Fragmentation (cell Biology) - DNA Cloning

DNA Cloning

DNA cloning can be performed spontaneously by the cell for reproductive purposes. This is a form of asexual reproduction where an organism splits into fragments and then each of these fragments develop into mature, fully grown individuals that are clones of the original organism (See reproductive fragmentation). DNA cloning can also be performed intentionally by laboratory researchers. Here, DNA fragmentation is a molecular genetic technique that permits researchers to use recombinant DNA technology to prepare large numbers of identical DNA molecules. In order for DNA cloning to be completed, it's necessary to obtain discrete, small regions of an organism's DNA that constitute specific genes. Only relatively small DNA molecules can be cloned in any available vector. Therefore, the long DNA molecules that compose an organism's genome must be cleaved into fragments that can be inserted into the vector DNA. Two enzymes facilitate the production of such recombinant DNA molecules:

1. Restriction Enzymes

Restriction enzymes are endonucleases produced by bacteria that typically recognize small base pair sequences (called restriction sites) and then cleave both strands of DNA at this site. A restriction site is typically a palindromic sequence, which means that the restriction-site sequence is the same on each strand of DNA when read in the 5' to 3' direction.

For each restriction enzyme, bacteria also produce a modification enzyme so that a host bacterium's own DNA is protected from cleavage. This is done by modifying the host DNA at or near each potential cleavage site. The modification enzyme adds a methyl group to one or two bases, and the presence of this methyl group prevents the restriction endonuclease from cutting the DNA.

Many restriction enzymes make staggered cuts in the two DNA strands at their recognition site, which generates fragments with a single stranded "tail" that overhangs at both ends, called a sticky end. Restriction enzymes can also make straight cuts in the two DNA strands at their recognition site, which generates blunt ends.

2. DNA ligase

During normal DNA replication, DNA ligase catalyzes end-to-end joining (ligation) of short fragments of DNA, called Okazaki fragments. For the purposes of DNA cloning, purified DNA ligase is used to covalently join the ends of a restriction fragment and vector DNA that have complimentary ends. They are covalently ligated together through the standard 3' to 5' phosphodiester bonds of DNA.

DNA ligase can ligate complimentary sticky and blunt ends, but blunt-end ligation is inefficient and requires a higher concentration of both DNA and DNA ligase than the ligation of sticky ends does. For this reason, most restriction enzymes used in DNA cloning make staggered cuts in the DNA strands to create sticky ends.

The key to cloning a DNA fragment is to link it to a vector DNA molecule that can replicate within a host cell. After a single recombinant DNA molecule (composed of a vector plus an inserted DNA fragment) is introduced into a host cell, the inserted DNA can be replicated along with the vector, generating a large number of identical DNA molecules. The basic scheme for this can be summarized as follows:

Vector + DNA Fragment

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Recombinant DNA

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Replication of recombinant DNA within host cell

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Isolation, sequencing, and manipulation of purified DNA fragment

There are numerous experimental variations to this scheme, but these steps are essential to DNA cloning in a laboratory.