mtDNAtest
-animal Kit

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mtDNAtest-animal

    The DNA sequence of mtDNA has been determined from a large number of organisms and individuals (including some organisms that are extinct), and the comparison of those DNA sequences represents a mainstay of phylogenetics, in that it allows biologists to elucidate the evolutionary relationships among species. It also permits an examination of the relatedness of populations, and so has become important in anthropology and field biology.

    Trace DNA analysis has become an integral part of a forensic laboratory's work and a key tool for investigators in forensic medicine. The low effective population size and rapid mutation rate (2-4% per million years) makes mtDNA also useful for assessing genetic relationships of various animals and also for identifying and quantifying the phylogeny (evolutionary relationships).The high mutation rate makes mtDNA most useful for comparisons of animals within species and among species. As the individual animals become more distantly related, the number of sequence differences becomes very large and changes begin to accumulate on changes until an accurate count becomes impossible.

    PRINCIPLE: Here, we would like to offer you mtDNAtest®-animal- a Kit based on our optimized technology intended for animal mitochondrial DNA analysis - hypervariable region I (HVI) II (HVII) and III (HVIII). The Kit was designed to cover the major issues and challenges related to routine and reproductible trace DNA analysis in forensic sciences but is also useful in population genetics, tracing along an animal's maternal line as well as animal identification on the basis of any biological materials. Kit includes specially selected reagents and primers, allowing amplification of mitochondrial DNA of any mammal (cat, dog, horse, cow, pig, deer, fox ...) and other animals, including many birds, amphibians and reptiles. The material for the study of biological samples are usually of unknown origin, which is made possible by the identification of mtDNAtest. The kit includes, among others primers for PCR and sequencing reactions, which were validated in international GEDNAP tests. BioTe21 team can help in the analysis and documentation of test results obtained through the use of mtDNAtest.

PRINCIPLE

       mtDNAtest®-animal Kit allows for individual identification of animals and animal remains, even when using higly degraded biological materials. Small nucleotide polymorphisms (SNPs) on the mitochondrial D-loop hypervariable regions can be also traced along an animal's maternal line. The Kit is designed to help scientists in forensic medicine (comparative analysis of evidence samples and reference materials) as well as migration analysis, pedigree generation for analysis of genetic linkage and association. The mtDNAtest®-animal is based on higly sensitive PCR method intended for routine testing applications.

      Sequence analysis may be performed automatically with our special software: SNPtester® that allows you for identyfication of any SNP without knowledge about correct determination of sequence variations and usage of IUPAC nomenclature. Just put your row sequence in the SNPtester® and wait for the results.

 Advantages of the Kits

  • Simple and quick implementation of the procedures,    
  • Simple method, independent of laboratory practice,
  • High yields even from higly-degraded samples
    containing small-amounts of nucleic acids, 
  • Standardized method for a variety of sample types,
  • Optimized protocols for a range of starting materials,
  • High sensitivity (DNA < 0.025 pg / reaction),
  • Optimized PCR primer set,
  • Excellent quality of results
    (obtained amplicons do not cause problems during analyses),
  • Continuous help during analyses,
  • Relatively low cost testing when compared to other methods.

Aplications:

  • accidents involving animals, animal deductions,
  • poaching and illegal hunting of animals,
  • theft of animals, especially those with a high unit price,
  • illegal sale of animals,
  • analysis of objects which had contact with animals (epidermis) associated with the illegal storage of animals,
  • intrusion on private premises,
  • bites by dogs,
  • damage caused by animals,
  • analysis and determination of pedigree animals,
  • identifying individual animals unique DNA profile,
  • and more ...,

Use of latest techniques of molecular biology, biotechnology and genetic engineering (polymerase chain reaction (PCR), sequencing), provides certainty of the results. See flowchart below:

mtDNAtest-animal Method scheme


 

 

 

 

 

 

 

 

 

 

 

 

 Why mitochondrial instead of nuclear DNA?

  • High copy number - quantitative majority - a single somatic cell contains ~ 10 - 1000 mitochondria and each mitochondrion carries 2 - 10 mtDNA copies, that result in about 100 - 10 000 copies of mitochondrial DNA per signle cell (containing nuclear DNA in one copy). This advantage is particularly significant for applications when using highly degraded biological material.

    Exemplary mtDNA SNP

  • High stability due to circular nature of mitochondrial DNA, making it easy to obtain for analysis,
  • High mutation rate (10 times higher mutation rate than nuclear DNA), espcecially in hypervariable regions (HVI, HVII) acumulating small nucleotide polymorphisms (SNP) in a relatively small fragment of mitochondrial genome, that allows for comparisons of individuals and simplify analyses. 
  • Fixed in time mutation rate of mtDNA (2-4% per million years) - the fairly high mutation rate of mtDNA is reasonably fixed, making it useful to trace DNA changes through time of human evolution.
  • No change in mtDNA from parent to offspring  (lack of recombination) - maternal and paternal mtDNA usually do not recombine. Unlike nuclear DNA, which is inherited from mother and father and in which genes are frequently rearranged in the process of recombination, there is usually no change in mitochondrial DNA (mtDNA) from mother to offspring, keeping the mtDNA sequence clean. This makes mtDNA a powerful tool for genetic identification (particularly when genomic DNA is degraded).
  • Maternal inheritance - mammalian mtDNA is known for its maternal inheritance.

Why mtDNAtest®-animal Kit ?

    Both, many years of professional experience in the field of forensic molecular medicine, as well as the work with mtDNA allowed us to design and introduce a new test, which is, in technical terms, an extremely easy way to differentiate SNPs (Single Nucleotide Polymorphism) in mtDNA, based on a simple high-resolution sequencing method (seq module) or by using a capillary electrophoresis separation of fluorescently-labeled DNA fragments (shown above SNP-module, available soon). mtDNAtest®-human Kit allows you to use a variety of biological materials containing low DNA amounts (see sensitivity tests below). Selected and optimized PCR primer set allows for amplification of mtDNA D-loop sequence (see mtDNA scheme) comprising of whole hypervariable HVI and HVII sequences, therefore it is useful especially in genetic identification and comparative mtDNA analysis of biological traces.The The Kit is closed in delivered 0.2mL PCR tubes (as ready to use capsules), that limits potential contaminations of the reagents, ensures high repeatability of the results in routine work with trace DNAs and allows for shortening the whole identification process even up to 24h.

 

mtDNAtest®-animal Kit sensitivity

    High sensitivity of mtDNAtest®-human Kitenables fragment amplification of mtDNA hypervariable regions, even using very degraded trace samples. Only 0.00025ng of pure total DNA is needed to be added to one PCR reaction tube (referred to as "capsule") to obtain positive results. Using a range of control systems and prevent the formation of false negatives and false positives, you can provide clarity and certainty of your studies.

Analysis of mtDNA using this technology does not require any special laboratory adaptations and can be performed in any genetic laboratory dealing with direct PCR product sequencing and/or STR systems (called: Short Tandem Repeats) of genomic DNA, equipped with access to any thermocycler. The seq-module of the mtDNAtest®-animal Kit operates using a variety of basic sequencing methods.

 

Materials & reagents

General information: mtDNAtest®-animal seq-module is based on an optimized and validated PCR reagents that produce amplicons ready to use in direct sequencing of PCR products and further comparison with a reference sequence. Using mtDNAtest®-animal seq-module you can obtain sequences that will allow you to score the sequence differences of your sample to the reference sequence.

The Kit includes reagents for PCR reactions and specific oligonucleotides for sequencing reactions. Optmized PCR reagents are closed in ready to use standard 0,2mL PCR tubes (referred to as capsules), which require only your template (total DNA) and an enzyme. Each PCR capsule contains a reaction mixture consisting of: an optimized PCR buffer, standarized PCR Primer Set, dNTPs, molecular biology grade water and other supplements improving the PCR reaction. Oligonucleotides for sequencing are provided in separate tubes.

For evaluation of your results, control DNA (positive control) and nuclease free water (non-template, negative control) are also provided.

The control mtDNA (NCBI, GeneBank accession no.: KC342222).

Equipment, reagents and materials required but not supplied:
You need only an enzyme 0,12uL/single capsule - a standard DNA polymerase with hot-start such as SuperHot Taq DNA Polymerase  5U/uL - Bioron (prefered) and typical laboratory equipment such as pipettes for the preparation of the PCR mixture and a thermocycler. If you have an instant access to any sequencer (genetic analyzer), you can perform all laboratory steps in 24h allowing for full identification of your samples in one day. There is also possibility to order the service (sequencing) in our lab or any other prefered lab. To sequence the PCR products in your laboratory you may need standard chemicals and materials used in the process (eg. BigDyeTerminator Sequencing Kit).  

If you have any queries or problems with the technology, please do not hesitate to contact us.

 

Product description

mtDNAtest®-animal seq-module is available in the following variants:

Product Name Cat. no. Number of capsules (reactions) Module Primer Set Kit allows for amplification of approximate length of PCR fragment [bp] Applications Features
mtDNAtest®-animal-sm-psN 1121030 30 seq N HVI-complete + HVII-complete + HVIII + flanking regions 2000 individual animal identification in maternal line High resolution and the largest range of analyzed  sequence (in one capsule)
mtDNAtest®-animal-sm-psN 1121060 60 seq N HVI-complete + HVII-complete + HVIII + flanking regions 2000 individual animal identification in maternal line High resolution and the largest range of analyzed  sequence (in one capsule)
mtDNAtest®-animal-sm-psO 1122030 30 seq O HVI-incomplete 450 very low amounts of trace DNA, species identyfication, individual animal identyfication (low resolution) the most sensitive, for stainsand degraded materials
mtDNAtest®-animal-sm-psO 1122060 60 seq O HVI-incomplete 450 very low amounts of trace DNA, species identyfication, individual animal identyfication (low resolution) the most sensitive, for stainsand degraded materials
mtDNAtest®-sm-psE 1115030 30 seq E beyond D-loop 250 very low amounts of trace DNA  very sensitive, allows for species identification
mtDNAtest®-sm-psE 1115030 60 seq E beyond D-loop 250 very low amounts of trace DNA  very sensitive, allows for species identification
mtDNAtest®-animal-sm-psP 1125030 30 seq P HVI-complete + flanking regions 1000 mixed primer sets 30 reaction for HVI only HVI region
mtDNAtest®-animal-sm-psR 1125030 30 seq R HVI-complete + HVII-complete + flanking regions 1000 mixed primer sets 30 reaction for HVII and HVIII only HVII & HVIII region

  Please contact us to ask about the Kits.


Optionally, it is possible to order a service:
•    Direct implementation of mitochondrial DNA analysis in your laboratory,
•    Performing a project of your lab adaptation to routine work with mtDNA samples,
•    Personnel training in the field of the technology and routine casework with various control systems,
•    Performance of an exemplary comparative analysis of personnel mtDNAs,
•    Lab validation and internal audits preparing for external validation,
•    Issuing a certificate stating the lab preparedness for performing mtDNA analysis,
•    6 months of results consultation.

 

As other IVD, the Kit is intended for applications utilizing molecular biology methods. This product is neither intended for the diagnosis, prevention, or treatment of a disease, nor has it been validated for such use either alone or in combination with other our products.