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Experiment V.
Recombinant DNA I:
Restriction Endonucleases, Plasmid Vectors & Cloning Genomic DNA Fragments from E. coli



Introduction

Recombinant DNA techniques have become essential to today's biologists. These techniques allow researchers to investigate topics such as nucleic acid structure and function, protein function, the nature of mutations, to name but a few. Recombinant DNA techniques are now also used extensively in forensic science, medicine, agriculture and anthropology.

Over the next three laboratory periods, we hope to introduce you to a number of common techniques used in recombinant DNA technology. Today, we will start with some of the most basic tools...

Our ultimate goal over the next few weeks is to show you how DNA is cloned and manipulated in the laboratory, and to show you how molecular techniques can be used to address basic questions in the biological sciences.

In many instances, recombinant DNA techniques are called "cloning" or "gene cloning", but this does not refer to the technology used to produce the famous sheep, Dolly.  "Cloning", in our useage, refers to the isolation some specific piece of DNA, physical attachment to a "vector" for transfer to bacteria for production of many identical copies of this piece of DNA.

For a good overview of the techniques used in molecular genetics:
The Basics of Molecular Genetics (http://www.cancergenetics.org/md2intro.htm)

Our specific goals in the next three weeks are to:

1. construct plasmid DNA molecules that contain DNA fragments from the genome of E. coli and

2. generate an ordered map of a (previously) cloned genomic DNA fragment isolated from the cellualr slime mold, Dictyostelium.

Flow Chart of Recombinant DNA Experiments I, II and III
 

Some Basic "Tools" of Recombinant DNA

There are a number of websites that discuss different aspects of recombinant DNA technology. Below are a few general sites that may be of interest:

A. Restriction Endonucleases

Some species of bacteria produce enzymes, called restriction endonucleases, that defend them from infection by viruses. These restriction enzymes catalyze the cleavage of double-stranded DNA molecules into smaller fragments, thus inactivating the invading viral chromosome and halting the infection.

A number of restriction endonucleases have been isolated from different species of bacteria. All of these enzymes cleave double-stranded DNA; however, they each recognize different DNA sequences, or recognition sites. A specific sequence of bases (usually 4-8 bases in length) defines the recognition site for the enzyme. For example, the restriction enzyme EcoRI (isolated from E. coli ) recognizes the sequence-

5'...GAATTC...3'

3'...CTTAAG...5'

and cuts each strand of the DNA between the G and A residues of the recognition sequence. The result of this cleavage reaction are DNA fragments that have "sticky" ends, that is the 5' end of one strand contains a short single-stranded overhang-
5'...G 3'

3'...CTTAA...5'

Note also that the recognition sequence is a palindrome; the recognition sequence is present in the 5' to 3' direction on both strands. In the genome of the average organism, one would expect the recognition sequence for EcoRI to occur on average every 4000 bp (every 46 bp).

Restriction enzymes have become useful tools for molecular biologists. In this context, restriction enzymes serve as "molecular scissors" for cutting DNA into smaller pieces, for preparing DNA for cloning, and the recognition sequences serve as "landmarks" along a piece of DNA for construction of maps.

B. Plasmids as Cloning Vectors

"Cloning" a piece of DNA simply means the act of making a chimeric piece of DNA by physically attaching (ligating) a fragment of DNA to another piece of DNA called a cloning vector. The cloning vector itself is a DNA molecule which posses DNA sequences that:

1. allow the chimeric DNA to replicate itself in the "host" (usually a bacterium, such as E. coli), and

2. allow selection of "host" cells that contain the cloning vector.

Additional properties of the vector DNA allow the experimenter to determine whether the cloning vector contains an "insert" (i.e., is a chimeric or "recombinant" DNA molecule). There are several types of cloning vectors available (plasmids, bacteriophages, cosmids etc.); the choice of a cloning vector usually depends on the size of the DNA fragment to be cloned and on what will be done with this cloned piece of DNA in later experiments.

Plasmids are small, circular, extrachromosomal DNA molecules which replicate independently of the "host" chromosome. There are several naturally occurring plasmids in bacterial species and a number of these plasmids encode genes which confer antibiotic resistance. A number of plasmids have been genetically engineered such that they contain several useful properties that can be exploited for use in cloning; an example of a plasmid, pBlueScript KS, used as a common cloning vector is shown below.

C. Agarose Gel Electrophoresis

The standard method used to separate, identify, and purify DNA fragments is electrophoresis through agarose gels.

For an overview of gel electrophoresis, see: Exp. IV: Quantitation & Separation of Proteins

Agarose gel electrophoresis is simple, rapid to perform, and capable of resolving mixtures of DNA fragments that cannot be separated adequately by other sizing procedures, such as density gradient centrifugation. Furthermore, the location of DNA within the gel can be determined directly: "bands" or fragments of DNA in the gel are stained with low concentrations of the fluorescent, intercalating dye ethidium bromide; as little as 1 ng of DNA can be detected by direct examination of the gel in ultraviolet light. Ethidium has an aromatic ring system that allows it to intercalate into the stacked base pairs and become bound via stacking interactions. When bound to DNA, ethidium fluoresces a bright orange when illuminated with ultra violet light.

The electrophoretic migration rate of DNA through agarose gels is dependent upon several parameters which are discussed below:

Amount of Agarose in Gel (%) Efficient Range of Separation of Linear DNA Molecules (kilobase pairs=kb=1000 base pairs)
0.3 60-5
0.6 20-1
0.7 10-0.8
0.9 7-0.5
1.2 6-0.4
1.5 4-0.2
2.0 3-0.1
Ready to take an inteactive quiz on topics related to molecular genetics?????
Recombinant DNA Tutorials (Cornell Univ.)


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