Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

DNA Cloning
Bacteriophage λ-Based Vectors

Bacteriophage λ-based vectors are approximately a thousand times more efficient than Plasmid Vectors when cloning large numbers of DNA fragments. For this reason, λ phage vectors have been widely used to construct DNA libraries—comprehensive collections of DNA fragments representing an Organism's genome or mRNA.

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Figure 88 - Plasmid vector pBR322

Two main factors account for the superior efficiency of phage λ as a cloning vector:

1) infection of E. coli Cells by λ virions occurs a thousand times more frequently than plasmid transformation,

2) a much larger number of λ clones, compared to transformed colonies, can be grown and identified on culture media in a Petri dish.

When a λ virion infects an E. coli Cell, it undergoes a lytic growth cycle during which the phage DNA replicates and assembles into more than 100 progeny phages, which are released upon lysis of the infected cell (see, for example, Figure 69). If λ phages are plated on an E. coli culture in a Petri dish, each virion infects a single cell. Subsequent cycles of phage growth lead to The formation of visible clear zones (devoid of cells) called plaques, in which E. coli cells are destroyed and progeny phage virions are released (see Figure 67).

The λ phage virion consists of a HEAD enclosing the phage DNA genome and a tail through which E. coli cells are infected. The genes of phage λ encoding the head and tail Proteins, as well as genes encoding various other proteins involved in METABOLISM/36.html">DNA Replication and cell lysis, are clustered into discrete Regions of the viral genome, which has a total length of 49 kb (Figure 89).

Figure 89 - Diagram of the bacteriophage λ genome

However, the central region of the phage λ genome contains genes that are non-essential for the lytic growth cycle. If these genes are excised and replaced with other DNA fragments, we obtain a recombinant DNA molecule that can be packaged in vitro to form a phage capable of infecting cells and forming plaques in cell culture (Figure 90).

Such in vitro packaging of recombinant DNA, which mimics the in vivo assembly process, requires preassembled heads and tails, along with two viral head proteins, NuI and A.

During the late stages of phage λ infection, a long DNA molecule known as a concatemer is formed. It consists of multiple copies of the 49 kb phage genome joined end-to-end and separated by COS sites—nucleotide sequences that occur only once per genome and to which the NuI and A proteins bind for virion assembly. The binding of NuI and A proteins to the COS site stimulates the insertion of the DNA segment lying between two COS sites into an empty head. Once the head is filled with DNA, the preassembled tail attaches to it, producing a λ virion capable of infecting an E. coli cell.

Figure 90 - Assembly of the bacteriophage λ virion

Such λ phages are convenient for constructing Genomic Libraries—collections of λ clones that collectively represent all DNA sequences of a given organism's genome.

However, constructing such genomic libraries for higher eukaryotes presents significant experimental challenges.

First, genes in these organisms typically contain introns and are therefore too long to fit entirely within a λ phage vector. Consequently, individual Gene sequences are fragmented and distributed across more than one λ clone (a characteristic also common to plasmid clones).

Furthermore, the presence of introns and long intergenic regions in eukaryotic genomic DNA often complicates the identification of the essential gene regions that actually encode protein sequences.

Therefore, for many studies, mRNA proves to be a more useful Starting Material for DNA library construction, as it lacks the non-coding regions characteristic of genomic DNA.

In this case, DNA copies of mRNA—known as complementary DNA (cDNA)—are synthesized and subsequently cloned into phage vectors. A large collection of such cDNA clones, representing all mRNAs expressed in a cell, is referred to as a cDNA library.