Replacement lambda Vectors, Cleavage Phage, Enzyme Generates, Three Fragments, Central Fragment, phage Genome

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Home >> Biotechnology and Genomics >> Cloning and Expression Vectors >> Replacement Lambda Vectors

Replacement vectors.

(i) EMBL3 and EMBI4. EMBL3 and EMBL4 are two vectors that were designed so that a central non-essential part of 44 kb long phage can bereplaced by a foreign DNA. Cleavage of the phage with an appropriate enzyme generates three fragments (left arm, right arm and a central fragment caIled stuffer).

Use of lambda phage as replacement vector for cloning a eukaryotic DNA fragment.

Replacement lambda vectors

Replacement lambda vectors EMBL3 and EMBL4 showing their essential elements

The central fragment representing 40% of the phage genome is nonessential for propagation of the phage and can be replaced by foreign DNA that may be as long as 20-23 kb. Non-recombinant DNA resulting from fusion of left arm and right arm wiII give a DNA molecule that is too small for packaging, so that there is automatic selection against non-recombinant phages. EMBL3 and EMBL4 can be used for preparing genomic libraries in eukaryotes with cloned fragments, 15-25 kb in size. The two vectors have polylinkers with reverse orders of restriction sites with respect to each other.

(ii) Charon 34 and Charon 35. Charon 34 and Charon 35 differ from each other only in their central fragments and will accept fragments up to 21kb (kilobases) long. They have more extensive range of restriction targets within their polylinkers than EMBL3 and EMBL4 or even Charon 4A and Charon 21 A.

In these vectors, physical separation of lambda arms from central fragment (stuffer) is required before ligation to donor fragment is attempted. In EMBL3 and EMBL4, removal of central fragment is not required, because there is a mechanism which ensures that 98% of the packagable DNA are recombinants.

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