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Universal primers for wildlife identification

A technology for universal primers and biological samples, applied in the field of identification of new universal primers, can solve problems such as impracticality

Inactive Publication Date: 2004-06-16
COUNCIL OF SCI & IND RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

We have no prior information in forensics about the species origin of confiscated animal parts and products, so these methods are virtually useless and impractical in wildlife identification

Method used

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  • Universal primers for wildlife identification
  • Universal primers for wildlife identification
  • Universal primers for wildlife identification

Examples

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Embodiment 1

[0108] Examples of identifying cytochrome b gene fragments that meet the requirements in columns 1, 2 and 3 mentioned in the subheading 'Objective of the invention' in the heading 'Summary of the invention'

[0109] Mitochondrial cytochrome b molecules have been widely used in molecular taxonomy research. As a slowly evolving gene, it has enormous information in its nucleotide sequence that distinguishes the family, genus, and species origin of animals 1-65 . Extensive databases of cytochrome b gene sequences from different animal species have been accumulated in NCBI's nr and mito databases. We have exploited these properties of the cytochrome b gene to determine the family, genus and species origin of any confiscated remains of unknown animal origin. To this end, we have identified a segment of the cytochrome b gene that is highly polymorphic between species but monomorphic in individuals of the same species, thus allowing individuals of unknown species to be grouped with ...

Embodiment 2

[0119] Example of developing universal primers to amplify the fragments mentioned in 'Example 1' A pair of universal primers were designed with the following characteristics:

[0120] 1. It targets the identified fragments (mentioned in 'Example 1') and amplifies them in the polymerase chain reaction (PCR).

[0121] 2. Its 3' and 5' ends are highly conserved in a wide range of animal species (marked with an asterisk (*) in Table 2), thus ensuring amplification of the above-mentioned fragments in a generic manner. Sequencing of the amplified fragments with these primer pairs revealed a molecular signature of the species of the analyzed material, which molecular signature compared to the sequence of a known reference animal revealed the species identity of the unknown biological material being studied.

[0122] 3. The tm (melting temperature) of the two primers are almost similar (about 58 degrees Celsius), ensuring significant annealing of both primers to their template, and th...

Embodiment 3

[0132] Example of developing universal PCR conditions to ensure amplification of any template of unknown origin in PCR and thus enhancing the versatility of the technique invented by us

[0133] The developed PCR conditions have the following unique characteristics:

[0134] 1. These conditions enable the amplification of any animal derived DNA template in a universal manner with the universal primers mentioned in 'Example 2'.

[0135]2. The conditions were chosen to ensure comparable annealing temperatures for the two primers, 'mcb398' and 'mcb869'.

[0136] 3. The standardized PCR conditions are therefore universal; thus the probability of PCR failure on templates of unknown origin due to non-standard conditions is excluded. This ensures the versatility of our technique in wildlife forensics.

[0137] 4. The above general conditions are:

[0138] Amplification reactions should be performed in a 20 μl reaction volume containing approximately 20 ng of template DNA, 100 μm o...

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Abstract

The invention provides novel universal primers that can amplify the fragment of cytochrome b gene of any animal species in polymerase chain reaction (PCR) and reveal the identity of the biological material of any unknown animal origin and a method for identification of the specific animal from a given biological sample.

Description

technical field [0001] The present invention relates to the identification of novel universal primers that can amplify cytochrome b gene fragments of any animal species in the polymerase chain reaction (PCR) and characterize biological material of any unknown animal origin at the species and subspecies level . The present invention also provides methods for identifying mitochondrial cytochrome b gene fragments in biological material of unknown origin. Background technique [0002] Numerous studies in evolutionary biology utilize phylogenetic information obtained from mitochondrial cytochrome b genes. This gene has been identified as an efficient molecule in molecular taxonomy to distinguish different lineages to the phylogenetic depth of families, genus and species 1-66 . A large number of sequence databases for cytochrome b genes of different animal species have been accumulated in public databases such as GenBank, NCBI (http: / / www.ncbi.nlm.nih.gov) and others. We have ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12Q1/68
CPCC12Q1/6888
Inventor S・K・维尔马L・辛格
Owner COUNCIL OF SCI & IND RES
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