Methodologies

The majority of molecularly-based identification methods rely on the interaction of unique enzymes and nucleic acids. Two extensively utilized classes of enzymes are restriction endonucleases and DNA polymerases.

Restriction endonucleases cleave DNA molecules at specific nucleotide sequences. Different endonucleases recognize and cut at different restriction recognition sequences. An example of two endonucleases and the specific sequence they recognize are Eco RI (GAATTC) and Bam HI (GGATCC). Eco RI cleaves between the guanine and the adenine, GAAATTC and Bam HI between the guanines GAGATCC. Restriction fragment length polymorphism (RFLP) is one molecular technique that requires restriction endonucleases to produce the unique DNA fragments which are the basis for this technique.

Polymerases are enzymes that catalyze the formation of DNA or RNA from nucleotide precursors. All DNA polymerases also require a preexisting template. For example, various DNA polymerases are utilized for different applications [DNA polymerase I (Kornberg polymerase), is used to label DNA; Taq DNA polymerase is used to amplify DNA fragments in the Polymerase Chain Reaction (PCR); etc.]; and some have been engineered for enhanced features, e.g., Sequenase™ which is used in DNA sequencing.

In RFLP the assorted DNA fragments resulting from the restriction endonuclease digestion are resolved by gel electrophoresis. Ethidium bromide staining is then used to reveal the fragments under UV (260 nm) light. Southern blotting of the gel can transfer the DNA fragments to a support membrane. The DNA is then fixed to the membrane and can be subjected to hybridization analysis. This enables identification of bands with sequence similarity to a labeled probe. Several printed reference laboratory manuals [e.g. Molecular cloning: A Laboratory Manual, 3rd Edition; Short Protocols in Molecular Biology, 4th Edition] and Internet web sites such as www.protocol-online.net and www.nwfsc.noaa.-gov/protocols detail these techniques. In dot blots, the target nucleic acid sample is affixed directly to the membrane without electrophoresis followed by hybridization with the labeled probe. Detailed information on these protocols can also be obtained by utilizing a search engine such as Google™ to search the internet.

DNA fingerprinting is an umbrella term that describes molecular identification techniques. As the term implies, the basic genetic material in the organism is utilized to determine its identity and relationship with other isolates. One of the earliest hybridization probes utilized was variable number tandem repeats (VNTRs) or minisatellite DNA (Jeffreys et al. 1985). The term "satellite" originated from buoyant density centrifugation studies in which the bulk of the sample DNA sediments as one main band, but smaller satellite bands were also observed. Satellites are composed of tandem repeats of DNA arranged in consecutive repeats. They sediment apart from the main band containing the bulk of the DNA due to their significantly different base composition. Minisatellites range in size from 1 kb to 20 kb. The VNTR are a type of minisatellite located in the noncoding regions of the genome. The size of the repeat units varies from 9 to 80 base pairs (bp). Smaller repeats consisting of only 1-6 bp are designated microsatellites, or short tandem repeats (STR). The STR have a repetitive region of less than 150 bp. Sequences of both mini- and microsatellites have been utilized in DNA fingerprinting. In Meyer et al. (1991), the DNA of various filamentous fungi were digested separately with different restriction endonucleases and the resulting fragments separated by electrophoresis. Selected VNTR oligonucleo-tides were labeled with 32P and hybridized to the dried gel. Results confirmed that the method based on these minisatellite DNA probes could differentiate between species and strains of fungi. Microsatellites have been used to discriminate strains of the aflatoxigenic species Aspergillus flavus and A. parasiticus (Tran-Dinh and Carter 2000).

The PCR technology provides researchers with a means to rapidly amplify small amounts of DNA, and frees them from the time-consuming task of having to isolate sufficient DNA for RFLP analysis. In PCR a thermostable polymerase is employed to enzymatically amplify a specific region of DNA sequence, defined by a set of two oligonucleotide primers. The target DNA is denatured and the primers are then annealed to the single-stranded DNA. The DNA polymerase then synthesizes the complementary DNA strand across the target region. This process is then repeated and the targeted DNA may be amplified a million-fold or more.

Randomly Amplified Polymorphic DNA (RAPD) is a PCR technique that yields genetic markers without the need to obtain prior nucleotide sequence data (Williams et al. 1990). Random nucleotide sequences are annealed to the template

DNA under low stringency. This is followed by PCR amplification and electrophoresis to produce a DNA fingerprint. The RAPD technique is relatively easy, fast, and requires only a minimum amount of starting material. However, the low stringency of the annealing process can produce PCR artifacts.

Several methods have been developed to reduce the number of artifacts produced by PCR amplification. One method is by using two pairs of PCR primers, or nested primers (Plikaytis et al. 1990). In this technique, the first pair produces a PCR fragment. Then this fragment and the second pair of primers (nested primers) binding to DNA sequence a few bases internal to the first pair of primers, are used in a further amplification. If the first pair of primers amplified the correct locus, then the second pair of primers will produce a slightly smaller PCR fragment. This method requires a knowledge of the sequence immediately adjacent to the first pair of PCR primers in order to synthesize the other "nested" pair of primers.

If the host DNA sequence is not known, Amplified Fragment Length Polymorphism (AFLP) will also reduce the number of PCR artifacts (Vos et al. 1995). In this technique, the target DNA is digested with restriction endonucleases to yield an assortment of different sized DNA fragments. Specific double-stranded adapter oligonucleotides are then ligated to these fragments. PCR primers specific to the adapter sequences with various selective 3' nucleotides are then utilized under high stringency PCR amplification and electrophoresis to produce a unique fragment profile. This technique is time-efficient and amplification is not completely random as in RAPD.

When the sequence of the target DNA is known, several other PCR procedures for identification are available, such as PCR amplification of internal transcribed spacer (ITS) of ribosomal DNA (rDNA). An example of the application of this PCR method is Beck and Ligon (1995) who designed PCR primers to detect Stagonospora nodorum and Septoria triticic in wheat. These primers were derived from species specific DNA sequences of the ITS of the pathogen's ribosomal DNA. The PCR amplification of ITS rDNA has also been employed to identify a wider variety of fungi that were potential pathogens and allergens (Makimura et al. 2001).

Knowledge of the specific sequence of polymorphic loci permits high-stringency PCR and thus circumvents the problem of artifacts and low reproducibility associated with random-primer methods (Scott and Straus 2000). One method of site-specific polymorphisms they reviewed was based on the sequence variability found in the introns of single copy metabolic and structural genes. Glass and Donaldson (1995) tested several such oligonucleotide primers for their ability to amplify segments of DNA that span introns in a selection of these genes. They identified primer sets that provided a useful tool for phylogenic studies of filamentous ascomycetes and related fungi. These oligonucleotide primers were utilized to differentiate Fusarium species (Donaldson et al. 1995). The PCR fragments generated were digested with several 4 bp recognition restriction enzymes. The short recognition site, four bases, of the restriction endonuclease increased the probability that a single base pair polymorphism could be detected.

Two other methods of high-stringency PCR are microsatellite based PCR-amplification and the use of the small subunit ribosomal DNA (SSU rDNA) based primers for DNA fingerprinting (Scott and Straus 2000). For example, Gargas and DePriest (1996) describe a list of PCR primers used to amplify and sequence the small subunit of fungal nuclear rDNA. This information identifies primers for special applications (intron-spanning, intron specific, etc) and represents a valuable resource for further research. Groppe et al. (1995) synthesized oligonucleotides corresponding to regions of the sequence of a microsatellite of the endophytic ascomycete Epichloe typhina. These were used for PCR amplification of DNA from different Epichloe isolates. The DNA from most isolates produced a single PCR product. This study pointed to a potentially useful role for microsatellite-containing loci as a molecular marker for population studies of Epichloe and other unrelated fungi.

DNA sequencing, while not yet extensively utilized because of the time and resources required, has been employed to a limited degree in species identification. Wang et al. (2001) recently reported on the use of mitochondrial cytochrome b gene to identify species of Aspergillus section Flavi. Mycelia of the Aspergillus isolates were harvested and their hyphae ruptured with glass beads and zymolyase. Their mitochondria were then collected by centrifugation and their mtDNA extracted. The sequence of the cytochrome b gene has also been used to distinguish species of Aspergillus section Fumigati (Wang et al. 2000) and for investigating the phylogentic relationships of other species of Aspergillus (Wang et al. 1998).

Single-Strand Conformation Polymorphism (SSCP) is another PCR-based system that requires knowledge of the target DNA sequence to generate specific oligonucleotide primers. In this technique, the target DNA is concurrently labeled and amplified by PCR using a labeled substrate. The PCR product is then denatured and resolved by electrophoresis. Any changes (mutations, etc.) in the target DNA are detected as altered mobility of separated single strands in autoradiograms. Precise information about the exact change can then be obtained by eluting the targeted DNA from the gel and amplifying it again for sequence determination (Hayashi 1991).

Heteroduplex analysis, like SSCP, is a recently developed technique that can detect a single base difference in target DNA (Keen et al. 1991). PCR amplification products from the isolates are combined after heat denaturation and then allowed to reanneal to form heteroduplexes. Any mismatched nucleotides, caused by substitutions, insertions, deletions, etc, will affect the DNA structure of the heteroduplex and lower its electrophoretic mobility. The heteroduplex is compared to duplexes with complete base complementarity by electro-phoresis. Kumeda and Asao (2001) employed this technique for the detection of intraspecific variation in isolates of Aspergillus Section Flavi. In their heteroduplex panel analysis (HAP), fragments of the internal spacer (ITS)

regions of the rDNA gene of the different isolates were first amplified. Heteroduplexes were then generated with the standard ITS reference fragment and then subjected to electrophoresis. The results of this HAP study corresponded well with the established taxonomy of the Section Flavi.

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