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Figure 14. Sample autoradiogram of DNA sequencing reaction pattern by the Sanger method. Source: Photography courtesy of J. M. MacPherson.

is a significant amplification of the desired segment of double-stranded DNA whose termini are defined by the 5' termini of the oligonucleotide primers.

The first experiments in PCR used the Klenow fragment of E. coli DNA polymerase I, which has a temperature optimum of 37°C. However, since the Klenow fragment is inactivated at temperatures required to denature DNA, it was necessary to add a fresh aliquot of the enzyme to the reaction repeatedly. Unfortunately, these reactions tended to only work well for the amplification of DNA fragments less than 200 bp. For larger fragments it was found that the yields were poorer and the products were often heterogeneous in size (36). These problems were solved with the discovery of the thermostable tag DNA polymerase, isolated from the cells of thermophilic bacterium Thermus aquaticus. Tag polymerase can survive extended incubation at 95°C; therefore, all the components can be added at the start of the reaction without any further replenishment. Also, since annealing and extension can be carried

Table 2. Enzymological Aspects of rDNA Work

Purpose

Enzyme

Function

Cell lysis Proteolysis RNA degradation Nick translation cDNA synthesis Process DNA

Joining of polynucleotides Amplifying DNA

Lysozyme, cellulase, mutanolysin Pronase, protease K RNase H

E. coli DNA Polymerase I Klenow fragment T4 DNA polymerase Reverse transcriptase Nuclease Bal31 Exonuclease digestion Mung-bean nuclease, Restriction endonucleases DNA methylases Phosphatases Polynucleotide ligase Taq polymerase

Removes cell walls Removes proteins Elimination of RNA Synthesize DNA

(cDNA) from mRNA

Endonuclease cuts Methylated bases Remove 5' phosphate Join ends of DNA or RNA

Produce oligonucleotides by polymerase chain reaction

DNA and the segment to be amplified

DNA and the segment to be amplified

Primer

PCR steps

Figure 15. PCR the double-stranded DNA template is first denatured by heating in the presence of a large molar excess of two specific oligonucleotides and four dNTPs.

Add taq polymerase, dTTP, dCTP, dGTP, dATP, magnesium chloride

3. DNA extends in the direction of the arrows at temperature of T = 70°C, 1 min

4. Repeat steps 1-4 for 30 or more cycles to get more new DNA

out at elevated temperatures, mispriming is reduced, thus resulting in improvements in the specificity and yield of the amplification reaction.

PCR amplification is currently used in a variety of needs in molecular cloning and analysis of DNA, for example, probes, generation of large amounts of DNA for sequencing, chromosome crawling, creation of mutant sequences, and the generation of cDNA from small amounts of mRNA. PCR is also extensively being used for (1) the specific amplification of cellular protein coding genes by differential or global expression, (2) detection of nucleic acid sequences of genetic modified plants or plant products, (3) pathogenic organisms in food and clinical samples, (4) the diagnosis of genetic disorders, and (5) in forensic cases.

Several modified versions of the PCR method have been developed. Compared with the original use of PCR, that is, to amplify segments of DNA located between two specific primer hybridization sites, a single-sided PCR method has been developed that initially requires specification of only one primer hybridization side. The second site is then defined by the ligation-based addition of a unique DNA linker. This method, referred to as ligase chain reaction (LCR), allows for exponential amplification of any fragment of DNA. Another modification of the basic PCR has led to the development of anchored PCR. By using anchored PCR it is possible to amplify full-length mRNA when only a small amount of the sequence information is available. PCR-based techniques such as Random Ampli fied Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), DNA Amplification Fingerprinting (DAF), and microsatellite/PCR have been developed and used for identifying DNA markers.

In many cases it becomes essential to have a PCR detection system that can identify desired gene sequences quickly, with high specificity and in large volumes. The best analytical tool available to do this is mass spectrometry (37). A mass spectrometer works by vaporizing DNA and then accelerating the molecules through a vacuum chamber with the help of an electric field. Tiny differences in the time that it takes the fragments to reach the detector reveal small differences in their mass and hence their sequence. This technique is known as matrix-assisted laser desorption ionization time-of-flight mass spectrometry, or MALDI-TOF MS, and has the capability to analyze hundreds of DNA samples for a variety of assays for point mutations and polymorphism analysis (38). Although originally used over a decade ago for protein analysis, it was not available for DNA analysis until 1993, when various matrices were developed that would work with DNA fragments as long as 100 base pairs. However, for practical sequencing MALDI-TOF would have to work with DNA fragments much longer than the current 100 base pair capacity. Currently, new matrices are being studied that could extend MALDI-TOF reach to 1000 bases, and if this works, then this technique would be a major breakthrough for high-throughput sequencing.

Cloned Genes In Vitro

Gene cloning is the use of experimental techniques that generate rDNA molecules in vitro with the desire for its incorporation and expression in a cell. With the use of rDNA technology one can produce a genomic DNA or cDNA library and hence a fragment containing any gene(s) from any source (39). Cloned genes are essential foundations of biotechnology. To clone a gene (Fig. 16), the genetic information, whether DNA or RNA, is processed by restriction endonucleases or reverse transcriptase or amplified by PCR, as the case may be. The steps involved in cloning foreign DNA into host cells include: (1) isolating RNA or DNA to be cloned; (2) choosing a suitable vector; (3) processing the DNA or RNA by restriction endonucleases or reverse transcriptase, respectively; (4) inserting the DNA fragments into the vector; (5) transferring of DNA into the desired host cell; (6) identifying those cells which have taken up the DNA, and (7) confirming that the clones are carrying the desired DNA fragment. These are the general steps within each of which several options are available. Figure 16 depicts the strategies involved in a gene cloning.

Several criteria are needed in deciding on a choice for cloning vector. Depending on fragment size to be cloned,

DNA fragments

Cellular DNA

Size separation (2-40Kb)

Joined to phage or plasmid

Desired size fragment w\

Transform the host directly

Recombinant vectors

Tested in expression systems o

In vitro packaging in A phage

Transduced, coli

Figure 16. A summary of the basic gene cloning experiment using E. coli as the ultimate host.

specific vectors must be employed. Thus, for fragments of DNA with sizes of <4 kb, <10 kb, <23 kb, and <46 kb, the recommended vectors are phage M13, bacterial plas-mids (pEMBL, pBR, etc), A phage (Fig. 11), and cosmids (33). Cosmids, which are a combination of a plasmid and a bacteriophage (lambda) based vector, can be made to contain large DNA fragments from the genomic library of an organism. The number of clones needed for fragments of 35 kb from E. coli genome would be 340; from S. cerevisiae, 6000; and from tomato plant cell, 60,000. Often the cloning strategies, for example, choice of available restriction sites, reporter genes (to indicate the expression of cloned gene), presence of one or more selectable phenotypes, autonomous replication in two or more hosts (shuttle vectors), nature of the experiment (eg, sequencing, preparation of probes, study of gene regulation), and exploitation and safety consideration (biohazard consideration, placement of nonconjugability functions, suicide vector system) decide the particular details of the system. Finally, in deciding about the choice for the vector, the question of cloned gene copy number (low or high) and ssDNA (used for hetero-duplexing and sequence analysis) versus dsDNA phages should be made in advance. In recent years more advanced vectors have been produced, for example, expression vectors and cassette vectors. They contain a promoter, a terminator, and ribosomal-binding sites, with the additional feature of a restriction site where a desired structural gene will be inserted. Presence of strong regulatory sequences aid significantly in the expression of the inserted gene in such a tailor-made expression vector system. This system offers great advantages for the production of rDNA-derived foreign products from cells. Additional uses of cassette vectors occur in those cases where partial removal of the reading frame from a structural gene has been made. After the start, the chimeric DNA, containing fragments of two different organisms, is inserted. The foreign gene product is recovered as a single (stand-alone) or fused (chimeric) polypeptides.

To examine the polypeptide products from rDNA, several in vitro (various transcription and translation systems) and in vivo (whole bacterial cell, minicell, and maxi-cell systems) can be used (40,41). Minicells are unique in that they are polar buds from rod-shaped bacteria (Fig. 2) lacking chromosomal DNA and therefore permit the screening of only those proteins that have been derived from the rDNA. After a recombinant DNA is generated, it is introduced into the new host through one of many techniques of gene transfer, such as electroporation, particle gun or biolistics, microinjection, microlaser technique, and liposome fusion (33,41,42). The initial choice of a bacterial host for rDNA work reflects the specific usage, for example, laboratory versus industrial; however, for food usage the GRAS (generally regarded as safe) status is important and strains of microorganisms with particular genetic markings are available. The transformed cell is then identified by the expression of the recombinant gene product under some selection or growth condition or by using variety of DNA, RNA, enzyme function related, fluorescent (eg, the green fluorescent protein), and immunological probes for colony or plaque hybridization (Fig. 17) and detection (33).

Figure 17. Autoradiogram of two halves of plates showing colony lift (left half) and plaque lift (right half). Source: Photograph courtesy of J. M. MacPherson.
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