US 2003/0143534 A1Published application

Probes for myctophid fish and a method for developing the same

AssigneeCouncil of Scientific and Industrial Research

InventorsUsha Goswami, Giacomo Bernardi, Subhash Chander Goswami, Robert K. Johnson

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Abstract

The DNA probes produced by molecular cloning and the characterization of specific gene region sequences is provided, these can be used as genetic markers for the genes such as Cytochrome b (cyt b); Mitochondrial control region (D-Loop); Inter Transcribed Spacers (ITS2) and Rhodopsin (ROD), 12S rRNA and 16S rRNA in mesopelagic lantern fishes which are found in the mesopelagic zones of the oceans where the photic regime is of dim light and associate themselves with the oxygen minimum layer, it also includes the recombinant DNA techniques for the preparation of specific gene probes and sequences of species specific primers of lantern fishes, novel gene probes and novel oligonucleotides for amplification of myctophid genes are disclosed.

Claims107

Dependent claims

A method for development of nucleotide probes for myctophid fishes, said method comprising the steps of:

(i) extracting the DNA from the muscle tissue of a myctophid fish,
(ii) selecting gene regions in the extracted DNA with the selected primers and the amplifying the same using polymerase chain reaction (PCR),
(iii) eluting the PCR amplified DNA,
(iv) reamplifying the gene regions from PCR amplified DNA and eluting the same,
(v) cycle sequencing of eluted DNA using a single primer,
(vi) purifying extension products,
(vii) sequencing the extension product on acrylamide gel,
(viii) confirming the sequences for the target gene by Blast-Email,
(ix) ligating the eluted PCR products in a vector,
(x) preparing the electro-competent cells for electro transformation,
(xi) electro transforming the host cells,
(xii) growing and harvesting of transformed host cells,
(xiv) confirming that the transformed bacteria has the plasmids with the gene inserts by PCR.
(xv) purifying recombinant plasmid DNA having the cloned gene probes from the transformed host cells,
(xvi) checking purity and specificity of the cloned DNA probe insert by cutting with restriction enzyme,
(xvii) confirming the molecular size of the DNA probe insert,
(xviii) PCR amplification of the gene insert from the probe using both primers,
(xix) eluting of the amplified gene region,
(xx) cycle sequencing of the gene region of the probe,
(xxi) sequencing of the cloned DNA insert on acrylamide gel,
(xxii) comparing the DNA sequence of the prepared DNA probes using “BLAST program” against the known sequences of similar genes in the genome data bases,
(xxiii) confirming the sequences of the cloned probe by aligning with sequences of the claim 1 (vii), and
(xxiv) designing species specific primers from the sequences.

20 more elements

The nucleotide base sequences of PSL CYTL (748 bp) comprising:

62 74. The nucleotide base sequences of PSL CYTL (748 bp) comprising:
5′ CTTNCCCATT TTGGGCGCTT NGGCNCGCTN CTCCNCGAGA CTCTGCGTAN TAATCCAANT CNCTNCGGGC CNCTCCCTAC CANTNCNCTA CACCNCAAAT TNCAACCCNG TTTCCTCATC ANTCAACCAC ATCTGTCGAA AACNTCAACT ACGGCTGACT AATCCGAAAA CATGCACGCT AACGGTGCCT CTTTCTTCTT CATCTGTATT TATCTNCNCN TTGGANGAGG ACTATNCTAC GGATCCTACC TCTACGAAGA GACGTGAGGT GTTGGTGTTA TTCTTCTCCT TCTAATAATG ATGACTGCNT TTGTTGGCTA TGTGCTNCCC NGAGGACAAA TGTCCTTTTG AGGTGCTACT GTCATTACAA NCCTACTCTC TGCTGTNCCG TNTGTTNGCG GCNCTCTANT TCAATGAATT TGAGGTGGCT TCTCCGTAAA CACGCAACGC TCACTCGTTT CTTCGCNTTC CACTTCTTGT TCCCATTTGT TGTCGCNGCT ATAACCNNGG TTCACCNGAT TTNCCGACAT CAAACAGGCT CTAAANCCCC CCCGGNTTGA CTCCATACAA CAAAACCCTC CACCCTATTC NCTATAAAAC TCTAGGTTCG TGCCCGTATT GGCTTACTTC ATGNCTATTT CCCNGNCGGA GGGACNAAAA TTCCTGCACC CCCTCCCCNC AAAATAAANA ATGTGTCTNT CCTACCANAA AACAACNNAN ACGGGGTNTG CNCTTCCATC ATCCACN 3′ 75. The nucleotide base sequences of PSLITS2F comprises:
(225 BP) 5′ TCTACGATCT ACCGGCNTTT NNTGTGGAAA GACGATCATG CATTTATGTG TGTCTTTCTA TGGATTTGAA CCGTGTGGTA CGTCTTTGCG TACTGCTTGG AAGGCTCAAC TTGCTTCTGT CCTTCTCTTG CAGTCTCGCA CTGTCTATGC AACGTGTTCT ACTTCGACTT CTGTCGAAAA ATCTTACTTT TGACCTCAGA TCAGACAAGA CTACCCGCTG AATTT 3′ 76. The nucleotide base sequences of PSL PROL comprises:
(749 BP) 5′ CCTTTTCGGN ATAGGCCCAN CTCAAATGAA TTCCTTCTCT CCTGGTCCAA GCCCAAACTG TGGACGGCAG GTTGACAATG GTTACAAATC GTGACAAATC GGCTACATAA TTGCCGATAG CGATGTCGTC AAACCAAGTC AAACAATGGC CGATGTATAT CGGCCAAACC CATATATGGG TCTGGCTGTA GTTTGTGTTG AGCAACGTCA CACCAGTGTC TGGTCAGCAT ATAAGATGTT GACATCTTGC AACATCTTAC CCACAGACAG ACAGTTACGG CTGCTTACGA ANGGCGCTAG TGTTGTGGTG AGAAACGAAG ATACATACGT CAAACAGACG CCGGTGCACT TGAAGACACT GTTTGAAGGT GCCGCACTAC TTGACAGACA GCCCATGATG CGCTGGACAG TGACCAAAGC TACNGGAGGA CCANATGGAA ATCCTGTTGG CGTTGCCGTG GGACTCAAGT TGTACACTTT TGGATGGTTG ATCACTANAN CCGCTGCCGG GAGAAGCACT CGCTCCTGGT TCACTAATCA GATTGAGGTT AACCANATTG ANGTAAACAT CTTCAACACA GTGTCTTTAT GCTGGATGAA ATTNAGCCCA CNGGACACCA NAAAAGAATT NCCNCTGGTT CTNNCGGGGG NCCCCNNNAA CGNNTNTTCC CCTTNTCTCN NNNGCGGNGA AGTTNCCCCC CCCCACTNAN NTCTTCCTTC AANANNTTTC CNCCNNNAGA GGTTTTCCCN 3′ 77. The nucleotide base sequences of ROD PSL SLMB comprises:
(748 BP) 5′ CCTGGTAGGG TTCCCCGTCA ACTTCCTCAC ACTGTACCTC ACNTTCGAGC ACAAGAAGCT ACTAACCCCC TTAAACTACA TCCTGCTCAA CCTGGCGGTC GGAGACCTCC TGATGGTGTA AGGAGGGTTC ACCACCACCA TCTACACCTC CATGCACGGC TACTTCGTCC TAGGGAAACT GGGCTGCGCC ATCGAAGGTT TCATGGCCAC CCATGGTGGT CAGGTCGCCC TTTGGTCCCT GGTGGTTTTG GCCGTGGAAA GGTGGCTGGT CGTCTGCAAN CCCATCTCCA GCTTCCGCTT CCAGGAGTCC CACTCCCTCA TGGGCCTGGC CGTGACCTGG GTGATGGCGA CGGCTTGTTC TGTGCCCCCC CTGGGTCGGC TGGTCTCGCT ACATCCCAGA AGGCATGCAG TGCTCATGCG GAATGGACTA CTACACTCCC GCGCCGGGCG TCAACAATGA ATCCTACGTN GTGTACATGT TCNTCANAAA AANAATNGGA CCNCNGGGCG ATCATNTTGN TANGNNAAGG CCAGNTGNTG NGAGCAGTCA AGGCGGCCGC CGCCGCCCAG CAAGAGTCCG AGACCACCCA GAGGGCCGAG AGGGAAGTCA CCCGNATGGT NATNANGATG GTNATNTCNT TCNTGGTAAG NAGGGNGCCA NACGCCAGCG TGGCCTGGTG GATCTTNNGN AACCAGGGNG CAGAATTAGG CCCNGTNTTC ATGACCCTGC CGGCNTTCTT TGCCAAGA 3′

2 more elements

Dependencies are inferred from the claim text.