**1.2 BAC applications**

The *de novo* synthesis of small DNA fragments has become routine although it remains expensive (Gibson et al., 2008, 2010; Itaya et al., 2008; Itaya, 2010). In that process, commercial enterprises customize desired DNA fragments based on nucleotide sequence information provided by the end-user. Larger DNA, equivalent to lengths clonable in BACs, can be prepared routinely by assembling small DNA fragments (Gibson 2011; Itaya & Tsuge, 2011). Reverse genetics methods applied in studies involving cultured cells and model animals are increasingly important in mutation research (Yang et al., 1997; Hardy et al., 2010). DNA can now be obtained by *de novo* synthesis using designed sequences or by flexible engineering of cloned DNA in BACs. Although BACs can accommodate DNA fragments longer than 100 kbp, the intrinsic physicochemical features of long-stretched polymer molecules render them fragile and their handling difficult. Due to the shearing force in liquids, DNA fragments easily break into small pieces and nuclease contamination may be introduced in the course of biochemical isolation procedures. Therefore care must be taken in the isolation and purification of large DNAs (Kaneko et al., 2005) and appropriate host cell systems are needed to nurture and protect the fragile DNA fragments to facilitate the preparation of undamaged DNA samples regardless of their size.

The integration of BAC inserts into the genome of *B. subtilis* is a starting point for subsequent manipulations. The BAC vector region (**BAC\*)** preinstalled in the host genome provides the cloning site for guest BAC clones via homologous recombination (identified by X).

their contribution to long-range sequence determinations has been demonstrated (Frengen et al., 1999; Osoegawa et al., 2001). Cutting-edge technologies that facilitate direct genome sequencing have dramatically reduced the need for BAC libraries as a sequencing resource.

The *de novo* synthesis of small DNA fragments has become routine although it remains expensive (Gibson et al., 2008, 2010; Itaya et al., 2008; Itaya, 2010). In that process, commercial enterprises customize desired DNA fragments based on nucleotide sequence information provided by the end-user. Larger DNA, equivalent to lengths clonable in BACs, can be prepared routinely by assembling small DNA fragments (Gibson 2011; Itaya & Tsuge, 2011). Reverse genetics methods applied in studies involving cultured cells and model animals are increasingly important in mutation research (Yang et al., 1997; Hardy et al., 2010). DNA can now be obtained by *de novo* synthesis using designed sequences or by flexible engineering of cloned DNA in BACs. Although BACs can accommodate DNA fragments longer than 100 kbp, the intrinsic physicochemical features of long-stretched polymer molecules render them fragile and their handling difficult. Due to the shearing force in liquids, DNA fragments easily break into small pieces and nuclease contamination may be introduced in the course of biochemical isolation procedures. Therefore care must be taken in the isolation and purification of large DNAs (Kaneko et al., 2005) and appropriate host cell systems are needed to nurture and protect the fragile DNA fragments to facilitate

The integration of BAC inserts into the genome of *B. subtilis* is a starting point for subsequent manipulations. The BAC vector region (**BAC\*)** preinstalled in the host genome provides the

cloning site for guest BAC clones via homologous recombination (identified by X).

the preparation of undamaged DNA samples regardless of their size.

Fig. 1b. *B. subtilis* as a new BAC dealer.

**1.2 BAC applications** 
