Patent ID: 7060434
Filing Date: 2006-06-13
Classification: C12Q

Abstract:
1. A method for developing nucleotide probes for myctophid fishes, said method comprising the steps of: (i) extracting DNA from the muscle tissue of a myctophid fish, (ii) selecting a gene region in the extracted DNA as a DNA template and amplifying the selected gene region with the a pair of forward and backward selected primers using polymerase chain reaction (PCR), (iii) eluting the amplified DNA containing the selected gene region, (iv) re-amplifying and re-eluting the amplified DNA in step (iii), (v) cycle sequencing the of eluted DNA containing the selected gene region using a single primer to produce an extension product, (vi) purifying the extension product containing the selected gene region, (vii) sequencing the nucleotides of the extension product of step (vi) on an acrylamide gel, (viii) confirming the nucleotide sequence of the selected gene region by Blast-Email, (ix) ligating the extension product containing the selected gene region as a DNA insert into a cloning vector, (x) preparing host cells for electro-transformation, (xi) electro-transforming the host cells with the vector-containing DNA insert, (xii) growing and harvesting of transformed host cells, (xiii) re-inoculating and growing transformed host cells that appear as white colonies and that express the DNA insert containing the selected gene region; (xiv) confirming the presence of the DNA insert containing the selected gene region by polymerase chain reaction, (xv) purifying the cloned DNA insert containing the selected gene region from the transformed host cells to produce a DNA probe, (xvi) checking the purity and specificity of the DNA probe by cutting with a restriction enzyme, (xvii) confirming the molecular size of the DNA probe, (xviii) amplifying the DNA probe using the selected set of forward and backward primers of step (ii), (xix) eluting the amplified DNA probe containing the selected gene region, (xx) cycle sequencing the eluted DNA probe in step (xix) using a single set of primer, (xxi) sequencing the eluted DNA probe in step (xix) on an acrylamide gel, (xxii) comparing the nucleotide sequence of the DNA probe using “BLAST program” against the known sequences of similar genes in the genome data bases, (xxiii) confirming the sequence of the DNA probe by aligning with sequence obtained in step (vii), and (xxiv) designing species specific primers based on the sequence of the DNA probe, wherein the DNA probe for the D-Loop gene is PSL PROL, wherein the nucleotide base sequence of PSL PROL comprises: (750 BP) (SEQ ID NO:44)