Bacterial Artificial Chromosome: Structure, Cloning, and Applications

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to Bacterial Artificial Chromosomes

What is a BAC?

A bacterial artificial chromosome (BAC) is a cloning vector derived from the naturally occurring F (fertility) plasmid of Escherichia coli. BACs are designed to carry large inserts of foreign DNA—typically 100 to 300 kilobases (kb)—making them indispensable tools for genomic research. The F plasmid itself is an episome that exists extrachromosomally in E. coli and is maintained at one or two copies per cell. This stringent copy number control is the defining feature that BACs inherit, and it is precisely what allows them to stably maintain large DNA fragments without the recombination and rearrangement problems that plague high-copy-number vectors.

The development of BACs in the early 1990s by Melvin Simon and colleagues addressed a critical gap in molecular cloning: the need for a vector that could handle fragments larger than what conventional plasmids (up to ~10 kb) or cosmids (up to ~45 kb) could accommodate, while avoiding the instability issues associated with yeast artificial chromosomes (YACs). Today, BACs are central to genome sequencing projects, physical mapping, and the construction of transgenic animals. Understanding their structure and mechanism is essential for any student of molecular biology.

BAC vs. Other Cloning Vectors

To appreciate BACs, one must first understand the landscape of cloning vectors. Standard plasmids are circular, double-stranded DNA molecules that replicate independently of the bacterial chromosome. They typically carry inserts of less than 10 kb because larger fragments become unstable and prone to deletion during replication. Cosmids are plasmids that incorporate the bacteriophage lambda cos site, allowing packaging into phage particles; they can carry inserts up to ~45 kb. YACs, which are linear vectors propagated in yeast, can theoretically carry megabase-sized inserts but suffer from high rates of chimerism (the joining of DNA fragments from different genomic regions) and rearrangement.

BACs occupy a middle ground: they accept inserts of 100–300 kb, are maintained in a well-characterized bacterial host, and exhibit far greater structural stability than YACs. Because BACs replicate in E. coli rather than yeast, they are also easier to manipulate using standard bacterial techniques. This combination of high capacity, stability, and ease of handling has made BACs the vector of choice for large-scale genomic projects.

Structure and Key Components of BAC Vectors

A BAC vector is a circular DNA molecule, typically 7–12 kb in size, that contains several essential functional elements derived from the F plasmid and standard cloning vectors. The most widely used BAC vector is pBAC108L and its derivatives, such as pBeloBAC11. All BAC vectors share a core set of components that govern replication, copy number, partitioning, and selection.

Origin of Replication and Copy Number Control

The replication of BACs is controlled by the oriS (origin of replication) and repE genes, both derived from the F plasmid. The repE gene encodes a replication initiator protein that binds to the oriS region to initiate DNA synthesis. This system is tightly regulated such that replication occurs only once per cell division cycle, maintaining the BAC at one or two copies per cell.

This low copy number is not a limitation but a feature. High-copy-number plasmids (e.g., pUC derivatives with 500–700 copies per cell) are prone to recombination between repeated sequences within large inserts, leading to deletions and rearrangements. By keeping the copy number low, BACs minimize the opportunity for such recombination events. Additionally, the repE–oriS system ensures that replication is synchronized with the bacterial cell cycle, preventing the accumulation of replication intermediates that could promote instability.

Partition Genes for Stable Inheritance

The parA and parB genes are essential for the faithful segregation of BAC molecules to daughter cells during cell division. These genes encode proteins that function analogously to the mitotic machinery of eukaryotes. ParA is an ATPase that forms a filamentous structure, while ParB binds to specific DNA sequences (the parS site) on the BAC. Together, they actively position the BAC molecules at the mid-cell and ensure that each daughter cell receives at least one copy after division.

Without functional parA and parB, BACs would be lost at a high frequency because random diffusion alone cannot guarantee that both daughter cells receive a copy when the plasmid is present at only one or two copies per cell. This active partitioning system is a major reason why BACs are so stably maintained over many generations, a property that is critical for constructing and propagating large genomic libraries.

Selectable Markers and Multiple Cloning Site

BAC vectors carry a selectable marker, most commonly the chloramphenicol acetyltransferase (cat) gene, which confers resistance to chloramphenicol. This antibiotic is used at a concentration of 12.5–25 µg/mL in culture media to select for bacteria that harbor the BAC. Chloramphenicol resistance is preferred over ampicillin or kanamycin resistance because the low copy number of BACs means that only low levels of the resistance enzyme are produced; chloramphenicol acetyltransferase is highly efficient and works well at these low concentrations.

The multiple cloning site (MCS) is a short DNA sequence containing several unique restriction enzyme recognition sites. In pBeloBAC11, the MCS is flanked by the lacZ gene, which encodes β-galactosidase. This allows for blue-white screening: when a DNA insert is successfully ligated into the MCS, the lacZ gene is disrupted, and colonies appear white on media containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside). Colonies with no insert retain a functional lacZ gene and appear blue. The MCS also contains recognition sites for rare-cutting restriction enzymes such as NotI, which are used to excise the entire insert from the vector for analysis.

How BAC Cloning Works: Step-by-Step Mechanism

BAC cloning is a multi-step procedure that requires careful preparation of both vector and insert DNA, followed by ligation, transformation, and screening. The entire process is summarized below.

Preparation of Vector and Insert DNA

1. Vector linearization: The BAC vector is digested with a restriction enzyme that cuts within the MCS. For pBeloBAC11, this is typically BamHI or HindIII. The linearized vector is then treated with calf intestinal alkaline phosphatase (CIP) or shrimp alkaline phosphatase (SAP) to remove 5′ phosphate groups. This prevents self-ligation of the vector and reduces the background of empty clones.

1. Insert DNA preparation: High-molecular-weight genomic DNA is isolated from the organism of interest. The DNA is then partially digested with a restriction enzyme that recognizes a 4-base-pair (bp) sequence, such as Sau3AI (recognizes GATC) or MboI. Partial digestion is used because a complete digestion would produce fragments too small for BAC cloning. The goal is to generate fragments in the 100–300 kb range. The reaction conditions (enzyme concentration and incubation time) are optimized empirically, typically using a titration series.

1. Size selection: The partially digested DNA is subjected to pulsed-field gel electrophoresis (PFGE). PFGE is essential because conventional agarose gel electrophoresis cannot resolve DNA molecules larger than ~50 kb. In PFGE, the electric field alternates between two directions, forcing large DNA molecules to reorient and migrate through the gel in a size-dependent manner. After electrophoresis, the gel region containing DNA of the desired size (e.g., 150–250 kb) is excised, and the DNA is recovered by electroelution or using a gel extraction kit designed for large fragments.

Ligation and Transformation into E. coli

1. Ligation: The linearized, dephosphorylated vector and the size-selected insert DNA are mixed at a molar ratio of approximately 1:5 to 1:10 (vector:insert). The ligation reaction is catalyzed by T4 DNA ligase in a buffer containing ATP and dithiothreitol (DTT). The reaction is incubated at 16°C for 12–16 hours. The low temperature and long incubation time favor the ligation of large DNA fragments, which have lower diffusion coefficients and require more time to encounter each other.

1. Transformation: The ligation products are introduced into competent E. coli cells by electroporation. Electroporation is used rather than chemical transformation because it is far more efficient for large DNA molecules. The cells (typically strain DH10B, which is recA1 and endA1 deficient to reduce recombination and nuclease activity) are mixed with the ligation reaction and subjected to a high-voltage electric pulse (typically 1.8 kV, 25 µF, 200 Ω in a 0.1 cm cuvette). This transiently permeabilizes the cell membrane, allowing the large BAC molecules to enter.

1. Recovery and plating: After electroporation, the cells are allowed to recover in SOC medium (super optimal broth with catabolite repression) at 37°C with shaking for 1 hour. The cells are then plated on LB agar containing chloramphenicol (12.5 µg/mL), X-gal, and IPTG. Plates are incubated at 37°C for 24 hours.

Selection and Screening of Recombinant Clones

After overnight incubation, colonies are visible on the plates. White colonies (indicating disruption of lacZ) are candidate recombinant clones. However, not all white colonies necessarily contain the correct insert; some may contain vector that has ligated to itself or to small contaminating fragments. Therefore, further screening is required:

1. Colony PCR: A small amount of each white colony is used as a template for PCR with primers that flank the MCS. This confirms the presence of an insert and provides a rough estimate of its size.

1. Restriction fingerprinting: BAC DNA is isolated from positive clones using a plasmid miniprep procedure adapted for large plasmids (see Plasmid Bacterial Miniprep). The DNA is digested with a restriction enzyme such as HindIII or NotI, and the resulting fragments are analyzed by PFGE. This confirms the insert size and reveals whether any rearrangements have occurred.

1. End sequencing: The ends of the insert are sequenced using primers that anneal to the vector sequences flanking the MCS. This provides sequence tags that can be used to map the clone to a specific genomic location.

Advantages and Limitations of BACs

High Capacity and Stability

The primary advantage of BACs is their ability to maintain large DNA fragments (100–300 kb) with high fidelity. This is achieved through the combination of low copy number, active partitioning, and the use of recombination-deficient E. coli host strains. BACs are remarkably stable: inserts are maintained without detectable rearrangement even after 100 or more generations of growth. This stability is essential for constructing genomic libraries that accurately represent the genome of the source organism.

BACs also have a low rate of chimerism compared to YACs. Chimerism occurs when two non-contiguous genomic fragments are ligated into a single clone, creating an artificial sequence that does not exist in the original genome. The rate of chimerism in BAC libraries is typically less than 5%, compared to 10–50% in YAC libraries. This makes BACs far more reliable for genome assembly and physical mapping.

Low Copy Number and Chimerism Issues

The low copy number of BACs is also their main limitation. Because each cell contains only one or two copies of the BAC, the yield of BAC DNA from a standard miniprep is very low—typically 0.5–2 µg from a 5 mL culture. This is insufficient for many downstream applications, such as DNA sequencing or transfection of mammalian cells. To obtain larger quantities of DNA, BAC-containing cells must be grown in larger volumes (100–500 mL), or the copy number must be induced.

Some BAC vectors include an inducible high-copy origin, such as the oriV from the RK2 plasmid, which can be activated by adding arabinose to the culture medium. However, inducing high copy number can increase the risk of recombination, so this approach must be used with caution.

Chimerism, while low, is not zero. Chimeric clones can arise from the co-ligation of two or more genomic fragments, particularly if the size-selection step is not stringent. Screening for chimerism requires comparing the restriction fingerprint of each clone to the predicted pattern from the genome sequence, which is not always feasible.

BAC Libraries and Their Construction

A BAC library is a collection of BAC clones that together represent the entire genome of an organism. The number of clones required depends on the genome size and the average insert size. For example, the human genome (~3.2 × 10⁹ bp) would require approximately 20,000 clones with an average insert size of 150 kb to achieve 10-fold coverage (10× genome equivalents). BAC libraries are essential resources for genome sequencing, positional cloning, and comparative genomics.

Genomic DNA Preparation and Partial Digestion

The first step in constructing a BAC library is the isolation of high-molecular-weight genomic DNA. This is typically done by embedding cells or nuclei in low-melting-point agarose plugs. The agarose matrix protects the large DNA molecules from mechanical shearing during subsequent manipulations. Cells are lysed within the plugs, and proteins are digested with proteinase K in the presence of EDTA to inactivate nucleases.

The agarose-embedded DNA is then partially digested with a restriction enzyme. Sau3AI is commonly used because it recognizes a 4-bp site (GATC) and produces fragments with cohesive ends compatible with BamHI-digested vectors. The partial digestion is performed by incubating the plugs with a limiting amount of enzyme for a short time (e.g., 15–30 minutes at 37°C). The reaction is stopped by adding EDTA and cooling on ice.

Size Selection and Cloning

The partially digested DNA is subjected to PFGE to separate fragments by size. The region of the gel containing DNA of 150–300 kb is excised, and the DNA is recovered by electroelution. This size-selected DNA is then ligated into the prepared BAC vector as described above.

The ligation mixture is transformed into E. coli by electroporation, and the cells are plated on selective media. Each colony represents a single BAC clone. The transformation efficiency for BAC ligations is typically 1,000–10,000 colonies per microgram of insert DNA, which is sufficient to generate a genome-wide library.

Arraying and Storage

Individual colonies are picked and arrayed into 384-well microtiter plates containing LB broth with chloramphenicol and glycerol (for cryopreservation). Each well contains a single BAC clone, and the plates are stored at −80°C. This arrayed format allows for efficient screening and retrieval of specific clones. Replica plates can be made for distribution to other laboratories.

The library is typically characterized by determining the insert size of a random sample of clones (e.g., 100–200 clones) using PFGE. This provides an estimate of the average insert size and the genome coverage of the library. The clones are also end-sequenced to generate sequence-tagged connectors that link the BACs to the genome sequence.

Applications of BACs in Genomics and Biotechnology

Genome Sequencing Projects

BACs were the workhorses of the Human Genome Project and remain important for sequencing complex genomes. In the hierarchical shotgun sequencing strategy, BAC clones are first mapped to specific chromosomal locations, and then each BAC is sequenced individually using the shotgun method. This approach reduces the complexity of genome assembly because each BAC provides a contiguous sequence of 100–200 kb that can be assembled independently before being ordered along the chromosome.

BACs are also used in next-generation sequencing projects to validate and finish genome assemblies. The long-range information provided by BAC end sequences helps to scaffold contigs and resolve repetitive regions. For organisms with highly repetitive genomes, such as plants, BAC-based approaches are often essential for obtaining a complete genome sequence. Recent advances in long-read sequencing technologies, such as those from Pacific Biosciences, have reduced the reliance on BACs for de novo assembly (see PacBio Only Bacterial Sequencing), but BACs remain valuable for closing gaps and resolving complex regions.

BAC-Based Transgenic Animals

BACs are widely used to generate transgenic animals, particularly mice. Because BACs contain large genomic fragments, they often include not only the gene of interest but also its native regulatory elements (promoters, enhancers, insulators) that are located far upstream or downstream of the coding sequence. This ensures that the transgene is expressed in a manner that closely mimics the endogenous gene.

To generate a BAC transgenic mouse, the BAC is first modified in E. coli using recombineering (homologous recombination mediated by the λ Red proteins). This allows for precise introduction of mutations, insertion of reporter genes (e.g., GFP), or deletion of specific exons. The modified BAC is then linearized and microinjected into the pronucleus of a fertilized mouse egg. The eggs are implanted into surrogate mothers, and the resulting offspring are screened for the presence of the transgene.

BAC transgenesis is particularly useful for studying genes that are regulated by distant enhancers, for expressing genes at physiological levels, and for creating disease models. For example, BACs carrying the human APP gene (amyloid precursor protein) with familial Alzheimer's disease mutations have been used to create mouse models of Alzheimer's disease.

Studying Gene Regulation

BACs are invaluable for studying gene regulation in its native chromosomal context. By introducing a BAC containing a gene of interest with a reporter tag (e.g., GFP or luciferase) into cells or animals, researchers can visualize when and where the gene is expressed. Because the BAC contains all the regulatory elements, the expression pattern is likely to be accurate.

BACs are also used to study chromosome structure and function. For example, BACs spanning the X-inactivation center have been used to investigate the mechanisms of X Chromosome Inactivation. Similarly, BACs containing telomeric sequences have been used to study Telomere Chromosome maintenance. In addition, BAC-based fluorescence in situ hybridization (FISH) probes are used to visualize specific chromosomal regions and to detect Chromosome Abnormality in clinical diagnostics.

BACs vs. Other Cloning Systems: YACs, Cosmids, and Fosmids

The choice of cloning system depends on the size of the DNA fragment to be cloned and the intended application. The table below summarizes the key differences between BACs and other major cloning vectors.

Insert Size and Stability

YACs can accommodate the largest inserts, up to 1 megabase (Mb) or more, which makes them attractive for cloning entire gene clusters or large genomic regions. However, YACs are notoriously unstable. The yeast host can rearrange or delete portions of the insert, and the high rate of chimerism makes it difficult to assemble contiguous sequences. YACs also have a high rate of coligation, where two or more unrelated genomic fragments are joined into a single YAC.

BACs, by contrast, are far more stable. The low copy number and the absence of homologous recombination in recA mutant E. coli strains minimize rearrangements. The chimerism rate is typically below 5%, making BACs the preferred choice for genome sequencing projects where accuracy is paramount.

Cosmids and fosmids are limited to inserts of ~40 kb, which is sufficient for many applications, such as cloning individual genes or small gene clusters. Fosmids are essentially BACs that contain the λ cos site, allowing them to be packaged into phage particles for efficient transduction. Fosmids have the same low copy number and stability as BACs but are easier to handle because of their smaller insert size.

Handling and Manipulation

BACs are manipulated in E. coli, which is the best-characterized organism in molecular biology. A vast array of genetic tools, mutant strains, and protocols are available. Recombineering allows for precise modification of BACs without the need for restriction enzymes or ligases. In contrast, YACs require growth in yeast, which is slower and more cumbersome. The large size of YAC DNA also makes it difficult to isolate and manipulate in vitro.

Cosmids and fosmids are the easiest to handle because their small size allows them to be purified using standard plasmid miniprep kits. However, their limited insert size means that many clones are needed to cover a genome, and long-range regulatory elements may not be included in a single clone.

Common Pitfalls and Troubleshooting in BAC Cloning

BAC cloning is technically demanding, and several failure modes are commonly encountered. Understanding these pitfalls and their solutions is essential for successful experiments.

Low Yield of Recombinant Clones

Symptom: Few or no colonies after transformation, or a high proportion of blue (empty) colonies.

Causes and solutions:

• Inefficient ligation: Large DNA fragments ligate poorly. Ensure that the vector:insert molar ratio is optimal (1:5 to 1:10) and that the ligation is incubated at 16°C for at least 12 hours. Use high-concentration T4 DNA ligase (e.g., 1–2 U/µL) and include PEG 4000 (5% w/v) in the reaction to promote macromolecular crowding.
• Poor transformation efficiency: Electroporation efficiency decreases with increasing DNA size. Use freshly prepared electrocompetent cells with an efficiency of at least 10¹⁰ CFU/µg for a small plasmid. Optimize the electroporation parameters (1.8 kV, 25 µF, 200 Ω) and ensure that the ligation mixture is desalted before electroporation, as salt ions cause arcing.
• Incomplete dephosphorylation of the vector: If the vector is not fully dephosphorylated, it will self-ligate, producing blue colonies. Increase the amount of phosphatase or extend the incubation time. Verify dephosphorylation by testing a control ligation with vector alone (should produce very few colonies).

Insert Instability or Rearrangement

Symptom: BAC clones show deletions, insertions, or rearrangements upon restriction analysis, or the insert size decreases after prolonged growth.

Causes and solutions:

• Recombination in the host: Use E. coli strains that are recA1 (deficient in homologous recombination) and endA1 (deficient in nonspecific endonuclease I). DH10B is the standard strain for BAC cloning.
• Toxic or repetitive insert sequences: Some genomic regions are inherently unstable in E. coli, particularly those containing long repeats or AT-rich sequences. If instability is observed, grow the cells at 30°C instead of 37°C to slow replication, and avoid growing cultures for more than 16 hours.
• High copy number induction: If using an inducible copy number system, avoid inducing high copy number unless absolutely necessary. When induction is required, minimize the induction time and immediately purify the DNA.

Contamination with Genomic DNA

Symptom: BAC DNA preparations contain contaminating E. coli chromosomal DNA, which interferes with downstream applications such as sequencing or transfection.

Causes and solutions:

• Incomplete lysis: Ensure that the cells are fully resuspended before lysis and that the lysis buffer is freshly prepared. Use the recommended volumes of buffers P1, P2, and P3 (from the alkaline lysis protocol) and do not vortex after adding P2.
• Insufficient centrifugation: After neutralization, centrifuge at high speed (≥15,000 × g) for at least 15 minutes at 4°C to pellet the genomic DNA and cell debris completely. If the supernatant is turbid, centrifuge again.
• Carryover from the gel: When isolating insert DNA from PFGE, ensure that the agarose plug is completely melted and that the DNA is thoroughly purified to remove agarose contaminants, which can inhibit ligation and transformation.

Summary and Key Takeaways

BACs are versatile cloning vectors derived from the F plasmid of E. coli. Their unique combination of large insert capacity, high stability, and ease of manipulation has made them indispensable in genomics and biotechnology. The key features of BACs—the oriS/repE replication system, the parA/parB partition genes, and the chloramphenicol resistance marker—work together to maintain large DNA fragments at low copy number with high fidelity.

BAC cloning involves the preparation of high-molecular-weight insert DNA, ligation into a linearized vector, transformation into E. coli by electroporation, and screening of recombinant clones. BAC libraries are constructed by arraying individual clones into microtiter plates, providing a renewable resource for genome-wide studies.

BACs are used in genome sequencing, the generation of transgenic animals, and the study of gene regulation. They offer significant advantages over YACs, cosmids, and fosmids in terms of insert size, stability, and ease of handling. While BAC cloning is technically demanding, understanding the common pitfalls and their solutions can greatly improve the success rate.

Frequently Asked Questions

What is a bacterial artificial chromosome (BAC)?

A bacterial artificial chromosome (BAC) is a circular DNA vector derived from the F plasmid of Escherichia coli. It is designed to carry large inserts of foreign DNA, typically 100–300 kb, and is maintained at one or two copies per cell. BACs are used to clone and manipulate large genomic fragments for applications such as genome sequencing, physical mapping, and transgenesis.

How does a BAC differ from a standard plasmid?

Standard plasmids are high-copy-number vectors that carry inserts of less than 10 kb. They replicate using a relaxed origin of replication and are present in hundreds of copies per cell. BACs, in contrast, are low-copy-number vectors (1–2 copies per cell) that use the F-plasmid oriS/repE replication system. The low copy number and the presence of partition genes (parA/parB) allow BACs to stably maintain much larger inserts (100–300 kb) without rearrangement.

What is the maximum insert size for a BAC?

The practical maximum insert size for a BAC is approximately 300 kb. While larger inserts have been reported, the efficiency of ligation and transformation decreases significantly with increasing insert size. The theoretical limit is set by the packaging capacity of the bacterial cell and the stability of the DNA molecule during replication.

Why are BACs used in genome sequencing?

BACs are used in genome sequencing because they provide long, contiguous DNA fragments that are essential for assembling large genomes. In the hierarchical shotgun sequencing strategy, BAC clones are mapped to specific chromosomal locations and then sequenced individually. This reduces the complexity of assembly and allows for the accurate ordering of sequence contigs. BACs are also stable and have a low rate of chimerism, making them reliable for constructing physical maps.

What are the main components of a BAC vector?

The main components of a BAC vector are: (1) the oriS origin of replication and the repE gene, which control replication; (2) the parA and parB genes, which ensure faithful segregation to daughter cells; (3) a selectable marker, typically the chloramphenicol acetyltransferase (cat) gene; and (4) a multiple cloning site (MCS) flanked by the lacZ gene for blue-white screening.

How are BAC libraries constructed?

BAC libraries are constructed by isolating high-molecular-weight genomic DNA, partially digesting it with a restriction enzyme such as Sau3AI, and size-selecting fragments of 100–300 kb by pulsed-field gel electrophoresis. The size-selected DNA is ligated into a linearized BAC vector, transformed into E. coli by electroporation, and plated on selective media. Individual colonies are picked and arrayed into 384-well plates for storage at −80°C.

What are the advantages of BACs over YACs?

BACs offer several advantages over yeast artificial chromosomes (YACs): (1) BACs are more stable, with a lower rate of insert rearrangement and deletion; (2) BACs have a much lower rate of chimerism (<5% vs. 10–50% for YACs); (3) BACs are manipulated in E. coli, which is easier and faster to work with than yeast; and (4) BAC DNA is easier to isolate and purify. The main disadvantage of BACs is their smaller maximum insert size (300 kb vs. 1 Mb for YACs).

Key Takeaways

• BACs are cloning vectors derived from the F plasmid of E. coli that carry inserts of 100–300 kb at low copy number.
• The essential components of a BAC vector are oriS, repE, parA, parB, a chloramphenicol resistance gene, and a multiple cloning site.
• BAC cloning involves partial digestion of genomic DNA, size selection by PFGE, ligation into a dephosphorylated vector, and electroporation into recA-deficient E. coli.
• BAC libraries are arrayed collections of clones that provide genome-wide coverage and are essential resources for sequencing and mapping.
• BACs are more stable and have lower chimerism rates than YACs, making them the preferred vector for large-scale genomic projects.
• BACs are used in genome sequencing, BAC-based transgenic animal production, and studies of gene regulation and chromosome biology.
• Common pitfalls in BAC cloning include low transformation efficiency, insert instability, and contamination with genomic DNA; these can be addressed by optimizing ligation conditions, using appropriate host strains, and careful DNA purification.

Further Reading

• Hao M et al. Bacterial-Artificial-Chromosome-Based Genome Editing Methods and the Applications in Herpesvirus Research. Microorganisms. 2023. PubMed 36985163
• Swaminathan S, Sharan SK. Bacterial artificial chromosome engineering. Methods in molecular biology (Clifton, N.J.). 2004. PubMed 15024162
• Chiem K et al. Bacterial Artificial Chromosome Reverse Genetics Approaches for SARS-CoV-2. Methods in molecular biology (Clifton, N.J.). 2024. PubMed 38064031
• Chung SK. Single Nucleotide Polymorphism-based Identification of Bacterial Artificial Chromosome-mediated Homologous Recombination. Frontiers in bioscience (Landmark edition). 2024. PubMed 39206912
• Narayanan K, Chen Q. Bacterial artificial chromosome mutagenesis using recombineering. Journal of biomedicine & biotechnology. 2011. PubMed 21197472
• Janes DE et al. Sex chromosome evolution in amniotes: applications for bacterial artificial chromosome libraries. Journal of biomedicine & biotechnology. 2011. PubMed 20981143

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