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AssigneeCheng-Taung Wang
InventorsHsiu-Luan Chang, Ren-Bao Liaw, Ming-Che Wu
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A method for rapidly identifying porcine estrogen receptor (ESR) marker comprises using published primers to amplify the target DNA fragment by polymerase chain reaction (PCR). The DNA fragment is cloned and then sequenced. The key positions of the sequence are modified to generate three primers which are used to amplify different DNA fragments with different genotypes by PCR to eliminate extra restriction cut reaction. A long one of the primers is to specifically amplify non-prolific allele, a short one of the primers is to specifically amplify prolific allele, and the remaining one is mutual and complimentary to the sequence. After PCR and electrophoresis, the sample with 90 bp band is identified as prolific genotype, the sample with 110 bp band is identified as non-prolific genotype, and the sample with 90 bp and 110 bp bands is identified as hetero-genotype.
A method for rapidly identifying porcine estrogen receptor marker by a mutagenically separated polymerase chain reaction, the method comprising the steps of:
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Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes: for detection or identification of organisms
Oligonucleotides characterized by their use: polymorphic or mutational markers
Measuring or testing processes involving enzymes, nucleic acids or microorganisms
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1. Field of the Invention
The present invention relates to a method for rapidly identifying porcine estrogen receptor (ESR) marker at a low cost. In particular, the present invention relates to a method for rapidly identifying porcine estrogen receptor marker by means of mutagenically separated polymerase chain reaction (MSPCR).
2. Description of the Related Art
FIG. 1 is a flowchart of a method for rapidly identifying porcine estrogen receptor marker in accordance with the present invention.
FIG. 2 is a diagram illustrating a fragment of a DNA sequence of a prolific allele of a pig, wherein the sequence of the sixty-second position through the sixty-seventh position is CAGCTG, which is to be identified and fragmented by the restriction enzyme Puv II.
FIG. 3 is a diagram illustrating a fragment of a DNA sequence of a non-prolific allele of a pig, wherein the sequence of the sixty-second position through the sixty-seventh position is CAACTT, which is cannot be identified and fragmented by the restriction enzyme Puv II.
The present invention provides a method for rapidly identifying porcine estrogen receptor marker by means of mutagenically separated polymerase chain reaction (MSPCR). As illustrated in FIG. 1, a first step of the method in accordance with the present invention comprises extracting a DNA sample from a pig to be identified. The DNA sample is extracted from blood, internal organs, muscular tissues, or semen. The DNA sample of 20˜25 ng is then combined with a solution comprising dNTP (deoxy-nucleotide-triphosphates) of 0.4 mM, a first primer A1 of 0.05 μM a second primer B1 of 0.1 μM, a third primer R1 of 0.2 μM, MgCl2 of 3 mM, 1×PCR buffer solution (including Tris-HCl of 20 mM, KCl of 50 mM, PH 8.4), and one unit of taq DNA polymerase. The total volume for the reactive substance is preferably 10 μL.
The first primer A1 has a DNA sequence as follows: 1 5′-GTCAGTCCAATTAGAATAGGGCGGGAATGGGGACTTGACAAG AAACGT-3′
The second primer B1 has a DNA sequence as follows: