CLONING LARGE DNA FRAGMENTS IN BAC AND YAC VECTORS

Bacterial Artificial Chromosome (BAC) and Yeast Artificial Chromosome (YAC) vectors are widely used in molecular biology for cloning large DNA fragments. These vectors are essential tools for studying complex genomes, constructing genomic libraries, and manipulating large DNA fragments. Cloning large DNA fragments in BAC and YAC vectors involves several key steps, including the isolation of high molecular weight DNA, preparation of vector DNA, ligation of the DNA fragments into the vectors, transformation into host cells, and screening for positive clones.

1) Isolation of High Molecular Weight DNA

The first step in cloning large DNA fragments in BAC and YAC vectors is the isolation of high molecular weight DNA from the source organism. This can be achieved using various methods such as phenol-chloroform extraction, cesium chloride gradient centrifugation, or commercial DNA isolation kits. The quality and integrity of the isolated DNA are crucial for successful cloning.

2) Preparation of Vector DNA

Once the high molecular weight DNA is isolated, the BAC or YAC vector needs to be prepared. The vector is typically isolated from a bacterial or yeast strain that contains the vector with an antibiotic resistance gene for selection. The vector DNA is then purified and linearized using restriction enzymes to create compatible ends for ligation with the target DNA fragment.

3) Ligation of DNA Fragments into Vectors

The next step involves ligating the large DNA fragments into the linearized BAC or YAC vectors. This process requires careful optimization of conditions to favor the ligation of a single insert per vector molecule. The ligated DNA is then transformed into competent bacterial or yeast cells.

4) Transformation into Host Cells

After ligation, the recombinant BAC or YAC molecules need to be introduced into host cells for replication. For BAC vectors, this typically involves transformation into Escherichia coli (E. coli) cells, while YAC vectors are often transformed into yeast cells such as Saccharomyces cerevisiae. The transformed cells are then plated on selective media containing antibiotics or other markers to identify positive clones.

5) Screening for Positive Clones

Once transformed cells have been plated, screening for positive clones is carried out. This can involve various methods such as colony hybridization, PCR screening, or direct sequencing to confirm the presence of the desired large DNA fragment within the BAC or YAC vector.

In summary, cloning large DNA fragments in BAC and YAC vectors involves several critical steps including isolation of high molecular weight DNA, preparation of vector DNA, ligation of DNA fragments into vectors, transformation into host cells, and screening for positive clones. These techniques have revolutionized genomic research by enabling the manipulation and study of large genomic regions.