to isolate BAC/PAC/P1 DNA in the 20-10,000 µg range (Mini to Giga scales); they are based on an anion-exchange column system (NucleoBond AX resin). If you are faced with a large number of BAC isolations to perform, QIAGEN's R.E.A.L.™ Prep 96 Plasmid Kits should provide some relief. R.E.A.L. Prep 96 Kits can either be used with the QIAvac manifold or automated with the BioRobot™ 9600 multipurpose workstation. Services provided by companies such as Ana-Gen Technologies Inc. and Commonwealth Biotechnologies Inc. include BAC/PAC DNA isolation and sequencing. Genome Systems also offers custom services for BAC/PAC/P1 DNA preparation. The yield is 10 µg DNA per BAC clone or 20-40 µg DNA per P1/PAC clone, confirmed either by PCR or hybridization. They can also shotgun sequence an entire BAC/PAC/P1 clone. Research Genetics offers BAC/PAC preparation with a yield of 10 µg DNA (PCR- or hybridization-verified) and first pass sequencing of P1/PAC/BAC clones. There are even automatic instruments from AutoGen such as AutoGen 850 (personal model) and AutoGen 740 (fully automatic), which can process BACs, PACs (AutoGen 850), and YACs (AutoGen 850 Alpha). Samples are processed by gentle alkaline lysis in four to six hours with a yield similar to the kits. Living Large Probably the most important future application of artificial chromosomes will lie in mammalian artificial chromosomes (MACs) and human minichromosomes. MACs can be constructed using YACs provided with human centromeres and mammalian selectable markers (Neomycin or Blasticidin resistance genes). Cellular multimerization events between YACs lead to huge MACs with 1-5 Mb inserts. MACs replicate and segregate as natural chromosomes. Larger human ACs (minichromosomes) can be constructed by introducing human centromeres (repetitive alpha satellite DNA in BACs) into human cells, together with telomeric and genomic DNA. This creates mitotically stable minichromosomes (6-10 Mb), which have been touted as the next generation of gene therapy vectors. Transformation-associated recombination (TAR) cloning in yeast is a powerful method of cloning genes from total human DNA. A linear TAR cloning vector is basically a YAC vector flanked by a small amount of sequence from the target gene on one end and a human repetitive Alu sequence on the other end. When this linear TAR cloning vector is transformed into yeast along with human genomic DNA, recombination results in generation of YAC clones containing the human gene insert. TAR vectors also contain F-factor sequences for transfer to E. coli as BACs. Powerful yeast genetics, our ability to manipulate bacterial genes, and the growing knowledge of human genetics have created a small universe of YACs, PACs, BACs, MACs, and a growing number of satellite molecular techniques. The use of these chromosome-like constructs for complex gene sequencing, gene product characterization, and eventually gene therapeutics, will keep many people busy for years to come. And upon entering this era of post-Genome Project biology, one of them may likely be you. Jorge D. Cortese can be contacted at jorcortese@aol.com. 1. A.W. Murray, J.W. Szostak, "Construction of artificial chromosomes in yeast," Nature, 305:189-93, 1983.

Getting to Megabase

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