What is Ribotyping?

Ribotyping involves the fingerprinting of genomic DNA restriction fragments that contain all or part of the genes coding for the 16S and 23S rRNA. Conceptually, ribotyping is similar to probing restriction fragments of chromosomal DNA with cloned probes (randomly cloned probes or probes derived from a specific coding sequence such as that of a virulence factor). Ribotyping is a method that can identify and classify bacteria based upon differences in rRNA. It generates a highly reproducible and precise fingerprint that can be used to classify bacteria from the genus through and beyond the species level. DNA is extracted from a colony of bacteria and then restricted into discrete-sized fragments. The DNA is then transferred to a membrane and probed with a region of the rRNA operon to reveal the pattern of rRNA genes. The pattern is recorded, digitized and stored in a database. It is variations that exist among bacteria in both the position and intensity of rRNA bands that can be used for their classification and identification. Databases for Listeria (80 pattern types), Salmonella (97 pattern types), Escherichia (65 pattern types) and Staphylococcus (252 pattern types) have been established. In addition over 35 different genera have been tested and RiboPrin t® patterns obtained. These data continue as more bacteria are ribotyped.

Examples of RiboPrint® Patterns

Why Ribotype?
Contamination source bacteria in finished products can originate from any one of the ingredients, processing personnel or the environment. Ribotyping allows the establishment of unequivocal relationships between bacterial isolates recovered from any of these sources and the finished product. Molecular epidemiology A number of diseases in animals that are caused by bacteria can be traced to the consumption of contaminated feed. Ribotyping can help to identify the contaminated feed source for elimination. Spread of infection within a group of animals can also be followed by ribotyping and routes of animal-to-animal transmission identified. Identification RiboPrint® patterns can be used to identify a bacterium if that pattern is in the database. Currently well over 600 patterns are in the database representing both Gram positive and Gram negative bacteria and this database grows continuously The largest number of RiboPrint patterns have been collected for Listeria, Salmonella, Staphylococcus and Escherichia coli. Custom identification databases can be developed for virtually any need.

How is the RiboPrinter® System's typing method superior?
Key to the utility of a typing method is its reproducibility and standardization. The most common technique for typing is using a panel of antisera which selectively react with a certain degree of nonoverlapping specificity. Establishing a serotyping scheme for a new microorganism is tedious and dependent upon the quality of the panel of antisera. Furthermore the dissemination of this technique to other laboratories is usually limited and the longevity of the assay dependent upon antisera stocks. Virtually no typing method has been developed to the stage that allows standards to be established especially when classifying below a species level. Although a number of techniques have been explored and practiced in different laboratories, there is no method whose output is easily standardized. Ribotyping has over the past 10-15 years been established as a robust classification and typing method. It was originally developed as a manual method, but took a quantum leap in reproducibility and standardization with the development of the RiboPrinter® Microbial Characterization System by Qualicon (a subsidiary of DuPont). This instrument automates all of the steps in the process from cell lysis to data capture and database comparisons. Further, reagent cassettes including the enzymes, enzyme conjugated -hybridization probe, electrophoretic gel and membrane have been developed with aim of delivering consistent performance. Data capture is accomplished via a CCD camera and the patterns stored in a digitized format. It is therefore relatively easy to compare results among laboratories and to exchange data electronically.

Ribotyping Protocol
Genomic DNA Isolation and Restriction Endonuclease Digestion Confluent growth was scraped with a sterile flat-head toothpick and suspended in 200 microliters ( L) 50mM Tris, 50mM EDTA (pH 8.0). Six hundred L more of 50mM Tris, 50 mM EDTA was then added and the suspension was thoroughly mixed by pipetting up and down. Then 45 L 20% sodium dodecyl sulfate (SDS) and then 10 L proteinase K (20 g/mL; Pharmacia, Piscataway, N.J.) were added. This solution was then incubated at 40°C for 1 hour. After an equal volume of phenol was added to each tube, s amples were vortexed, and centrifuged for 5 minutes. The top layer was extracted, and an equal volume of chloroform was added. The preparation was vortexed again, centrifuged, and extracted. Two and a half volumes of absolute ethanol were added and the DNA was precipitated out and spooled onto a glass capillary pipette. The DNA was washed with a few drops of absolute ethanol, dried, and re-suspended in 50 L dH2O. Separate restriction endonuclease digestion reactions were set up using EcoR1 and PvuII, 10 units/ L (Boehringer Mannheim, GmbH, Germany) as instructed by the manufacturer using 2 L DNA. They were incubated at 37°C overnight. The samples were then centrifuged and 0.5 l of enzyme was added. The samples were re-incubated at 37°C for a minimum of 3 hours. They were centrifuged again and 3 L stop dye was added. Every batch of restriction enzyme reaction contained two reactions with a positive control strain that was included on two lanes on each gel. Gel Electrophoresis and Southern Blot Hybridization Samples were run on a 0.8% agarose gel in 1X Tris-borate-EDTA at 22 volts and 17 milliamps, for 17 hours. HindIII was used as a size standard along with a known E. coli isolate designated as 3915. Each agarose gel was assigned a number, and when more than one gel was run, the position of the first standard reference strain was changed in each gel (1st lane on the first gel, to the Nth lane on the Nth gel). Electrophoresis gels were stained in ethidium bromide. If two gels were stained in a single container, one corner of the gel with the higher number was clipped. The label for each gel was also transferred to the staining container. Each gel was then photographed and a hard copy of the print was labeled with the gel sheet (containing the isolate numbers loaded on each lane, and the enzyme used to cut the DNA, plus date, gel number, voltage, current, gel strength, buffer strength, and electrophoresis time information). After photography each gel was returned to the same staining container. The DNA fragm ents were then transferred to a Nitran filter (Schleicher & Schuell, Keene, N.H.), baked at 80°C for 1 hour, and probed with 32P-labeled copies of E. coli rRNA, which were made by extension of random hexanucleotide primers using Avian Technical Support Document Upper Oyster Creek (Segment 1245) Appendix C C-2 Myeloblastosis Virus reverse transcriptase (Stratagene, La Jolla, California) under conditions specified by the supplier. Each membrane filter was labeled with the gel number, restriction enzyme designation, date, and technician¶s initials. Hybridization was done in 5X SSC (1X SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% SDS, 1mM EDTA, and 50% formamide at room temperature overnight. Salmon sperm DNA and blocking reagent (Boehringer Mannheim GmbH, Germany) were used to block nonspecific binding. Three washes were done with a solution of 2X SSC and 0.1% SDS, once at 25°C for 20 minutes and twice at 65°C for 20 minutes to wash off low-homology, non-specific binding. Blots were then exposed with an intensifying screen to x-ray film (Kodak, Rochester, New York) for 24 hours at -70°C. Two to three exposures were done to ensure all possible bands would show up. All reagents and buffers were made according to formulas in the MEI standard operating proce dures. Reagents and buffers were tested for sterility. Restriction Fragment Length Polymorphism Analysis Each ribotype was then analyzed by assigning an alphanumeric pattern based on the distance between the bands. Bands more than 3 mm apart were counted as singles while bands that were within 3 mm of each other were counted as doubles or triples. For example, two bands that were closer than 3 mm were designated ³2´ and a group of three bands with 3 mm or less between each band were designated ³3.´ A ³1´ designated a single band more than 3 mm distant from another band. Each unique banding pattern was called a ribotype and assigned an alphanumeric pattern. Two isolates that had the same numeric value but different banding patterns were assigned letters to differentiate the two ribotypes. For example, two isolates with an identical numerical pattern of 2122111, but with the bands shifted so the two isolates did not have identical banding patterns, were labeled 2122111A and 2122111B. The ribotypes were then entered into a Microsoft Access® database and compared to the other ribotypes of known source in the library database. Ribotype patterns that numerically appeared to be similar were compared side -byside visually to judge matching. Isolates with the same PvuII and EcoR1 ribotypes are deemed to be members of the same ribogroup. Using this approach only isolates with two identical ribotypes were grouped together.

variable number tandem repeats
variable number tandem repeats (VNTR; minisatellite DNA) A family of genetic loci found in eukaryotes consisting of short (15±100 bp) sequences of DNA repeated in tandem arrays; in humans these arrays are typically 1±5 kb long. The alleles for any particular locus all have the same sequence but differ as to how many times the sequence is repeated. VNTR loci contribute to repetitive DNA and have proved valuable in DNA fingerprinting, especially in human forensic science. The VNTR sequences can be released intact from a DNA sample using restriction endonuclease enzymes and identified using gene probes with Southern blotting. Alternatively, the VNTR alleles can be amplified by the polymerase chain reaction, separated by gel electrophoresis, and the resultant patterns compared without the need for special gene probes. Since each VNTR locus typically has many different alleles, the likelihood of two individuals having identical sets of alleles for even a few such loci is very remote. Hence, a DNA sample can be ascribed to a particular person with a high degree of certainty.

A Variable Number Tandem Repeat (or VNTR) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length between individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting.

fig:schematic of a variable number tandem repeats in 4 alleles

VNTR structure and allelic variation
In the schematic above, the rectangular blocks represent each of the repeated DNA sequences at a particular VNTR location. The repeats are tandem - they are clustered together and oriented in the same direction. Individual repeats can be removed from (or added to) the VNTR via recombination or replication errors, leading to alleles with different numbers of repeats. Flanking the repeats are segments of non-repetitive sequence (shown here as thin lines), allowing the VNTR blocks to be extracted with restriction enzymes and analyzed by RFLP, or amplified by the polymerase chain reaction (PCR) technique and their size determined by gel electrophoresis.

Use of VNTRs in genetic analysis
VNTRs were an important source of RFLP genetic markers used in linkage analysis (mapping) of genomes. Now that many genomes have been sequenced, VNTRs have become essential to forensic crime investigations, via DNA fingerprinting and the CODIS database. When removed from surrounding DNA by the PCR or RFLP methods, and their size determined by gel electrophoresis or Southern blotting, they produce a pattern of bands unique to each individual. When tested with a group of independent VNTR markers, the likelihood of two unrelated individuals having the same allelic pattern is extremely improbable. VNTR analysis is also being used to study genetic diversity and breeding patterns in populations of wild or domesticated animals.

Chromosomal locations of the 13 VNTR loci in the CODIS panel.

VNTR Inheritance
In analyzing VNTR data, two basic genetic principles can be used:

Identity Matching- both VNTR alleles from a specific location must match. If two samples are from the same individual, they must show the same allele pattern. Inheritance Matching- the VNTR alleles must follow the rules of inheritance. In matching an individual with his parents or children, a person must have an allele that matches one from each parent. If the relationship is more distant, such as a grandparent or sibling, then matches must be consistent with the degree of relatedness.


Relationship to other types of repetitive DNA
Repetitive DNA, representing over 40% of the human genome, is arranged in a bewildering array of patterns. Repeats were first identified by the extraction of Satellite DNA, which does not reveal how they are organized. The use of restriction enzymes showed that some repeat blocks were interspersed throughout the genome. DNA sequencing later showed that other repeats are clustered at specific locations, with tandem repeats being more common than inverted repeats (which may interfere with DNA replication). VNTRs are the class of clustered tandem repeats that exhibit allelic variation in their lengths.

Classes of VNTRs
There are two principal families of VNTRs: microsatellites and minisatellites. The former are repeats of sequences less than about 5 base pairs in length (an arbitrary cutoff), while the latter involve longer blocks. Confusing this distinction is the recent use of the terms Short Tandem Repeat (STR) and Simple Sequence Repeat (SSR), which are more descriptive, but whose definitions are similar to that of microsatellites. VNTRs with very short repeat blocks may be unstable - dinucleotide repeats may vary from one tissue to another within an individual, while trinucleotide repeats have been found to vary from one generation to another (see Huntington's disease). The 13 assays used in the CODIS database are usually referred to as STRs, and most analyze VNTRs that involve repeats of 4 base pairs.

"Minisatellites" consist of repetitive, generally GC-rich, variant repeats that range in length from 10 to over 100 bp. These variant repeats are tandemly intermingled, which makes minisatellites ideal for studying DNA turnover mechanisms. A minisatellite is a section of DNA that consists of a short series of bases 10±60 bp. These occur at more than 1,000 locations in the human genome. Some minisatellites contain a central (or "core") sequence of letters ³GGGCAGGANG´ (where N can be any base) or more generally a strand bias with purines (adenosine (A) and guanine (G)) on one strand and pyrimidines (cytosine (C) and thymine (T)) on the other. It has been proposed that this sequence per se encourages chromosomes to swap DNA. In alternative models, it is the presence of a neighbouring cis-acting meiotic double-strand break hotspot which is the primary cause of minisatellite repeat copy number variations. Somatic changes are suggested to result from replication difficulties (which might include replication slippage, among other phenomena). When such events occur, mistakes are made, thus causing minisatellites at over 1000 locations in a person's genome to have slightly different numbers of repeats, thereby making each individual unique. The most highly mutable minisatellite locus described so far is CEB1 (D2S90) described by Vergnaud.

Since the fortuitous discovery of the first human minisatellite in 1980 by A.R. Wyman and R. White[4] and especially the discovery that the extreme polymorphism of minisatellites made them superb for DNA fingerprinting by Alec Jeffreys,[5] this class of repeats has been an intense focus of studies that have addressed the turnover mechanisms that provoke their instability. Due to their high level of polymorphism, minisatellites have been extensively used for DNA fingerprinting as well as for genetic markers in linkage analysis and population studies. Minisatellites have also been implicated as regulators of gene expression (e.g., at levels of transcription, alternative splicing, or imprint control) or as part of bona fide open reading frames.

Minisatellites have been associated with chromosome fragile sites and are proximal to a number of recurrent translocation breakpoints. Some human minisatellites (~1%) have been demonstrated to be hypermutable, with an average mutation rate in the germline higher that 0.5% up to over 20%, making them the most unstable region in the human genome known to date. While other genomes (mouse, rat and pig) contain minisatellite-like sequences, none was found to be hypermutable. Since all hypermutable minisatellites contain internal variants, they provide extremely informative systems for analyzing the complex turnover processes that occur at this class of tandem repeat. Minisatellite variant repeat mapping by PCR (MVR-PCR) has been extensively used to chart the interspersion patterns of variant repeats along the array, which provides details on the structure of the alleles before and after mutation. Studies have revealed distinct mutation processes operating in somatic and germline cells. Somatic instability detected in blood DNA shows simple and rare intra-allelic events two to three orders of magnitude lower than in sperm. In contrast, complex inter-allelic conversion-like events occur in the germline.[6] Additional analyses of DNA sequences flanking human minisatellites have also revealed an intense and highly localized meiotic crossover hotspot that is centered upstream of the unstable side of minisatellite arrays. Repeat turnover therefore appears to be controlled by recombinational activity in DNA that flanks the repeat array and results in a polarity of mutation. These findings have suggested that minisatellites most probably evolved as bystanders of localized meiotic recombination hotspots in the human genome.

Evolutionary aspects
Studies have shown that the evolutionary fate of minisatellites tends towards an equilibrium distribution in the size of alleles, until mutations in the flanking DNA affect the recombinational activity of a minisatellite by suppressing DNA instability. Such an event would ultimately lead to the extinction of a hypermutable minisatellite by meiotic drive.

Microsatellites, also known as Simple Sequence Repeats (SSRs), or sometimes Short Tandem Repeats (STRs), are repeating sequences of 1-6 base pairs of DNA.Microsatellites are typically neutral and codominant. They are used as molecular markers in genetics, for kinship, population and other studies. They can also be used to study gene duplication or deletion.

1 Introduction 2 Amplification of microsatellites o 2.1 Development of microsatellite primers o 2.2 ISSR-PCR 3 Limitations of microsatellites 4 Role in evolution o 4.1 Mechanisms for change o 4.2 In proteins o 4.3 Gene regulation o 4.4 Within introns o 4.5 Within transposons

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One common example of a microsatellite is a (CA)n repeat, where n is variable between alleles. These markers often present high levels of inter- and intra-specific polymorphism, particularly when tandem repeats number ten or greater.[2] The repeated sequence is often simple, consisting of two, three or four nucleotides (di-, tri-, and tetranucleotide repeats respectively), and can be repeated 10 to 100 times. CA nucleotide repeats are very frequent in human and other genomes, and are present every few thousand base pairs. As there are often many alleles present at a microsatellite locus, genotypes within pedigrees are often fully informative, in that the progenitor of a particular allele can often be identified. In this way, microsatellites are ideal for determining paternity, population genetic studies and recombination mapping. It is also the only molecular marker to provide clues about which alleles are more closely related.[3] Microsatellites owe their variability to an increased rate of mutation compared to other neutral regions of DNA. These high rates of mutation can be explained most frequently by slipped strand mispairing (slippage) during DNA replication on a single DNA strand. Mutation may also occur during recombination during meiosis.[4] Some errors in slippage are rectified by proofreading mechanisms within the nucleus, but some mutations can escape repair. The size of the repeat unit, the number of repeats and the presence of variant repeats are all factors, as well as the frequency of transcription in the area of the DNA repeat. Interruption of microsatellites, perhaps due to mutation, can result in reduced polymorphism. However, this same mechanism can occasionally lead to incorrect amplification of microsatellites; if slippage occurs early on during PCR, microsatellites of incorrect lengths can be amplified.

Amplification of microsatellites
Microsatellites can be amplified for identification by the polymerase chain reaction (PCR) process, using the unique sequences of flanking regions as primers. DNA is repeatedly denatured at a high temperature to separate the double strand, then cooled to allow annealing of primers and the extension of nucleotide sequences through the microsatellite. This process results in production of enough DNA to be visible on agarose or polyacrylamide gels; only small amounts of DNA are needed for amplification as thermocycling in this manner creates an exponential increase in the replicated segment[5]. With the abundance of PCR technology, primers that flank microsatellite loci are simple and quick to use, but the development of correctly functioning primers is often a tedious and costly process.

Development of microsatellite primers
If searching for microsatellite markers in specific regions of a genome; for example within a particular exon of a gene, primers can be designed manually. This involves searching the genomic DNA sequence for microsatellite repeats, which can be done by eye or by using automated tools such as repeat masker. Once the potentially useful microsatellites are determined (removing non-useful ones such as those with random inserts within the repeat region), the flanking sequences can be used to design oligonucleotide primers which will amplify the specific microsatellite repeat in a PCR reaction. Random microsatellite primers can be developed by cloning random segments of DNA from the focal species. These are inserted into a plasmid or bacteriophage vector, which is in turn implanted into Escherichia coli bacteria. Colonies are then developed, and screened with fluorescently±labelled oligonucleotide sequences that will hybridise to a microsatellite repeat, if present on the DNA segment. If positive clones can be obtained from this procedure, the DNA is sequenced and PCR primers are chosen from sequences flanking such regions to determine a specific locus. This process involves significant trial and error on the part of researchers, as microsatellite repeat sequences must be predicted and primers that are randomly isolated may not display significant polymorphism.[2][6] Microsatellite loci are widely distributed throughout the genome and can be isolated from semi-degraded DNA of older specimens, as all that is needed is a suitable substrate for amplification through PCR. More recent techniques involve using oligonucleotide sequences consisting of repeats complimentary to repeats in the microsatellite to "enrich" the DNA extracted. The oligonucleotide probe hybridizes with the repeat in the microsatellite, and the probe/microsatellite complex is then pulled out of solution. The enriched DNA is then cloned as normal, but the proportion of successes will now be much higher, drastically reducing the time required to develop the regions for use. However, which probes to use can be a trial and error process in itself.[7]

ISSR (for inter-simple sequence repeat) is a general term for a genome region between microsatellite loci. The complementary sequences to two neighboring microsatellites are used as PCR primers; the variable region between them gets amplified. The limited length of amplification cycles during PCR prevents excessive replication of overly long contiguous DNA sequences, so the result will be a mix of a variety of amplified DNA strands which are generally short but vary much in length. Sequences amplified by ISSR-PCR can be used for DNA fingerprinting. Since an ISSR may be a conserved or nonconserved region, this technique is not useful for distinguishing individuals, but rather for phylogeography analyses or maybe delimiting species; sequence diversity is lower than in SSR-PCR, but still higher than in actual gene sequences. In addition, microsatellite sequencing and ISSR sequencing are mutually assisting, as one produces primers for the other.

Limitations of microsatellites
Microsatellites have proved to be versatile molecular markers, particularly for population analysis, but they are not without limitations. Microsatellites developed for particular species can often be applied to closely related species, but the percentage of loci that successfully amplify may decrease with increasing genetic distance.[6] Point mutation in the primer annealing sites in such species may lead to the occurrence of µnull alleles¶, where microsatellites fail to amplify in PCR assays.[6][8] Null alleles can be attributed to several phenomena. Sequence divergence in flanking regions can lead to poor primer annealing, especially at the 3¶ section, where extension commences; preferential amplification of particular size alleles due to the competitive nature of PCR can lead to heterozygous individuals being scored for homozygosity (partial null). PCR failure may result when particular loci fail to amplify, whereas others amplify more efficiently and may appear homozygous on a gel assay, when they are in reality heterozygous in the genome. Null alleles complicate the interpretation of microsatellite allele frequencies and thus make estimates of relatedness faulty. Furthermore, stochastic effects of sampling that occurs during mating may change allele frequencies in a way that is very similar to the effect of null alleles; an excessive frequency of homozygotes causing deviations from Hardy-Weinberg equilibrium

expectations. Since null alleles are a technical problem and sampling effects that occur during mating are a real biological property of a population, it is often very important to distinguish between them if excess homozygotes are observed. When using microsatellites to compare species,homologous loci may be easily amplified in related species, but the number of loci that amplify successfully during PCR may decrease with increased genetic distance between the species in question. Mutation in microsatellite alleles is biased in the sense that larger alleles contain more bases, and are therefore likely to be mistranslated in DNA replication. Smaller alleles also tend to increase in size, whereas larger alleles tend to decrease in size, as they may be subject to an upper size limit; this constraint has been determined but possible values have not yet been specified. If there is a large size difference between individual alleles, then there may be increased instability during recombination at meiosis.[6] In tumour cells, where controls on replication may be damaged, microsatellites may be gained or lost at an especially high frequency during each round of mitosis. Hence a tumour cell line might show a different genetic fingerprint from that of the host tissue.

Tandem repeat
Tandem repeats occur in DNA when a pattern of two or more nucleotides is repeated and the repetitions are directly adjacent to each other.
Example An example would be:

in which the sequence A-T-T-C-G is repeated three times.

When between 10 and 60 nucleotides are repeated, it is called a minisatellite. Those with fewer are known as microsatellites or short tandem repeats. When exactly two nucleotides are repeated, it is called a "dinucleotide repeat"; when three are repeated, it is called a "trinucleotide repeat" (as in trinucleotide repeat disorders.) When the number is not known, variable, or irrelevant, it is sometimes called a variable number tandem repeat (VNTR). MeSH classifies variable number tandem repeats under minisatellites.[2]

Tandem repeat describes a pattern that helps determine an individual's inherited traits. Tandem repeats can be very useful in determining parentage. Short tandem repeats are used for certain genealogical DNA tests. DNA is examined from microsatellites within the chromosomal DNA. Minisatellite is another way of saying special regions of the loci. Polymerase chain reaction (or PCR) is performed on the minisatellite areas. The PCR must be performed on each organism being tested. The amplified material is then run through electrophoresis. By checking the percentage of bands that match, parentage is determined. In the field of Computer Science, tandem repeats in strings (e.g. DNA sequences) can be efficiently detected using suffix trees or suffix arrays. Studies in 2004 linked the unusual genetic plasticity of dogs to mutations in tandem repeats.[3]

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