Multiple loci VNTR analysis

Last updated
Tandum (top) versus interspersed (bottom) repeated nucleic acid sequence Tandem and interspersed repeat schematic.png
Tandum (top) versus interspersed (bottom) repeated nucleic acid sequence

Multiple loci VNTR analysis (MLVA) is a method employed for the genetic analysis of particular microorganisms, such as pathogenic bacteria, that takes advantage of the polymorphism of tandemly repeated DNA sequences. A "VNTR" is a "variable-number tandem repeat". This method is well known in forensic science since it is the basis of DNA fingerprinting in humans. When applied to bacteria, it contributes to forensic microbiology through which the source of a particular strain might eventually be traced back, making it a useful technique for outbreak surveillance.

Contents

In a typical MLVA, a number of well-selected and characterised (in terms of mutation rate and diversity) loci are amplified by polymerase chain reaction (PCR), so that the size of each locus can be measured, usually by electrophoresis of the amplification products together with reference DNA fragments (a so-called DNA size marker). Different electrophoresis equipment can be used depending on the required size estimate accuracy, and the local laboratory set-up, from basic agarose gel electrophoresis up to the more sophisticated and high-throughput capillary electrophoresis devices. [1] From this size estimate, the number of repeat units at each locus can be deduced. The resulting information is a code which can be easily compared to reference databases once the assay has been harmonised and standardised. [2] [3]

MLVA has become a major first line typing tool in a number of pathogens where such an harmonisation could be achieved, including Mycobacterium tuberculosis , [4] Bacillus anthracis, [5] Brucella. [6] [7]

Some MLVA-associated web sites

Software for analysis of MLVA data

Related Research Articles

<span class="mw-page-title-main">Gel electrophoresis</span> Method for separation and analysis of biomolecules

Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.

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.

A microsatellite is a tract of repetitive DNA in which certain DNA motifs are repeated, typically 5–50 times. Microsatellites occur at thousands of locations within an organism's genome. They have a higher mutation rate than other areas of DNA leading to high genetic diversity. Microsatellites are often referred to as short tandem repeats (STRs) by forensic geneticists and in genetic genealogy, or as simple sequence repeats (SSRs) by plant geneticists.

Tandem repeats occur in DNA when a pattern of one or more nucleotides is repeated and the repetitions are directly adjacent to each other. Several protein domains also form tandem repeats within their amino acid primary structure, such as armadillo repeats. However, in proteins, perfect tandem repeats are unlikely in most in vivo proteins, and most known repeats are in proteins which have been designed.

A minisatellite is a tract of repetitive DNA in which certain DNA motifs are typically repeated 5-50 times. Minisatellites occur at more than 1,000 locations in the human genome and they are notable for their high mutation rate and high diversity in the population. Minisatellites are prominent in the centromeres and telomeres of chromosomes, the latter protecting the chromosomes from damage. The name "satellite" refers to the early observation that centrifugation of genomic DNA in a test tube separates a prominent layer of bulk DNA from accompanying "satellite" layers of repetitive DNA. Minisatellites are small sequences of DNA that do not encode proteins but appear throughout the genome hundreds of times, with many repeated copies lying next to each other.

<span class="mw-page-title-main">Haplotype</span> Group of genes from one parent

A haplotype is a group of alleles in an organism that are inherited together from a single parent.

<span class="mw-page-title-main">Variable number tandem repeat</span>

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.

Variable number of tandem repeat locus is any DNA sequence that exist in multiple copies strung together in a variety of tandem lengths. The number of repeat copies present at a locus can be visualized by means of a Multi-locus or Multiple Loci VNTR Analysis (MLVA). In short, oligonucleotide primers are developed for each specific tandem repeat locus, followed by PCR and agarose gel electrophoresis. When the length of the repeat and the size of the flanking regions is known, the number of repeats can be calculated. Analysis of multiple loci will result in a genotype.

A null allele is a nonfunctional allele caused by a genetic mutation. Such mutations can cause a complete lack of production of the associated gene product or a product that does not function properly; in either case, the allele may be considered nonfunctional. A null allele cannot be distinguished from deletion of the entire locus solely from phenotypic observation.

A Y-STR is a short tandem repeat (STR) on the Y-chromosome. Y-STRs are often used in forensics, paternity, and genealogical DNA testing. Y-STRs are taken specifically from the male Y chromosome. These Y-STRs provide a weaker analysis than autosomal STRs because the Y chromosome is only found in males, which are only passed down by the father, making the Y chromosome in any paternal line practically identical. This causes a significantly smaller amount of distinction between Y-STR samples. Autosomal STRs provide a much stronger analytical power because of the random matching that occurs between pairs of chromosomes during the zygote making process.

<span class="mw-page-title-main">Electropherogram</span>


An electropherogram, or electrophoregram, can also be referred to as an EPG or e-gram. It is a record or chart produced when electrophoresis is used in an analytical technique, primarily in the fields of forensic biology, molecular biology and biochemistry. The method utilizes data points that correspond with a specific time and fluorescence intensity at various wavelengths of light to represent a DNA profile.

<span class="mw-page-title-main">Paramutation</span>

In epigenetics, a paramutation is an interaction between two alleles at a single locus, whereby one allele induces a heritable change in the other allele. The change may be in the pattern of DNA methylation or histone modifications. The allele inducing the change is said to be paramutagenic, while the allele that has been epigenetically altered is termed paramutable. A paramutable allele may have altered levels of gene expression, which may continue in offspring which inherit that allele, even though the paramutagenic allele may no longer be present. Through proper breeding, paramutation can result in siblings that have the same genetic sequence, but with drastically different phenotypes.

<span class="mw-page-title-main">STR analysis</span> Biological DNA analysis for allele repeats

Short Tandem Repeat (STR) analysis is a common molecular biology method used to compare allele repeats at specific loci in DNA between two or more samples. A short tandem repeat is a microsatellite with repeat units that are 2 to 7 base pairs in length, with the number of repeats varying among individuals, making STRs effective for human identification purposes. This method differs from restriction fragment length polymorphism analysis (RFLP) since STR analysis does not cut the DNA with restriction enzymes. Instead, polymerase chain reaction (PCR) is employed to discover the lengths of the short tandem repeats based on the length of the PCR product.

Single-strand conformation polymorphism (SSCP), or single-strand chain polymorphism, is defined as a conformational difference of single-stranded nucleotide sequences of identical length as induced by differences in the sequences under certain experimental conditions. This property allows sequences to be distinguished by means of gel electrophoresis, which separates fragments according to their different conformations.

Mycobacterium canettii, a novel pathogenic taxon of the Mycobacterium tuberculosis complex (MTBC), was first reported in 1969 by the French microbiologist Georges Canetti, for whom the organism has been named. It formed smooth and shiny colonies, which is highly exceptional for the MTBC. It was described in detail in 1997 on the isolation of a new strain from a 2-year-old Somali patient with lymphadenitis. It did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence. It had shorter generation time than clinical isolates of M. tuberculosis and presented a unique, characteristic phenolic glycolipid and lipo-oligosaccharide. In 1998, Pfyffer described abdominal lymphatic TB in a 56-year-old Swiss man with HIV infection who lived in Kenya. Tuberculosis caused by M. canettii appears to be an emerging disease in the Horn of Africa. A history of a stay to the region should induce the clinician to consider this organism promptly even if the clinical features of TB caused by M. canettii are not specific. The natural reservoir, host range, and mode of transmission of the organism are still unknown.

<i>Bacillus anthracis</i> Species of bacterium

Bacillus anthracis is a gram-positive and rod-shaped bacterium that causes anthrax, a deadly disease to livestock and, occasionally, to humans. It is the only permanent (obligate) pathogen within the genus Bacillus. Its infection is a type of zoonosis, as it is transmitted from animals to humans. It was discovered by a German physician Robert Koch in 1876, and became the first bacterium to be experimentally shown as a pathogen. The discovery was also the first scientific evidence for the germ theory of diseases.

DNA Specimen Provenance Assignment (DSPA) also known as DNA Specimen ProvenanceAssay, is a molecular diagnostic test used to definitively assign biopsy specimen identity and establish specimen purity during the diagnostic testing cycle for cancer and other histopathological conditions. The term first appeared in the 2011 scientific paper, “The Changing Spectrum of DNA-Based Specimen Provenance Testing in Surgical Pathology,” published in the American Journal of Clinical Pathology, which built upon concepts described in an earlier paper published in the Journal of Urology.

<span class="mw-page-title-main">Forensic DNA analysis</span>

DNA profiling is the determination of a DNA profile for legal and investigative purposes. DNA analysis methods have changed numerous times over the years as technology improves and allows for more information to be determined with less starting material. Modern DNA analysis is based on the statistical calculation of the rarity of the produced profile within a population.

DXZ4 is a variable number tandemly repeated DNA sequence. In humans it is composed of 3kb monomers containing a highly conserved CTCF binding site. CTCF is a transcription factor protein and the main insulator responsible for partitioning of chromatin domains in the vertebrate genome.

References

  1. Vergnaud G, Pourcel C (2009). "Multiple locus variable number of tandem repeats analysis". Molecular Epidemiology of Microorganisms. Methods Mol. Biol. Vol. 551. pp. 141–58. doi:10.1007/978-1-60327-999-4_12. ISBN   978-1-60327-998-7. PMID   19521873.
  2. Grissa I, Bouchon P, Pourcel C, Vergnaud G (2008). "On-line resources for bacterial micro-evolution studies using MLVA or CRISPR typing". Biochimie. 90 (4): 660–8. doi:10.1016/j.biochi.2007.07.014. PMID   17822824.
  3. Nadon CA, Trees E, Ng LK, Møller Nielsen E, Reimer A, Maxwell N, Kubota KA, Gerner-Smidt P, the MLVA Harmonization Working Group (2013). "Development and application of MLVA methods as a tool for inter-laboratory surveillance". Euro Surveill. 18 (35): 20565. doi: 10.2807/1560-7917.es2013.18.35.20565 . PMC   5667538 . PMID   24008231.
  4. Blouin Y, Hauck Y, Soler C, Fabre M, Vong R, Dehan C, Cazajous G, Massoure PL, Kraemer P, Jenkins A, Garnotel E, Pourcel C, Vergnaud G (2012). "Significance of the identification in the Horn of Africa of an exceptionally deep branching Mycobacterium tuberculosis clade". PLOS ONE. 7 (12): e52841. doi: 10.1371/journal.pone.0052841 . PMC   3531362 . PMID   23300794.
  5. Thierry S, Tourterel C, Le Flèche P, Derzelle S, Dekhil N, Mendy C, Colaneri C, Vergnaud G, Madani N (2014). "Genotyping of French Bacillus anthracis strains based on 31-loci multi locus VNTR analysis: epidemiology, marker evaluation, and update of the internet genotype database". PLOS ONE. 9 (6): e95131. doi: 10.1371/journal.pone.0095131 . PMC   4046976 . PMID   24901417.
  6. Scholz HC, Vergnaud G (2013). "Molecular characterisation of Brucella species". Rev. Sci. Tech. 32 (1): 149–62. doi:10.20506/rst.32.1.2189. PMID   23837373.
  7. Lindstedt BA, Torpdahl M, Vergnaud G, Le Hello S, Weill FX, Tietze E, Malorny B, Prendergast DM, Ní Ghallchoir E, Lista RF, Schouls LM, Söderlund R, Börjesson S, Åkerström S (2013). "Use of multilocus variable-number tandem repeat analysis (MLVA) in eight European countries, 2012". Euro Surveill. 18 (4): 20385. doi: 10.2807/ese.18.04.20385-en . PMID   23369388.