BACs and PACs as vectors for cloning large DNA segments (100-300 kb), Phage Vectors, Physical Maps, Artificial Chromosomes

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BACs and PACs as vectors for cloning large DNA segments (100-300 kb)

Genome analysis and map based cloning in higher plants and animals required isolation, of large pieces of DNA, which can not be cloned in plasmid or phage vectors. A variety of vectors are now available; which are suitable for cloning such large DNA fragments suitable for preparation of high-resolution physical maps of higher eukaryotes (both animals and plants).

These vectors, 0.5 to 2 megabase (Mb) in length, particularly include yeast artificial chromosomes, (YACs) capable of accepting fragments of 100-2000 kbp and PI vectors capable of cloning fragments of upto 100 kbp (kilobase pairs).

Mammalian artificial chromosome (MAC) vectors have also been designed, which' are capable of cloning fragments of more then 1000 kilobase pairs. Plasmids based on F-factors were also designed which could accept fragments upto 120kbp in length (for YAC and MAC vector, see later in this chapter). Of these vectors, YACs are most commonly used, but following difficulties have been encountered while using YACs

(i) In Y AC libraries, a fraction of clones result due to co-cloning events giving single clones with non-contiguous fragments-these are described as chimeric vectors. (ii) Y AC clones also exhibit some degree of instability due to deletions/rearrangements in the cloned inserts. (iii) YACs are similar to yeast normal chromosomes in size thus making it difficult to separate them by simple methods.

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BACs and PACs as Vectors for Cloning large DNA Segments (100-300 kb) Bacterial Artificial Chromosomes (BACs) P1-derived Artificial Chromosomes (PACs) Plant and Animal viruses as Vectors Plant Viruses (Cauliflower Mosaic virus or CaMV and Geminiviruses) Animal Viruses Transposons as Vectors Transposons of Higher Plants (Ac/Dc or Mul of Maize) Transposons of drosophila (P elements) YAC and MAC Vectors for Cloning very large DNA Segments (1000 kb) Yeast Artificial Chromosomes (YACs) Mammalian Artificial chromosomes (MACs) Expression Vectors Promoters Nopaline Synthase (nos) Promoter from T-DNA Dual Promoter of Mannopine Synthase (mas) genes 1 and 2 35S Promoter of CaMV Polyhedrin Promoter from Baculovirus Expression Cassettes Baculovirus and Expression Vector System for Insect Cells Virus Expression Vectors for Mammalian Cells Binary and Shuttle Vectors