Introduction and Goals
Recombinant DNA technology includes the techniques developed for the isolation, manipulation and alteration of DNA in a test tube, as well as the transfer of this DNA back into cells. This tutorial describes commonly used techniques for fragmenting DNA, separating DNA fragments, and identifying and isolating specific DNA fragments. These approaches have made it possible for scientists to engineer specific DNA sequences, and even novel combinations of DNA sequences in a test tube, which can then be introduced back into cells. By the end of this tutorial you should know:
- How restriction endonucleases cleave DNA
- How agarose gel electrophoresis separates DNA molecules
- How Southern blotting and Northern blotting can be used to identify specific DNA fragments or mRNA, respectively
- How a recombinant molecule is generated, including the characteristic of a plasmid vector
- The differences between genomic and cDNA libraries
- How recombinant DNA is used to generate transgenic organisms
Restriction Nucleases Cleave DNA
One of the first steps in being able to manipulate and study discrete DNA sequences is the ability to cleave large molecules of DNA into smaller defined fragments. This can be accomplished by the use of restriction endonucleases, which are enzymes that can cleave (or digest) double-stranded DNA in a site-specific fashion. These enzymes are found naturally in a variety of different bacteria, and are used by the bacteria to degrade foreign DNA; their own DNA is modified to be protected from degradation. Generally, restriction endonucleases recognize a short nucleotide sequence (4-8 bp) and cleave the DNA in a precise fashion. Cleavage breaks the phosphodiester bond in both of the two strands of double-stranded DNA. Many restriction endonuclease recognition sites are palindromic, meaning the sequence is identical whether reading from the top or bottom strand (but always reading 5' to 3'). For example, the restriction endonuclease Eco RI, which cleaves the sequence 5'GAATTC3', was initially isolated from bacteria (Escherichia coli). This restriction endonuclease will cleave DNA isolated from any cell, prokaryotic or eukaryotic. There are dozens of different restriction endonucleases that recognize and cleave different sequences. Examples of two restriction endonucleases and their recognition sites are illustrated in Figure 1.
Some enzymes (e.g. Eco R1) cleave DNA in such a fashion as to leave short, single-stranded overhangs (sticky ends); others (e.g. Alu I) leave blunt ends(illustrated in Figure 1). If DNA, from any source (e.g. bacteria, plants or animals), is cleaved with a single restriction enzyme, the generated DNA fragments will have identical sequences at their ends. The frequency of cleavage of DNA is simply the frequency of a particular site occurring in the DNA. For an enzyme that has a 6 bp recognition site, the probability that the four nucleotides (G, A, T, C) will make up the recognition sequence is 1 in 4096 bp (1 in 46).
Agarose Gel Electrophoresis Separates DNA Fragments
DNA fragments of differing lengths can be separated by agarose gel electrophoresis. A mixture of DNA fragments is loaded onto a gel, the gel is covered in buffer, and an electric field is generated when the gel box is connected to a power supply (negative electrode on top of the gel, and positive on the bottom). The DNA migrates through the gel toward the positive electrode. Larger DNA molecules migrate more slowly, and smaller fragments migrate more quickly. Typically a molecular weight marker (MW marker) is included, which is essentially a mixture of DNA fragments of known lengths. The migration of an unknown fragment is compared to the migration of the MW marker and thereby the length of an unknown fragment is determined. The distance of migration is proportional to the length of the DNA fragment. Also, in order to visualize the DNA, the gel must be either cast with, or soaked in, a fluorescent dye. The most common dye used is ethidium bromide, which binds to DNA and fluoresces under UV light.
Nucleic Acid Hybridization
When the total nuclear DNA from a human cell is cleaved with Eco R1, it generates so many fragments of different sizes that when the fragments are separated by agarose gel electrophoresis they appear as a smear of DNA rather than individual bands. It would be impossible to determine if a particular band was present or perhaps altered. The technique of Southern blottingpermits the detection of a single fragment of DNA using a sequence-specific probe. DNA fragments are separated by gel electrophoresis and the bands are transferred to a special membrane that binds single-stranded nucleic acid. Typically the DNA is first denatured in the gel, by soaking it in alkaline buffer, and then it is rapidly neutralized. The gel is laid flat, the membrane is laid over the gel, and a stack of absorbent paper towels is placed on top of the membrane. The absorbent paper will wick the liquid from the gel through the membrane and, as it does so, the DNA will travel from the gel onto the membrane and be bound there. The relative positions of the DNA in the gel are preserved when the DNA is transferred to the membrane. The membrane, also referred to as the blot, is removed and then mixed with a specific probe. The probe is a single strand of DNA of a specific sequence, typically labeled fluorescently or radioactively. The membrane is placed in a solution with the probe and left for several hours. The probe will anneal (hybridize) to the fragments bound to the membrane that contain complementary DNA sequences. The unbound probe is washed away, and what remains on the blot is the labeled probe hybridized to the fragments that possess complementary DNA. Using this technique, one can detect the presence of a unique fragment of DNA in an entire fragmented genome.
Another related technique is Northern blotting. In this procedure, isolated mRNA is separated by gel electrophoresis and then transferred to membrane, where it is hybridized to a labeled, single-stranded DNA probe. This permits the detection of mRNAs that are complementary to a specific sequence of DNA and allows one to monitor the expression and differential splicing of a single gene in many different tissues.
Although the techniques described allow one to distinguish a region of DNA from the genome at large, it is even more useful to be able to isolate just the gene of interest and produce sufficient quantities of that particular region of the genome for additional investigations and manipulations. DNA cloning is the process of extracting just one region of the genome and producing many identical molecules of this DNA sequence. In order to do so, a recombinant DNA molecule (the DNA sequence of interest joined to a vector sequence) is constructed. The vectorsequence supports the replication and transmission of the recombinant DNA (usually in bacteria). Recombinant DNA molecules are created by mixing DNA fragments (e.g. fragments generated by Eco R1) from different sources (e.g. human and bacteria) that have compatible ends and then adding the enzyme ligase, which will join the two fragments of DNA. You may remember that DNA ligase joins the Okazaki fragments in DNA replication. In the test tube, isolated ligase will also join DNA fragments in a reaction termed ligation, as long as the ends are compatible (the same sticky ends or two blunt ends). Once the recombinant molecule is made it can be introduced into bacteria, where it will replicate and make many copies of this novel combination of DNA.
Most recombinant DNA molecules are made using bacterial plasmids as vectors. Plasmids are small (3,000-12,000 bp), circular DNA molecules that replicate in bacteria (Figure 5). All plasmids have an origin of replication (ORI), and many also encode for an antibiotic resistance (typically ampicillin resistance). Most plasmid vectors also have a number of different restriction endonucelase sites so that foreign DNA can be easily recombined with the plasmid. A recombinant DNA molecule can be introduced into bacteria through the process of transformation. The bacteria can be manipulated to take up DNA from the media in which they reside. Each bacterium takes up only one recombinant molecule. Typically the product of a ligation will be used to transform the bacteria. The frequency of transformation is low, but the few bacteria that have taken up recombinant DNA can be selected for easily by placing the bacteria in media with antibiotics (e.g. ampicillin) - only those with the recombinant DNA molecule (including the plasmid vector) will grow. The recombinant plasmid is present in hundreds of copies per bacterium, and is stably replicated and segregated to daughter cells each time the bacterium divides. Starting from a single bacterium, one can produce a large culture of bacteria that carries the identical recombinant plasmid. The bacterial culture can be lysed and the recombinant plasmid isolated from the rest of the bacterial DNA, providing a renewable source of pure recombinant plasmid.
The cloning of genes through this method has been supplemented by amplification of specific DNA sequences using the polymerase chain reaction (PCR, see the tutorial on DNA replication). Due to the availability of the complete genomic sequence of many organisms, it is relatively easy to design PCR primers specific for the amplification of any portion of the genome.
Plasmids have been used to create DNA libraries, a collection of recombinant plasmids each carrying a different DNA sequence. There are two important types of libraries: genomic libraries and cDNA libraries. A genomic library is composed of DNA from a nuclear genome fragmented by restriction endonuclease digestion and recombined with vector DNA. Each recombinant plasmid (or clone) has the same vector sequence, but has a different region of the genome (sometimes referred to as the insert) with which it is recombined. A complete genomic library would contain every region of the genome represented as a clone.
A cDNA library is composed of the "expressed" genome. It is made to DNA copies of the mRNA expressed in a particular cell. The mRNA is isolated from a specific tissue or cell type and is copied into DNA using the enzyme reverse transcriptase. Reverse transcriptase is normally encoded by double-stranded RNA viruses. It will copy RNA into DNA. The cDNA libraries are specific for the tissue from which the mRNA is isolated. The distribution of sequences in the cDNA library is also tissue specific. A gene that is highly expressed will be copied into DNA more often and therefore compose a larger fraction of the library than a gene that is poorly expressed. The cDNAs, since they are made from mature mRNAs, will only include the exons and not the introns. In fact, early comparisons of cDNAs and genomic sequences first alerted scientists to the existence of exons and introns.
Isolation and Identification of Individual Recombinant Clones
The cDNA libraries and genomic libraries, once made, are used to transform bacteria, and each transformed bacterium (transformant) contains a different clone from the library. This collection of bacteria can be screened to isolate the individual colonies that carry the recombinant clone of interest. This can be achieved through a colony filter liftand hybridization. A colony filter lift is analogous to a Southern blot, however, instead of fragmented DNA being transferred to the membrane, recombinant plasmid DNA from different bacteria are transferred to the membrane. Bacteria are typically grown in petri dishes, and each colony is a growth of bacteria originating from a single bacterium. The colonies are transferred to membrane, lysed and the DNA is denatured. The membrane is then hybridized with a specific probe for the gene of interest. The region of the membrane that contains DNA from the colony bearing the desired clone is then labeled. This membrane is aligned with the original petri dish containing the colonies, and the correct one is selected.
Individual recombinant clones, either a genomic clone or a cDNA clone, can be sequenced using the dideoxy chain termination method (described in the tutorial on DNA replication). For any given library, the vector sequence is identical and is known; therefore, primers derived from the vector sequence can be used to determine the unknown DNA sequence inserted into the vector. The human genome project of the 1990s was carried out by generating thousands of random genomic clones, sequencing all of the inserts and assembling the sequence into overlapping stretches of contiguous genomic sequence.
Transgenic Plants and Animals
Once a gene has been cloned (as either a cDNA clone or genomic clone), it is often desirable to introduce a specific mutation in the DNA and determine the effects of the mutations in the organism. For instance, one might hypothesize that a particular enhancer is critical for expression of a gene in muscle cells. To test this hypothesis, one might want to create a version of this gene in which the enhancer has been removed, reintroduce it into the genome and determine if this modification alters the levels of gene expression. Alternatively, one might fuse this enhancer to the promoter of another gene that is not normally expressed in muscle cells and determine if the presence of the enhancer is sufficient to drive gene expression in these cells. To perform either of these experiments, one would need to create a transgenic organism- an organism whose genome has been altered using recombinant DNA techniques. Many different transgenic fruit flies and mice have been generated to study the regulation of gene expression (as described above), as well as many other aspects of cell biology.
In mice, recombinant DNA is injected directly into developing embryos, and with some frequency the DNA inserts randomly into the genome. After injection, the embryos are implanted in a female and the resulting mice can be screened for the ones carrying the desired transgene (the particular recombinant injected). In addition, there are methods for targeting DNA insertion into the mouse genome. At a low frequency, DNA can be targeted so that it recombines with the homologous region of the genome, replacing the normal gene with the introduction transgene. This procedure is referred to as "knocking out" or gene replacement. A gene can be investigated by "knocking out" the gene and examining the impact on the growth and behavior of that knockout organism. Gene replacement has also been used to create mouse models of human genetic disorders by creating a similar mutation in mice.
The examples described above would likely be transgenic organisms, to be used in the laboratory. However, transgenic plants (and potentially, animals) can be used for agricultural purposes as well. Transgenic corn, soy and other crops have been engineered to express a variety of different traits, including insect resistance, herbicide resistance, slower ripening and increased nutrition content. These are referred to as *genetically modified (GM)*crops. Currently it is estimated that about half of the corn and soy used in the U.S. for food production is derived from GM crops. A small number of livestock (sheep and goats) have been genetically modified to express human proteins (e.g. clotting factor) in their milk.
The manipulation of DNA is possible through the use of restriction endonucleases that cleave DNA in a sequence-specific fashion. DNA fragments can be separated by length using agarose gel electrophoresis. Southern blotting is a technique that allows the detection of a specific region of DNA. The DNA fragments in an agarose gel are transferred to a membrane that binds the DNA and the membrane is hybridized with a specific probe; thus, a specific sequence of DNA is distinguished from all the fragments generated. Northern blotting is the transfer of mRNA to a membrane and hybridization with a specific probe to detect a specific mRNA. DNA cloning involves making a recombinant DNA molecule of the gene or DNA of interest and a vector sequence. Commonly, bacterial plasmids are used as vectors. The DNA fragments to be cloned are mixed with vector (with compatible ends) and a ligation is performed. The products of the ligation are used to transform bacteria and colonies are selected that bear the recombinant DNA molecule. A genomic library is a collection of thousands of bacteria, each bearing a different genomic sequence cloned into the same vector. A cDNA library is a collection of bacteria, each bearing a DNA copied from mRNA and cloned into the same vector. Colony filter lifts can be hybridized with a specific DNA sequence to identify the bacteria harboring a particular clone. DNA alterations of all sorts (loss of sequence, change of sequence and new combinations of sequence) can be generated in a test tube. Transgenic organisms can be generated to test the effects of the alterations in DNA. In mice, homologous recombination is used to target the disruption of genes by replacing the normal gene with an engineered mutant version. Genetically modified plants are used in agriculture; these have been engineered to be resistant to a pest or to have some other desirable trait.