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

DNA Cloning
Recombinant DNA Technology

To investigate the Structure and function of a Gene at THE MOLECULAR LEVEL, it is essential to obtain a large amount of a specific gene in pure form. A variety of Methods, collectively known as "Recombinant DNA technology," are used for DNA Cloning—generating A large number of identical copies of a target gene.

A recombinant DNA molecule is defined as a DNA molecule assembled from DNA segments derived from different sources.

The primary objective in cloning a DNA fragment of interest is to insert it into a so-called vector.

A vector is a DNA molecule fragment that possesses all the necessary components for its Replication within the host Cell used for propagation.

Once the recombinant DNA molecule—consisting of the vector and the inserted DNA fragment to be cloned—is introduced into The Cell, the inserted DNA fragment replicates along with the vector, ultimately producing a large number of identical DNA molecules. The general cloning workflow is illustrated in Figure 83.

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Figure 83 - General scheme of DNA cloning

Although numerous experimental techniques have been devised to adapt each individual step, DNA cloning generally incorporates all of these essential stages.

Below we will examine Two Types of vectors most commonly used in E. coli Cells:

1) Plasmid Vectors, which replicate within the cell alongside the cellular genome,

2) bacteriophage X-based vectors, which replicate as lytic Viruses, ultimately destroying the cell and packaging the replicated DNA into virions.

The main challenge in DNA cloning is to obtain short DNA segments of an Organism that contain specific genes. Furthermore, only relatively small DNA molecules can be incorporated into existing vectors. Therefore, extremely long genomic DNA molecules of an organism must be cleaved into fragments of the required length, which can then be joined to vector DNAs.

Two types of Enzymes are used to synthesize such recombinant DNAs:

1) restriction enzymes,

2) DNA ligases.

9.1.1. DNA Fragmentation. Restriction enzymes are endonucleases synthesized by Bacteria that recognize specific 4–8 nucleotide DNA sequences, known as restriction sites, and cleave both DNA strands at these locations.

A restriction site typically forms a palindromic sequence—a region of a double-stranded DNA molecule in which both strands have the same nucleotide sequence when read from the 5' to the 3' end. In other words, a palindromic sequence is an inverted tandem repeat, for example:

For each restriction enzyme, the bacterium synthesizes a corresponding modifying enzyme that protects the bacterium's own DNA from Cleavage by modifying it at potential cleavage sites. The modifying enzyme adds a methyl group to one or two bases, typically within the restriction site. Once this DNA Methylation has taken place, the restriction endonuclease is unable to cut the DNA.

Modifying (methylating) enzymes, together with Restriction Endonucleases, form a restriction-modification system that protects the host's own DNA and destroys foreign DNA (such as bacteriophage DNA or DNA introduced during transformation) by cleaving it at all restriction sites.

Most restriction enzymes make a staggered cut across the two DNA strands at the restriction site, generating fragments with single-stranded "sticky ends" (Figure 84, Table 5).

Figure 84 - DNA cleavage by the EcoRI restriction enzyme

These fragment overhangs, generated by cleavage at a given restriction site, are complementary to all other overhangs produced by the same restriction enzyme.

Table 5 - Selected restriction enzymes and their restriction sites

At room Temperature, these single-stranded regions from different fragments generated by the same restriction enzyme can spontaneously pair with one another. Consequently, they are frequently referred to as "sticky ends".

Certain restriction enzymes, such as AluI and SmaI, cleave both DNA strands at the exact same position within the restriction site, yielding fragments with "blunt ends". At the blunt ends of such fragments, all NUCLEOTIDES in both DNA strands are paired with their complementary counterparts.

The DNA of any given organism features a completely unique set of restriction sites. Therefore, restriction enzymes cut a given DNA molecule into a defined, reproducible set of fragments known as restriction fragments.

Restriction enzymes isolated from several hundred different bacterial species make it possible to cleave DNA molecules into a large number of distinct fragments corresponding to the recognition sites of these enzymes (Table 5).

Restriction enzyme names are typically italicized because they are Abbreviations of the Latin names of the bacteria from which they were isolated.

9.1.2. Insertion of DNA Fragments into Vectors. DNA Restriction fragments are inserted into vectors using DNA ligases (Figure 85). During normal METABOLISM/36.html">DNA replication, DNA ligases catalyze the joining (sealing) of the ends of short DNA segments known as Okazaki fragments. In DNA cloning, purified DNA ligases are utilized to covalently link a genomic restriction fragment and a vector DNA that possess complementary ends.

The vector DNA and the restriction fragment are covalently joined together via standard 3'—5' phosphodiester bonds of DNA.

Notably, bacteriophage T4 DNA ligase can join any blunt DNA ends In addition to sealing complementary sticky ends. However, blunt-end ligation is extremely inefficient, requiring significantly higher concentrations of both DNA and DNA ligase compared to sticky-end ligation.

Figure 85 — Ligation of restriction fragments with complementary sticky ends by T4 DNA ligase