V

Figure 4-13: A typical Southern blot with distinct bands. Each vertical lane consists of DNA from a separate individual. All the individual DNAs were digested with the same restriction endonuclease. Following separation on electrophoresis and transfer to a nylon membrane, hybridization was performed with the selected radioactive probe, and thus only those fragments complementary to the probe are visualized. This is an analysis of a family with hypertrophic cardiomyopathy, and the different patterns reflect restriction fragment length polymorphisms (RFLPs) characteristic of the marker locus, which is linked to the disease locus. (From Mares A Jr, Towbin J, Bies RG, Roberts R. Molecular biology for the cardiologist. Curr Probi Cardiol 1992; 17:9-72. Reproduced with permission from the publisher and authors.)

Table 4-1: Separation and Identification oF Molecular Species

Procedures Molecule Labeled Probe

Southern blotting DNA DNA or cDNA Northern blotting RNA DNA or cDNA Western blotting Protein Antibody

Cloning of a Gene

DNA cloning is a technique used to produce large quantities of a specific DNA fragment of interest.35 It generally is quite feasible to produce a billion copies of a DNA fragment by routine bacterial cloning techniques. The DNA fragment of interest (insert) is inserted into the DNA of a vector, and the vector is amplified in an appropriate host cell. The host provides amplification of the DNA of both the vector and the foreign insert. The prerequisites for cloning are (1) isolation of the DNA fragment of interest, (2) a vector, which is often an extrachromosomal segment of DNA

with the ability to propagate independently of the host DNA, (3) a restriction endonuclease to digest both the insert and the vector so the DNA ends will be compatible for ligation (as illustrated in Fig. 4-14), (4) a DNA ligase to ligate the insert into the vector, (5) a means to introduce the vector into the host cell, and (6) a means to differentiate the host cells that have incorporated the vector from those which have not. Standard vectors used in cloning have circular DNA and fall into three classes: (1) plasmids harvested from bacterial cells (a plasmid is an extrachromosomal segment of DNA present in bacteria that is self-replicating and on which are located certain genes that express resistance to ampicillin or other antibiotics), (2) bacteriophages (commonly referred to merely as phages, they are viruses that invade and multiply in bacterial cells), and (3) an artificially developed vector (referred to as a cosmid). The insert and vector are enzymatically ligated together by DNA ligase into circular DNA, and the recombinant product (hence the name recombinant) is incorporated into a host such as a bacterium or a mammalian cell for amplification Fig. 4-15). In order to identify whether or not the particular DNA of interest has been replicated in the host, a so-called selection gene, such as one responsible for ampicillin resistance, is incorporated into the vector. The bacteria are grown in media containing ampicillin so that only those with the resistance gene will survive. Since the resistance gene is attached to the DNA fragment of interest, it indicates that colonies (bacteria) or plaques (phage) that survive must contain the gene of interest. The size of the insert is a limitation in cloning. Plasmids can only accommodate inserts up to approximately 15,000 base pairs, phages up to 25,000 base pairs, and cosmids up to 45,000 base pairs. Recently, a new vector has been developed, namely, bacterial artificial chromosomes (BACs), that accommodates DNA fragments of up to 200,000 base pairs. The yeast artificial chromosome (YAC),36 developed several years ago, accommodates DNA inserts of up to 2 million base pairs but is extremely difficult to work with on a routine basis; in contrast, the BACs are as convenient as plasmids or phages. This has markedly accelerated the cloning of large fragments of DNA. Cloning, as discussed, is performed to obtain multiple copies of DNA, and unless specifically designed, the DNA is neither transcribed into mRNA nor translated into protein. If one desires to express a particular DNA fragment or gene, one must then use what is referred to as an expression vector. It is imperative to provide a promoter element that is appropriate for the host, and the gene must contain the appropriate 5' untranslated region for binding to the ribosome as well as the appropriate 3' region for stability of the message. An example would be the expression of rt-PA in mammalian cells, whereby the protein is expressed and secreted to be harvested and processed commercially for use as a thrombolytic agent.

Blunt End Sticky Ends

Figure 4-14: Restriction endonucleases recognize specific sequences and cut in a specific manner. The sequences recognized may be anywhere from 3 to 8 base pairs long and may cut to give a blunt end or a staggered end (EcoRl). Enzymes that provide staggered ends (cohesive or sticky ends) have unpaired bases that are easy to ligate together because they are complementary to each other, as shown in this illustration. This feature is exploited in cloning or in the formation of any recombinant DNA molecule. For cloning purposes, the fragment of DNA to be inserted is digested with the same restriction enzyme as is used to digest the DNA of the vector into which it will be inserted. Thus the sticky ends of the DNA insert and the vector will be complementary and easy to ligate together in the presence of the enzyme DNA ligase, as illustrated in Fig. 4-15.

Development of Gene Libraries

Gene libraries are usually called either genomic or cDNA libraries. A genomic library refers to one made from genomic DNA. A library is a collection of DNA fragments that have been cloned in an appropriate vector and grown in a particular host, usually bacteria. A major difference between a genomic and a cDNA library is that a genomic library contains DNA fragments composed of introns and exons, whereas a cDNA library is made from mRNA that represents genes expressed in a particular organ and does not have introns. The cDNA library contains genes specifically expressed in a particular tissue only. In contrast, a genomic library, whether derived from the heart or another tissue, will have the same genes. To make a human genomic library, one must first isolate the whole genome of a cell, cut it into fragments with a restriction enzyme, and insert the fragments into a vector replicated in an appropriate host, usually bacteria.37 To increase the odds that enough fragments are cloned to represent the whole genome, certain calculations are necessary. It is assumed that the recognition site for a particular restriction enzyme occurs at random. For the restriction enzyme EcoRl, with a 6-base-pair recognition site, the average size of each fragment will be 46 = 4096 base pairs. In contrast, if the recognition site involves 4 base pairs, each fragment would be 44 = 256 base pairs long. If the 6-base-pair cutter were used for the human genome, the result would be the 3 billion base pairs of the human genome divided by 4096 to produce roughly 750,000 fragments requiring 750,000 colonies or clones. However, the recognition sites are not evenly or randomly distributed. Thus some fragments are larger and others are smaller, so to be certain, at least l million colonies would be required. Other factors also must be considered, such as the choice of vector with respect to insert size. Any part of the library that is used can be replaced by regrowing it, and thus the library is a permanent, renewable source of DNA. cDNA libraries of the whole heart and specific structures of the heart such as the Purkinje system are now available. To isolate a particular gene or fragment of DNA or cDNA from a library generally requires a radioactive cDNA probe.

Polymerase Chain Reaction

The PCR has revolutionized application of the techniques of molecular biology. This technique was not developed until 1985,38,39 but its impact already has been felt throughout medicine and biotechnology. This procedure, conveniently and without the tedium of cloning, can provide l million copies of a DNA fragment in 3 to 4 h and 1 billion copies within 24 h. PCR simply and ingeniously takes advantage of the natural DNA replication process. One must know the sequence of the two ends of the DNA fragment that is to be amplified, but short sequences of 15 to 30 base pairs are adequate, and fragments in between these sequences as large as 20 kb can be amplified. The sequence is used to make two oligonucleotides, referred to as primers, with one for each end of the DNA fragment. The sequence of one primer is complementary to the sense direction, and the sequence of the other is made complementary to the antisense direction. The primers are used to prime the synthesis of cDNA strands and are designed such that the DNA between the primers is the fragment of interest to be amplified. If mRNA is to be amplified, it is first converted to a cDNA using the enzyme reverse transcriptase. The primers (oligonucleotides) and the necessary bases are added in excess, together with the enzyme Taq DNA polymerase (which catalyzes DNA synthesis) and a sample containing the DNA to be amplified. There are three steps to each cycle.

Initially, one must denature the DNA (separate the primers and the native DNA) into separate strands, which is done by increasing the temperature to 95°C. The temperature is then decreased to 50°C so that the primers and native DNA will reanneal to their complementary base sequences. The native DNA strands will bind not only to each other but also to the primers. The temperature is now increased to 65°C for synthesis of the new DNA fragments. Synthesis in the presence of Taq1 polymerase is initiated at the 5' end, and further nucleotides are added in the 5' to 3' direction to provide the desired double-stranded DNA fragment. Taq1 DNA polymerase, isolated from Thermus aquaticus, is thermostable, which is of tremendous advantage in performing the PCR reaction. Since the high temperatures of up to 95°C do not destroy this polymerase, it negates the need to add DNA polymerase between each cycle. Furthermore, since Taq polymerase has an optimal activity at around 70°C, one can significantly accelerate DNA synthesis. The cycle is then repeated, and after about 30 cycles over 3 h, one should have about 1 million copies. There are many clinical applications for PCR. To make a diagnosis of viral myocarditis, for example, one can use PCR to amplify from a myocardial biopsy any specific viral RNA or DNA for which primers can be made. The sensitivity of most conventional techniques is inadequate to detect molecules unless present in 50,000 to 100,000 copies per cell. In contrast, only one copy of RNA or DNA is needed for detection by PCR, and in 3 to 4 h, up to 1 million copies can be generated, which is adequate abundance for detection by most conventional techniques. PCR offers exquisite diagnostic sensitivity and specificity for determining the etiology of cardiac disorders such as myocarditis, and in patients undergoing cardiac transplantation, it is used for detecting infection or immunologic rejection. Another application of PCR is to detect and amplify mutations associated with hereditary disorders. One also can sequence DNA directly from PCR without the need for cloning.

Electrophoretic Mobility Shift Assay (Band-Shift Assay)

This technique is used routinely to study transcriptional factors. On gel electrophoresis, DNA exhibits a certain migratory pattern owing to the large fragments moving more slowly and thus being detected as the stained bands closer to the negative electrode. If a transcription factor is bound to its DNA-binding site, migration is slowed, and the decreased mobility will be detected as a shift in the migrating band through the gel (hence the name). Using an antibody to the protein, one also can study the protein specifically. It was this technique that identified a unique family of DNA- and RNA-binding proteins that are specific for the triplet repeat CTG (or CUG) and are thought to play a role in the pathogenesis of myotonic dystrophy.40

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