Amplified Ribosomal DNA Restriction Analysis

Last updated

Amplified rDNA (Ribosomal DNA) Restriction Analysis is the extension of the technique of RFLP (restriction fragment length polymorphism) to the gene encoding the small (16s) ribosomal subunit of bacteria. The technique involves an enzymatic amplification using primers directed at the conserved regions at the ends of the 16s gene, followed by digestion using tetracutter Restriction enzymes. The pattern obtained is said to be representative of the species analysed. Patterns obtained from several restriction enzymes can be used to phylogenetically characterize cultured isolates and 16s genes obtained through cloning from community DNA [1]

ARDRA rationale and procedure

Vaneechoutte et al., [2] were among the first to use this method and applied it to characterize Mycobacterium species and Acinetobacter species [3] Numerous other studies have applied ARDRA for different bacterial taxa. Based on the simple formula for the frequency of random occurrence of a Restriction site, a 4-bp sequence can occur once every 256 bp. A restriction enzyme having a recognition site of more than 4 bp would be irrelevant with respect to a gene of approximately 1500 bp such as that coding for the 16s ribosomal subunit. A number of tetracutter Restriction enzymes are available in the market. For the sake of statistical significance, at least three restriction enzymes must be used for the analysis to overcome the probability of certain restriction enzymes to yield similar patterns for not so unrelated organisms.

The amplicon to be analysed must preferably correspond to a size of greater than 1000 bp, purely for the sake of encountering a greater possibility of the restriction site. The amplicons must be preferably purified before digestion. This can be done by numerous commercially available purification kits. Care should be taken to choose the restriction enzymes. Certain restriction enzymes recognize the same sites, and cannot contribute productively to the analysis. Overnight digestion (10–16 hours) of about 300-500 ng of amplicon DNA in a 20 μL system with 4-5 units of Restriction Enzyme along with the recommended buffer at the prescribed temperature is recommended. Following digestion, the reaction is stopped and the entire digest run in a 2-3% agarose gel at 90-100 V. The gel should be of a length that would allow proper resolution of minor fragments, as small as 100–200 bp.

Analysis of the patterns is done with methods used for RAPD patterns. Clusters of related bacteria can be represented in the form of a cladogram or phylogram. After initial analysis a multivariate analysis program such as NT-SYS [4] or PAST [5] furnishing details about the presence or absence of bands, marked as 1(for presence) and 0(for absence). The data can be subsequently used for generating a phylogram or cladogram. The data table can be used to plot a phylogenetic tree that would indicate the relationship of the organisms based on the restriction pattern obtained from their respective 16s genes. Commercial software is also available for the analysis of these patterns, most used are the packages GelCompar II and BioNumerics.

Related Research Articles

In molecular biology, restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, in order to distinguish individuals, populations, or species or to pinpoint the locations of genes within a sequence. The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.

Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It is also a product and services market, with an estimated value of $168 billion by 2017.

<i>Acinetobacter</i> Genus of bacteria

Acinetobacter is a genus of gram-negative bacteria belonging to the wider class of Gammaproteobacteria. Acinetobacter species are oxidase-negative, exhibit twitching motility, and occur in pairs under magnification.

Variable number tandem repeat

A variable number tandem repeat 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 among 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.

Multilocus sequence typing (MLST) is a technique in molecular biology for the typing of multiple loci, using DNA sequences of internal fragments of multiple housekeeping genes to characterize isolates of microbial species.

Temperature gradient gel electrophoresis

Temperature gradient gel electrophoresis (TGGE) and denaturing gradient gel electrophoresis (DGGE) are forms of electrophoresis which use either a temperature or chemical gradient to denature the sample as it moves across an acrylamide gel. TGGE and DGGE can be applied to nucleic acids such as DNA and RNA, and proteins. TGGE relies on temperature dependent changes in structure to separate nucleic acids. DGGE separates genes of the same size based on their different denaturing ability which is determined by their base pair sequence. DGGE was the original technique, and TGGE a refinement of it.

Amplified fragment length polymorphism

AFLP-PCR or just AFLP is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic engineering. Developed in the early 1990s by Keygene, AFLP uses restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the sticky ends of the restriction fragments. A subset of the restriction fragments is then selected to be amplified. This selection is achieved by using primers complementary to the adaptor sequence, the restriction site sequence and a few nucleotides inside the restriction site fragments. The amplified fragments are separated and visualized on denaturing on agarose gel electrophoresis, either through autoradiography or fluorescence methodologies, or via automated capillary sequencing instruments.

Gene map Spatial arrangement of genes on a chromosome

Gene maps help describe the spatial arrangement of genes on a chromosome. Genes are designated to a specific location on a chromosome known as the locus and can be used as molecular markers to find the distance between other genes on a chromosome. Maps provide researchers with the opportunity to predict the inheritance patterns of specific traits, which can eventually lead to a better understanding of disease-linked traits.

Ribotyping is a molecular technique for bacterial identification and characterization that uses information from rRNA-based phylogenetic analyses. It is a rapid and specific method widely used in clinical diagnostics and analysis of microbial communities in food, water, and beverages.

Terminal restriction fragment length polymorphism is a molecular biology technique for profiling of microbial communities based on the position of a restriction site closest to a labelled end of an amplified gene. The method is based on digesting a mixture of PCR amplified variants of a single gene using one or more restriction enzymes and detecting the size of each of the individual resulting terminal fragments using a DNA sequencer. The result is a graph image where the x-axis represents the sizes of the fragment and the y-axis represents their fluorescence intensity.

SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. A SNP is a single base pair mutation at a specific locus, usually consisting of two alleles. SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.

Multiplex ligation-dependent probe amplification (MLPA) is a variation of the multiplex polymerase chain reaction that permits amplification of multiple targets with only a single primer pair. It detects copy number changes at the molecular level, and software programs are used for analysis. Identification of deletions or duplications can indicate pathogenic mutations, thus MLPA is an important diagnostic tool used in clinical pathology laboratories worldwide.

Loop-mediated isothermal amplification (LAMP) is a single-tube technique for the amplification of DNA and a low-cost alternative to detect certain diseases. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP with a reverse transcription step to allow the detection of RNA.

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.

MAGIChip

MAGIChips, also known as "microarrays of gel-immobilized compounds on a chip" or "three-dimensional DNA microarrays", are devices for molecular hybridization produced by immobilizing oligonucleotides, DNA, enzymes, antibodies, and other compounds on a photopolymerized micromatrix of polyacrylamide gel pads of 100x100x20µm or smaller size. This technology is used for analysis of nucleic acid hybridization, specific binding of DNA, and low-molecular weight compounds with proteins, and protein-protein interactions.

Diversity arrays technology

Diversity Arrays Technology (DArT) is a high-throughput genetic marker technique that can detect allelic variations to provides comprehensive genome coverage without any DNA sequence information for genotyping and other genetic analysis. The general steps involve reducing the complexity of the genomic DNA with specific restriction enzymes, choosing diverse fragments to serve as representations for the parent genomes, amplify via polymerase chain reaction (PCR), insert fragments into a vector to be placed as probes within a microarray, then fluorescent targets from a reference sequence will be allowed to hybridize with probes and put through an imaging system. The objective is to identify and quantify various forms of DNA polymorphism within genomic DNA of sampled species.

Ribosomal RNA (rRNA) intergenic spacer analysis (RISA) is a method of microbial community analysis that provides a means of comparing differing environments or treatment impacts without the bias imposed by culture- dependent approaches. This type of analysis is often referred to as community fingerprinting. RISA involves PCR amplification of a region of the rRNA gene operon between the small (16S) and large (23S) subunits called the intergenic spacer region ISR.

Combined bisulfite restriction analysis

Combined Bisulfite Restriction Analysis is a molecular biology technique that allows for the sensitive quantification of DNA methylation levels at a specific genomic locus on a DNA sequence in a small sample of genomic DNA. The technique is a variation of bisulfite sequencing, and combines bisulfite conversion based polymerase chain reaction with restriction digestion. Originally developed to reliably handle minute amounts of genomic DNA from microdissected paraffin-embedded tissue samples, the technique has since seen widespread usage in cancer research and epigenetics studies.

Fibrolytic bacteria constitute a group of microorganisms that are able to process complex plant polysaccharides thanks to their capacity to synthesize cellulolytic and hemicellulolytic enzymes. Polysaccharides are present in plant cellular cell walls in a compact fiber form where they are mainly composed of cellulose and hemicellulose.

Community fingerprinting is a set of molecular biology techniques that can be used to quickly profile the diversity of a microbial community. Rather than directly identifying or counting individual cells in an environmental sample, these techniques show how many variants of a gene are present. In general, it is assumed that each different gene variant represents a different type of microbe. Community fingerprinting is used by microbiologists studying a variety of microbial systems to measure biodiversity or track changes in community structure over time. The method analyzes environmental samples by assaying genomic DNA. This approach offers an alternative to microbial culturing, which is important because most microbes cannot be cultured in the laboratory. Community fingerprinting does not result in identification of individual microbe species; instead, it presents an overall picture of a microbial community. These methods are now largely being replaced by high throughput sequencing, such as targeted microbiome analysis and metagenomics.

References

  1. ↑ Sklarz, M., R. Angel, O. Gillor, and MIM. Soares. 2009. Evaluating amplified rDNA restriction analysis assay for identification of bacterial communities. Ant van Leeuwenhoek 96:659–664.
  2. ↑ Vaneechoutte, M., H. De Beenhouwer, G. Claeys, G. Verschraegen, A. De Rouck, N. Paepe, A. Elaichouni, and F. Portaels. 1993. Identification of Mycobacterium species with amplified rDNA restriction analysis. J. Clin. Microbiol. 31:2061–2065.
  3. ↑ Vaneechoutte, M., L. Dijkshoorn, I. Tjernberg, A. Elaichouni, P. De vos, G. Claeys, and G. Verschraegen. 1995. Identification of Acinetobacter Genomic Species by Amplified Ribosomal DNA Restriction Analysis. J. Clin. Microbiol. 33:11–15.
  4. ↑ Exeter Software: NTSYSpc http://www.exetersoftware.com/cat/ntsyspc/ntsyspc.html
  5. ↑ PAlaeontological STatistics: http://folk.uio.no/ohammer/past/
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.