New primer set for DNA barcording of haptophytes and uses thereof

Abstract

The present invention relates to a novel primer set for PCR amplification reactions and base sequence decoding for DNA barcoding of phylogenetic dwarfism, i.e., specific microalgae taxa, and its uses. This primer set, which targets the chloroplast rbcL gene region, offers higher success rates and efficiency in PCR amplification reactions than existing primers, thereby providing a technology that enables faster and more accurate identification of various strains of phylogenetic dwarfism. Furthermore, the primers of the present invention can decode DNA base sequences (approx. 1,650 bp) that are longer than existing primers (approx. 1,500 bp), enabling more detailed analysis and species identification. This offers significant advantages in various studies and applications, including not only microalgae species identification but also the evaluation of their industrial applicability, quality control, analysis of phylogenetic characteristics, and life history analysis. Additionally, the primer set of the present invention can be utilized as an important tool for monitoring microalgae at an ecological level, that is, for tracking changes in microalgae communities due to environmental changes or pollution.

Claims

Claim 1 A primer set for clonal DNA barcoding comprising a primer consisting of a nucleotide sequence represented by SEQ ID NO. 1; and a primer consisting of a nucleotide sequence represented by SEQ ID NO.

2. Claim 2 A primer set for DNA barcoding of clonal diffractors according to claim 1, characterized in that the primer set binds to the chloroplast RuBisCo large subunit (rbcL) gene region of the clonal diffractors. Claim 3 In paragraph 1, the above-mentioned clonal algae is Chrysotia ( Chrysotila )inside, Dicrateria Dicrateria ) genus, Diacronema( Diacronema Emilia ( Emiliania ) within, pseudoisocracy (Pseudoisochrysis ) inside, Isocrisis Isochrysis ) genus, Pavlova( Pavlova ) genus, pleurocrisis( Pleurochrysis ) genus, Primnesium( Prymnesium ) genus and thysocrasis ( Tisochrysis A primer set for clonal DNA barcoding, characterized by being one or more selected from a group consisting of ) genera. Claim 4 A kit for coding DNA of clonician algae comprising a primer set according to any one of claims 1 to 3. Claim 5 A kit for DNA barcoding of clonal algae according to claim 4, characterized in that the kit further comprises DNA polymerase, dNTPs, buffer, or sterile distilled water. Claim 6 (1) a step of isolating DNA from a sample; (2) a step of performing PCR amplification using a primer set comprising a primer having a base sequence represented by SEQ ID NO. 1 and a primer having a base sequence represented by SEQ ID NO. 2, using the isolated DNA from step (1) as a template; and (3) a step of analyzing the PCR amplification product from step (2); comprising a method for barcoding DNA of clonal algae. Claim 7 A method for barcoding DNA of clonal algae according to claim 6, wherein the amplification of step (2) is one or more methods selected from the group consisting of Polymerase Chain Reaction, Nested Polymerase Chain Reaction, Multiplex Polymerase Chain Reaction (multiplex PCR), Competitive Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Real-time Quantitative Polymerase Chain Reaction, DNA chip, and Loop-mediated isothermal amplification. Claim 8 A method for barcoding DNA of clonal algae according to claim 6, wherein the analysis of step (3) above is one or more methods selected from the group consisting of next-generation sequencing, Sanger sequencing, single-molecule real-time sequencing, ion semiconductor analysis, pyrosequencing, SBS (sequencing by synthesis), SBL (sequencing by ligation), and chain termination. Claim 9 A method for coding DNA of clonal algae according to claim 6, characterized in that the sample is a clonal algae culture medium or cell. Claim 10 In paragraph 6, the above-mentioned clonal algae is Chrysotia ( Chrysotila )inside, Dicrateria Dicrateria ) genus, Diacronema( Diacronema Emilia ( Emiliania ) within, pseudoisocracy (Pseudoisochrysis ) inside, Isocrisis Isochrysis ) genus, Pavlova( Pavlova ) genus, pleurocrisis( Pleurochrysis ) genus, Primnesium( Prymnesium ) genus and thysocrasis ( Tisochrysis A method for barcoding DNA of clonal algae, characterized by being one or more selected from a group consisting of ) genera.

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