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

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Problems solved by technology

We have no prior information in forensics about the species origin of confiscated animal parts

Method used

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

Examples

Experimental program
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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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PUM

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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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