U.S. patent application number 10/981507 was filed with the patent office on 2005-06-23 for probes for myctophid fish and a method for developing the same.
This patent application is currently assigned to Council of Scientific & Industrial Research. Invention is credited to Bernardi, Giacomo, Goswami, Subhash Chander, Goswami, Usha, Johnson, Robert K..
Application Number | 20050132970 10/981507 |
Document ID | / |
Family ID | 27613830 |
Filed Date | 2005-06-23 |
United States Patent
Application |
20050132970 |
Kind Code |
A1 |
Goswami, Usha ; et
al. |
June 23, 2005 |
Probes for myctophid fish and a method for developing the same
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.
Inventors: |
Goswami, Usha; (Goa, IN)
; Bernardi, Giacomo; (Santa Cruz, CA) ; Goswami,
Subhash Chander; (Goa, IN) ; Johnson, Robert K.;
(Charleston, SC) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Council of Scientific &
Industrial Research
|
Family ID: |
27613830 |
Appl. No.: |
10/981507 |
Filed: |
November 5, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10981507 |
Nov 5, 2004 |
|
|
|
09782604 |
Feb 14, 2001 |
|
|
|
Current U.S.
Class: |
119/217 |
Current CPC
Class: |
C12Q 1/6876
20130101 |
Class at
Publication: |
119/217 |
International
Class: |
A01K 061/00 |
Claims
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
SEQ ID NO:42, 43 or 45.
2. A polynulcleotide sequence comprising SEQ ID NO: 42, 43 or 45.
Description
CROSS REFERENCE TO RELATED PATENT APPLICATION
[0001] This application is a Divisional application of Ser. No.
09/782,604, filed Feb. 14, 2001. The entire specification of the
aforementioned application is incorporated herein by reference.
FIELD OF INVENTION
[0002] This invention relates to the molecular cloning and
characterization of specific gene region sequences. More
particularly the invention relates to genetic markers which have
been identified in several genes such as Cytochrome b (cyt b);
Mitochondrial control region (D-Loop); Inter Transcribed Spacers
(ITS2) and Rhodopsin (ROD) in a mesopelagic lantern fish which are
found in the mesopelagic zones and associate themselves with the
oxygen minimum layer. The invention is also concerned with the
recombinant DNA techniques for the preparation of specific gene
probes useful for identification of larval and adult life history
stages of myctophids i.e. Lantern fishes. The invention also deals
with construction of species specific primer sequences.
BACKGROUND OF THE INVENTION
[0003] Among the mesopelagic fishes, the lantern fishes (Family
Myctophidae) are extremely common and numerous in both species and
individuals in the open ocean midwaters of the world oceans. The
myctophid fishes are generally found associated with the oxygen
minimum layer of the midwaters and are active vertical migrators.
They have adapted themselves to low oxygen conditions and low/dark
light intensity conditions.
[0004] Most studies on midwater fish have been restricted to
investigations of the oxygen minimum layer in the eastern tropical
pacific and/or morphological adaptations (Kinzer et al. Deep Sea
Research II, 40 (3): 783-800 (1993).
[0005] Little is known about the biology and ecology of this group
particularly the dynamics of the interaction between
mesozooplankton stocks and abundant migratory myctophids.
[0006] The extreme oxygen deficiency of the Arabian Sea at mid
depth has been well documented. Dissolved oxygen slowly increases
below 1000 meter depth from about 0.2 ml per liter to 2.5
milliliter per liter at 2000 meter depth. Near the bottom at 3000
meter oxygen values average 3.5 ml per liter (SenGupta, R and S. W.
A. Naqvi, Deep Sea Research, 31: 671-706, (1984).
[0007] The myctophid fish are adapted to low oxygen conditions and
low/dark light intensity conditions. Their physiological
adaptations to these varying abiotic and biotic factors have
enabled them to survive in the vast suboxia below 150 meter depth
zone.
[0008] The myctophids are strong vertical migrators and active
swimmers. Most of them are black/dark brown in color and possess
fluorescent organs called photophores. They constitute food for
higher fishes of commercial importance such as Tuna, Sharks and
marine mammals.
[0009] In the northern and western Arabian Sea itself the US GLOBEC
Report No. 9 (1993) reported a biomass of about 100 million metric
tonnes per year (present world fishery catch is around seventy
million metric tonnes). Their dominance could be due to their
ability to live in the suboxic middle depths and avoid
predators.
[0010] Due to high protein content vast myctophid populations can
form an excellent fish meal and poultry feed to support aquaculture
and other farming activities. The myctophids can facilitate the
assessment of water bodies, estimation of genetic resources,
genetic variability and the level of gene flow between various
stocks and populations in the world oceans.
[0011] Traditionally, various investigators including; Gunther, A.
Report on the deep-Sea Fishes collected by H.M.S. Challenger during
the years 1873-1876, Reprint 1963, Text volume, J. Cramer,
Weinheim, Hafner, New York, 1-135 (1887); Gilbert, Proceedings of
the U.S. National Museum, 48: 305-380 (1915), and Fraser-Brunner,
proceedings of the Zoological society of London, 118 (4) :
1019-1106 (1949), classified the species in the family Myctophidae
by using differences in the position and numbers of photophores
located on head and body of fish.
[0012] While photophore differences are still of primary
importance, the morphometry is also used statistically (Paxton,
Bulletin of the natural history museum of Los Angeles County,
Science, No. 13 (1972).
[0013] The methods are incompetent to identify the closely allied
species, populations and the life history stages. This is the major
bottleneck for the proper identification, population dynamics and
stock assessment of these fishes leaving their systematics
unjustified.
[0014] There are several reasons for this confusion. In the first
place, the fish are particularly fragile and easily damaged in
collecting nets, so most specimens collected tend to be in poor
condition.
[0015] Second, the group contains many morphologically similar
species from all parts of the world.
[0016] Third though there is only small amount of variability in
most characters, there is enough in some cases for similar species
to overlap even in those characters wherein they differ most
significantly (Zahuranec, B, Zoogeography and Systematics of the
lantern fishes of the genus Nannobrachium (Lampanyctini:
Myctophidae) PhD Thesis , Scripps Institution of Oceanography,
University of California, San Diego, 1995).
[0017] The problems have been compounded since specimens of many
forms have not been numerous. It is difficult to collect ample
material for the taxonomic studies using the existing conventional
methods.
[0018] The midwater habitat of the fish make them a uncommon and
difficult material to get for work as special gears are required on
board of ship.
[0019] The mesopelagic habitats of the north Arabian sea support
rich and varied fauna. Most of these fishes belong to Myctophidae,
Gonostomitids, Stemoptychids etc. The richness of this resource in
the mesopelagic regime of the oceans all the world over has been
well documented (Boltachev, A. R. J. Ichthyol, 27 (4): 539-547
(1987); Hussain and Ali Khan, Deep Sea Research, 34 (7A): 1293-1299
(1987); Alikhan and Aftab, Marine Research, 2 (1-2): 1-9
(1993).
[0020] US Globec, 1993 has reported a biomass of 100 million Tonnes
of Myctophids in the Arabian Sea believed to be constituted by
Benthosema pterotum and Diaphus sp. The true identity of stocks is,
however, unknown.
[0021] In class Pisces there is a large body of population genetic
data available on CD-ROMs and fish data bases (Agustin and
Palomares, 5.sup.th International Symposium on Genetics in
aquaculture, 19-25 , 1994). However, very little information
pertains to the families of lantern fishes. There is only one paper
on myctophids by Afanas-yev et al. , J.Ichthiology, 30 (1):28-37
(1990).
[0022] The earlier workers have described genetic diversity amongst
conspecific and closely related species using protein
electrophoresis. However, in the recent years, the molecular
techniques using DNAs as the genetic markers have almost replaced
these traditional methods with many shortcomings and providing
information about only 1% of the genome.
[0023] Several workers have applied the new methods of DNAs for
quantifying genetic relatedness among group of fish in stock
assessments among wild and cultured populations and in studies of
taxonomy and population genetic (Datta et al. , Gene, 62: 331- 336.
1988; Devlin et al. Canadian Journal of Fishery and Aquatic
Sciences, 48 (9): 1606-1612,1991; Berson et al. Molecular
Microbiology, 5 (9), 2261-2264, 1991; Martinez et al. Genome, 36:
1119-1123, 1993; Du Jun et al. DNA and cell Biology, 12 (8):
739-751, Cytogenetic and cellular genetics, 65: 233-237, 1993;
Pogson et al. Genetics, 139: 375-3851994 and Carr et al. Molecular
Ecology,4:79-88, 1995.
[0024] There is a great lacunae in our knowledge of myctophid
resource and stock assessment using genetic techniques. The only
information so far available in related isozyme studies on 3
species endemic to the eastern pacific (Afanas-yev et al.
1990).
[0025] There is absolutely no literature available on genetic
assessment of stocks from the other oceans.
[0026] A judicious exploitation and resource management requires
proper identification of the larval and adult stages of huge
myctophid populations in the world oceans. The genetic resource
assessment, species identification, characterzation of life history
stages, estimation of genetic variability and level of gene flow
between various stocks for proper utiliztion of this vast fishery
potential and for assessment and evaluation of trophic dynamics at
the top end of the marine food chain.
[0027] Various authors have described methods of making DNA probes
and their use as genetic markers in various organisms using mostly
cDNA approach. Some patents pertain to improvement of DNA
amplification related methods and formulation of different
primers.
[0028] Weissman et al. 1983 patented Method for cloning genes (U.S.
Pat. No. 4,394,443 published on Jul. 19, 1983). Mullis et al. In
U.S. Pat. No. 4,683,195 published on Jul. 28, 1987 describe process
for amplifying, detecting, and/or cloning nucleic acid sequences.
The methods provided recombinant clones coding for human
histocompatibility antigens, in particular clones for HLA-B
antigens. The recombinant DNA expression system is developed for
use in control of larval and adult insects and conferring pesticide
resistance to crop plants.
[0029] Erlich et al. (U.S. Pat. No. 5,314,809 published on May 24,
1994) provide methods for enhanced specificity and sensitivity of
nucleic acid amplification. The methods are simplified nested
amplification procedures wherein both inner and outer primer pairs
are present in the amplification reaction mixture.
[0030] Grosz, et al. In U.S. Pat. No. 5,340,728 published on Aug.
23, 1994 describe method for amplification of targeted segments of
nucleic acid using nested polymerase chain reaction. Nuovo et al.
(1996) in U.S. Pat. No. 5,538,871, published on Jul. 23, 1996
describe in improved In situ polymerase chain reaction. Barry et
al. In U.S. Pat. No. 5,574,145 published on Nov. 12, 1996 isolated
nucleic acid molecules targeted to the region intermediate to the
16 S and 23 S rRNA genes useful as probes for determining bacteria.
They describe a method for generating DNA probes specific for an
organism and capable of distinguishing in a non-empirical manner
between species.
[0031] Cossart et al. (U.S. Pat. No. 5,523,205 published on Jun. 4,
1996) describe DNA probes specific for hemolytic listeria bacteria.
Trent et al. In the U.S. Pat. No. 5,693,464 published on Dec. 2,
1997 report rapid reproducible procedures for generating chromosome
region-specific (CRSPs) for diagnostic and research
applications.
[0032] Scott and Tomita (1998) give uses of cytochrome P450.
Sub.Ipr gene (U.S. Pat. No. 5,734,086, published on Mar. 31, 1998)
. Harris et al. (1998, U.S. Pat. No. 5,849,544, Dec. 15, 1998 )
give method of characterization and provide method for the
detection of diagnostic base sequences in one or more nucleic acids
contained in a sample.
[0033] Jeffreys et al (1998, U.S. Pat. No. 5,853,989, published on,
Dec. 29, 1998) describe method of characterisation of genomic DNA.
They used primers which selectively prime specific type of internal
repeat unit in a tandemly repeated region. Ryder et al. (1998, U.S.
Pat. No. 5,786,183, published on Jul. 28, 1998) give methods of
enhancing nucleic acid amplification. Dandliker et al. (1998, U.S.
Pat. No. 5,707,813, published on Jan. 13, 1998 ) report nucleic
acid probes and methods.
[0034] Kuhns (1999, U.S. Pat. No. 5,981,171, published on Nov. 9,
1999) describe diagnostic assays using nucleic acid probes. He
describes methods and compositions for a rapid quantitative nucleic
acid hybridization assay fpr detecting a DNA or RNA sequence in a
biological sample.
[0035] Caetano-Anolles (1999, U.S. Pat. No. 5,962,221, published on
Oct. 5, 1999) give oligonucleotide constructs and methods for the
generation of sequence signatures from nucleic acids.
[0036] Bebbington et al (1999, U.S. Pat. No. 5,891,693, published
on Apr. 6, 1999) describe recombinant DNA methods vectors and host
cells.
[0037] Rothschild et al. In U.S. Pat. No. 5,939,264 published on
Aug. 17, 1999) describe genetic markers in pigs for reproductive
traits using polymorphism in the reproductive genes.
[0038] Molecular cloning and characterization of a further gene
sequences coding for human relaxin is given in U.S. Pat. No.
4,758,516 by Hudson et al. published on Jul. 19, 1988.
[0039] Some patents related to the genes reported by us is
available on humans and other organisms. Aguirre et al. (1998)
developed progressive rod-cone degeneration disease genetic markers
and assays in a canine (U.S. Pat. No. 5,804,388). Reports on
diagnosis of hereditary retinal degenerative diseases is given by
Dryja et al. In U.S. Pat. No. 5,262,529 published on Nov. 16, 1993
They developed a probe for identifying region of photoreceptor
protein of humans and also made the primers . Dryja et al in Mar.
1996, U.S. Pat. No. 5.498,521 published on Mar. 12 , 1996 later
report the method which involves analyzing the DNA of the subject
to determine the presence or absence of a mutation in a gene for
photoreceptor protein. Shassere, et al. (1997 U.S. Pat. No.
5,698,398 published on Dec. 16, 1997) disclosed quality control
compositions suitable as sample specimens to measure performance of
DNA probe tests which determine cytogenetic abnormalities, such as
chromosome copy number, of cells in a tissue sample.
[0040] However, all of these are related to making cDNA probes and
the work is mostly on humans, canines, bacteria and other
organisms, mostly of terrestrial origin.
[0041] No patent is available on fish DNA probes and sequences as
genetic markers particularly in the myctophid fish. Nor are primer
sequences designed for these fishes.
[0042] Earlier Mitochondrial DNA control region and Ribosomal
Internal transcribed spacer (ITS2) are used for systematic
relationships. Reed K. M, et al. (1998) studied sequence analysis
of the mitochondrial DNA control region of ciscoes (genus
Coregonus) with taxonomic implications for the Great Lakes species
flock.
[0043] Molecular cloning of rod opsin (rhodopsin) cDNA from retinas
of various teleost fishes, octopus, squids, shrimps, Lamprey and
screening of cDNA libraries of rhodopsin is done by several workers
(O'Brien and Al-Ubaidi, M. R., Gene, 193 (2): 141-150 ;
Crescitclli, F, et al. Journal of Comparative Physiology, 1985: 157
(3): 323-333; Tsai, H. J. et al., Biochemical molecular Biology,
109, 91: 81-88; Hara-Nishimura et al. FEBS-LETT, 317 (1-2): 5-11,
1993). Crandall, K. A. and Hillis, D. M. Nature, 387 (No. 6634),
667-668,1997, describe rhodopsin protein evolution in the dark in
cave dwelling cray fish. Fitzgibbon, j et al., Gene, 1995,164 (2),
273-277. Harada, Y et al. Journal of Biochemistry,110 (4), 501-
507,1991 report synthesis and expression of rhodopsin gene in
Octopus. But all these reports deal with the protein rhodopsin, not
much has been said about its gene even in these species.
[0044] Douzery E, et al. Molecular Biology and Evolution,
14(11):1154-66 (1997) use the mitochondrial control region of
Cervidae: evolutionary patterns and phylogenetic content. Barreto
G, et al. American Journal of Human Genetics 58(3):609-16 (1996)
report Mitochondrial D-loop "signatures" produced by low-stringency
single specific primer PCR constitute a simple comparative human
identity test. Brown J R, et al. Genetics.142(2):525-35. 1996
describe Length variation, heteroplasmy and sequence divergence in
the mitochondrial DNA of four species of sturgeon (Acipenser). Lee
W J, et al. J Mol Evol. 1995 July;41(1):54-66 report structure and
evolution of teleost mitochondrial control regions. No reports of
DNA probes for genetic marking of any marine species is come
across.
[0045] Jobst J, et al (1998) give information on Molecular
evolution of the internal transcribed spacers (ITS1 and ITS2) and
phylogenetic relationships among species of the family
Cucurbitaceae. Odorico D M, et al. (1997) describe Variation in the
ribosomal internal transcribed spacers and 5.8S rDNA among five
species of Acropora (Cnidaria; Scleractinia): patterns of variation
consistent with reticulate evolution. Despres L, et al (1995)
describe ITS2 ribosomal RNA indicates Schistosoma hippopotami is a
distinct species. Crabtree M B, et al. (1995) study development of
a species-diagnostic polymerase chain reaction assay for the
identification of Culex vectors of St. Louis encephalitis virus
based on interspecies sequence variation in ribosomal DNA spacers.
Mukhamedov R S, et al. (1994) report [Nucleotide sequence of
internal transcribed spacers and 5.8S rDNA for the ribosomal operon
from alfalfa Medicago sativa and cotton Gossypium hirsutum L]. No
reports of DNA probes in open ocean marine fish are seen.
[0046] Goswami and Bernardi (1999) described nucleotide gene
sequences of 16 S and 12 S genes of five myctophid species viz:
Tarletonbeania crenularis; Protomyctophum crockeri; Lampanyctus
regalis; Diaphus theta; Stenobrachius leucosparus in the National
center of Biotechnology information GenBank. These sequences were
published on 20 Apr. 1999 and can be screened at www period ncbi
period nlm period nih period gov Genbank databases. The respective
accession numbers are AF134250; AF 134249; AF 134248; AF 134247; AF
134246; AF 134245; AF 134244; AF134243; AF 134242 and AF
134241.
[0047] As such, in order to obcriate the draw backs listed in the
use of conventional taxonomic methods for identification of open
ocean midwater fish, the applicants developed a novel method and
approach, using molecular biology.
[0048] The invention relates to the molecular cloning and
characterization of specific gene region sequences. More
particularly the invention relates to genetic markers which have
been identified in several genes such as Cytochrome b (cyt b);
Mitochondrial control region (D-Loop); Internal Transcribed spacer
between nuclear ribosomal genes (ITS2) & Rhodopsin gene of
visual pigments (Rod) and mitochondrial ribosomal RNA genes for
myctophid fishes.
[0049] As discussed above, prior art patents and reports deal with
different groups of organisms. The invention on the other hand
concerns an economically important group of open ocean midwater
fish found in all the world oceans which has adapted itself to the
oxygen minimum conditions and to low/dark light intensities for
vision, the genetic information of which has not been reported so
far.
[0050] The genomic DNA probes are sequence specific and are ideal
for identification of complimentary regions of species specific
genes. Cytochrome b is an important component of mitochondrial
electron transport chain and plays important role in respiratory
physiology (William, F. Ganong, Chapter 17 199-235 in Review of
medical physiology pp. 599; 1977). The gene encoding cytochrome b,
termed Cyt b of myctophid fishes plays an important role in
adaptations and survivals of these fishes in the suboxic
regions.
[0051] The Rod gene encoding for rhodopsin of the retinal rods is
important for adapations of these fishes to varying light
intensities which varies with the depth.
[0052] The mitochondrial control region D-Loop an Internal
transcribed spacer ITS2 gene regions are highly prone to rapid
mutations and can be suitable gene candidates for identification of
variants at the intra species and population level.
[0053] The invention discloses species specific DNA probes for
detection of genes such as cyt b, Rod, D-Loop, ITS2. This invention
is useful for identification of early and adult life history stages
of myctophids i.e. Lantern fishes.
[0054] This is the first report of preparation of DNA probes useful
for molecular genetic identification of myctophid fish. Novelty of
the invention is in use of novel gene region DNA probes for
identification of eggs and early larvae of mesopelagic fish which
live in oxygen minimal and low light conditions.
OBJECTS OF THE INVENTION
[0055] The main object of the invention is to provide an effective
method for identifying stock and population of myctophids.
[0056] Another object is to provide a method using polymerase chain
reaction with specific primers to detect and quantify the
expression of genes like cytochrome b, Rod, D-Loop, ITS.sub.2 etc.
in myctophid fishes.
[0057] Another object of the present invention is to develop
specific DNA probes for identification of populations of
mesopelagic fish of the type myctophids.
[0058] Yet another object of the invention is to develop DNA probes
comprising specific nucleotide sequences which specifically can
detect mitochondrial genes such as Cyt b, D-Loop in myetophid
fishes.
[0059] Still another objective is to develop DNA probes comprising
specific nucleotide sequences which specifically can detect nuclear
genes such as Rod and ITS2 in fishes.
[0060] Another objective of the invention is design specific
primers for specific gene regions.
[0061] Still another object is to develop kits containing primer
sets.
DETAILED DESCRIPTION OF THE INVENTION
[0062] The present invention seeks to overcome the drawbacks
inherent in the prior art by providing the highly efficient and
selective means for identifying the stocks and populations of
myctophids using specific nucleic acid probes of certain genes. As
used herein the, term `probes` and `probe` are used to designate
the collection of DNA segments produced by amplification of
specific regions.
[0063] Accordingly, the invention provides a method for development
of DNA probes for myctophids which comprises:
[0064] (i) Extracting the DNA from the tissue of an identified
myctophid fish,
[0065] (ii) Selecting primers for species and population level,
[0066] (iii) Amplifying the extracted DNA using polymerase chain
reaction (PCR) using the selected primers,
[0067] (iv) Eluting the amplified DNA from the PCR reactions,
[0068] (v) Cycle sequencing of 5 mu L. eluted DNA using single
primer,
[0069] (vi) Purifying extension products,
[0070] (vii) Sequencing on acrylamide gel in an automated
sequencer.
[0071] (viii) Cloning a part of the eluted DNA (IV) from the
resulting PCR product into a suitable vector,and growing in
competent transformed host cells,
[0072] (ix) Purifying recombinant plasmid DNA having the cloned
gene probes from the transformed host cells.
[0073] (x) Checking purity and specificity of the cloned DNA probe
insert,
[0074] (xi) Confirming the molecular size of the DNA probe
insert,
[0075] (xii) Sequencing of the cloned DNA probe.
[0076] (xiii) Comparing the DNA sequences of the prepared DNA probe
with the sequences of the same gene region done in the automated
sequencer,
[0077] (xiv) Comparing the DNA sequence of the DNA probes using
"BLAST" program against the known sequences of similar genes in the
genome data bases.
[0078] Thus, the invention provides nucleotide sequences, primers
and probes which exhibit myctophid specificity in nucleic acid
amplification reactions. The invention relates to nucleotide
sequences specific to mitochondrial genes in myctophid fishes. The
invention also provides nucleotide sequences specific for the
nuclear genes of myctophid fishes. Thus, the invention provides
oligonucleotide primers that can be used for amplification of
target sequence in myctophid fishes. These primers can amplify
specific regions of gene by PCR.
[0079] In an embodiment the mitochondrial genes are Cyt b and
D-loop genes.
[0080] In another embodiment the nuclear genes are Rod and ITS-2
genes.
[0081] In yet another embodiment the myctophid fishes are selected
from the group comprising the steps of Stenobrachius leucospara,
Diaphus theta, Protomyctophum crockeri, Tarletonbeania crenularis,
Lampanyctus regalis, Symbolophorus sp., Triphoturus sp. and
Lampanyctus sp.
[0082] In still another embodiment the primer set (forward and
backward primers) used for amplification and detection of Cyt b
gene contains oligonucleotides with the sequences (SEQ ID NOS
1-2):
1 CYT 1: 5' TGA YTT GAA RAA CCA YCG TTG 3' CYT 2: 5' CTC CAR TCT
TCG RYT TAC AAG 3'
[0083] In another embodiment (forward and backward primers) used
for reamplification and detection of Cyt b gene contains
oligonucleotides with the sequences (SEQ ID NOS 3 and 2):
2 CBI-L: 5' CCA TCC AAC ATC TCA GCA TGA TGA AA 3' CYT 2: 5' CTC CAR
TCT TCG RYT TAC AAG 3'
[0084] In another embodiment the primer set (forward and backward
primers) used for PCR amplification and detection of D-Loop gene
contains oligonucleotides with the sequences:
3 (SEQ ID NO 4) PRO-L: 5' CTA CC 3' D-LOOP H: 5' CCT GAA GTA GGA
ACC AGA TG 3'
[0085] In another embodiment (forward and backward primers) used
for PCR amplification of ITS2 gene contains oligonucleotides with
the sequences (SEQ ID NOS 5-6):
4 ITS2-F: 5' CTA CGC CTG TCT GAG TGT C 3' ITS2-R: 5' ATA TGC TTA
AAT TCA GCG GG 3'
[0086] In yet another embodiment the primer set (forward and
backward primers) used for PCR amplification of Rhodopsin gene Rod
contains oligonucleotides with the sequences (SEQ ID NOS 7-8):
5 ROD-R: 5' TCT TTC CGC AGC ACA ACG TGG 3' ROD-F: 5' CAT ATG AAT
ACC CTC AGT ACT ACC 3'
[0087] In still another embodiment the primer set (forward and
backward primers) used for PCR amplification of 12S RNA gene
contains oligonucleotides with the sequences (SEQ ID NOS 9-10):
6 12 SA-L: 5' AAA CTG GGA TTA GAT ACC CCA CTA T 3' 12 SB-H: 5' AGA
GTG ACG GGC GGT GTG T 3'
[0088] In another embodiment the primer set (forward and backward
primers) used for PCR amplification of 16S RNA gene contains
oligonucleotides with the sequences (SEQ ID NOS 11-12):
7 16 SAR-L: 5' CGC CTG TTT ATC AAA AAC AT 3' 16 SBR-H: 5' CCG GTC
TGA ACT CAG ATC ACG T 3'
[0089] In yet another embodiment the vector used for cloning was
Bluescript KS.sup.- phagemid.
[0090] In still another embodiment the vector used for cloning had
ampicillin resistance gene for selection.
[0091] In another embodiment the vector used for cloning had Lac Z
gene for blue white colony selection.
[0092] In another embodiment the CoI E 1 was the origin for
replication of phagemid in the absence of helper phage.
[0093] In another embodiment F 1 (-) origin for recovery of
antisense strand of lac Z gene when a host strain containing the
bluescript II phagemid (FIG. 1).
[0094] In another embodiment the host cells used for transformation
were E. coli blue
[0095] bacteria (Bacteria Strain XL 1 blue ) XL1-Blue
:--F'::Tn10,pro A.sup.+B.sup.+lacl.sup.q
[0096] (lacZ)M15/recA1enda1gyrA96(Nal.sup.r)thi
hsdR17(r.sub.k.sup.-m.sub.- k.sup.+)supE44relA1 lac.
[0097] The invention also relates to specific DNA sequences for the
cloned DNA probe inserts for the Cyt b, D-Loop, Rod, ITS2 genes.
The invention provides species specific primer sequences for
amplification and detection of Cyt b, D-Loop, Rod, ITS2, 12S RNA
and 16 S RNA genes of Stenobrachius leucopsarus (SLMB) myctophid
fish. The sequences of the species specific primer 12S rRNA -H and
12S rRNA -L of Stenobrachius leucopsarus (SLMB) designed were such
as (SEQ ID NOS 13-14):
8 12S-H 5' CCC ACT CAC TGC TAA CTC C 3' 12S-L 5' GGC TAA CTA CAA
TCA TCT GCT 3'
[0098] The sequences of the species specific primer 16S rRNA -H and
16S rRNA -L of Stenobrachius leucopsarus (SLMB) designed were such
as (SEQ ID NOS 15-16):
9 16S-H 5' TAC GCA TAA CGG CTC TGG 3' 16S-L 5' CTA CTA CAC CTC AAC
TAC ATC T 3'
[0099] The sequences of the species specific primer Cyt -H and Cyt
-L of Stenobrachius leucopsarus (SLMB) designed were such as(SEQ ID
NOS 17-18):
10 Cyt-H 5' GCT CGG GCT GCT GGA ATC TT 3' Cyt-L 5' CAA CCT CAT CTG
TCG TAA AC 3'
[0100] The sequences of the species specific primer ITS2 -H and
ITS2 -L (Forward) of Stenobrachius leucopsarus (SLMB) designed were
such as (SEQ ID NOS 19-20):
11 ITS2-H 5' ATA CTC TGC GGA CAT ACT TGA CTG 3' ITS2-F 5' ACT TGA
CTG ACC TTC TTA CT 3'
[0101] The sequences of the species specific primer Pro-L and D
Loop -H of Stenobrachius leucopsarus (SLMB) designed were such as
(SEQ ID NOS 21-22):
12 Pro-L 5' CAG TCT CGT CAA ACC AAG TCA AAC 3' D loop-H 5' ATA ATC
ATC CAG CAT AAA CAC AC 3'
[0102] The sequences of the species specific primer ROD -L and
ROD-H of Stenobrachius leucopsarus (SLMB) designed were such as
(SEQ ID NOS 23-24):
13 ROD-L 5' CCT GGT AGA GTT CGC CGT CA 3' ROD-H 5' CGT GTT CCT TAT
CAT TGT GCC T 3'
[0103] The sequences of the species specific primer 16S rRNA -H and
16S rRNA-L of yet another myctophid Lampanyctus regalis (LRMB)
designed were such as (SEQ ID NOS 25-26):
14 16S-H 5' TCG TAG TTC AGC AGT CAG 3' 16S-L 5' CAC CAG CCA AGT ATG
TTT CTC 3'
[0104] The sequences of the species specific primer 12S rRNA -H and
12S rRNA -L of yet another myctophid Lampanyctus regalis (LRMB)
designed were such as(SEQ ID NOS 27-28):
15 12S-H 5' GCC TCC ATC ATC CCT CAC CTT AC 3' 12S-L 5' CTA TTC GCC
TCG CTC AGA C 3'
[0105] The sequences of the species specific primer 16S rRNA -H and
16S rRNA -L of yet another myctophid Diaphus theta (DTMB) designed
were such as (SEQ ID NOS 29-30):
16 16S-H 5' CTC CGT CCG TCT CGC CTC TG 3' 16S-L 5' AAA TCC GCC CTT
ATG TGT GTT C 3'
[0106] The sequences of the species specific primer 12S rRNA -H and
12S rRNA -L of yet another myctophid Diaphus theta (DTMB) designed
were such as (SEQ ID NOS 31-32):
17 12S-H 5' CAT CGG CTT GCT CTA TTC CTT G 3' 12S-L 5' TCT ATC GGC
GGC GTA TCA C 3'
[0107] The sequences of the species specific primer 16S rRNA -H and
16S rRNA -L of yet another myctophid Tarletonbaenia crenularis
(TCMB) designed were such as (SEW ID NOS 33-34):
18 16S-H 5' GGC GAT TCT ACG GCA CGG GCG 3' 16S-L 5' AAA CTG GTC CTC
AAC TAT GTC A 3'
[0108] The sequences of the species specific primer 12S rRNA -H and
12S rRNA -L of yet another myctophid Tarletonbaenia crenularis
(TCMB) designed were such as (SEQ ID NOS 35-36):
19 12S-H 5' CCG ATT CAG CCA CGA TTC CCT C 3' 12S-L 5' CCT AAA GCC
CAG ATA ACT ACA 3'
[0109] The sequences of the species specific primer 16S rRNA -H and
16S rRNA -L of yet another myctophid Protomyctophum crockeri (PCMB)
designed were such as (SEQ ID NOS 37-38):
20 16S-H 5' CGT GTT CTG ATG ATG ATG TGC T 3' 16S-L 5' ATT CCT TCC
TCT TAG TAT G 3'
[0110] The sequences of the species specific primer 12S rRNA -H and
12S rRNA -L of yet another myctophid Protomyctophum crockeri (PCMB)
designed were such as (SEQ ID NOS 39-40):
21 12S-H 5' GCT GAA CTT ACT ATG CCC TAC T 3' 12S-L 5' CCG ATT GAC
GCC GAA CTA TG 3'
[0111] Table 1--Forward primer (SEQ ID NO: 18) designed for
cytochrome b gene of Stenobrachius leucopsarus (slmb primer cyt L)
with 5' to 3' end sequences (SENSE) and summaries of
oligonucleotide and structural analyses.
[0112] Table 2--Backward primer (SEQ ID NO: 17) designed for
cytochrome b gene of Stenobrachius leucopsarus (slmb primer cyt H)
with 5' to 3' end sequences (ANTISENSE) and summaries of
oligonucleotide and structural analyses.
[0113] Table 3--Forward primer (SEQ ID NO: 20) designed for
Internal Transcribed Spacer (ITS2) of Stenobrachius leucopsarus
(slmb primer ITS2 F) with 5' to 3' end sequences (SENSE) and
summaries of oligonucleotide and structural analyses.
[0114] Table 4--Backward primer (SEQ ID NO: 19) designed for
Internal Transcribed Spacer (ITS2) Stenobrachius leucopsarus (slmb
primer ITS2-H) with 5' to 3' end sequences (ANTISENSE) and
summaries of oligonucleotide and structural analyses.
[0115] Table 5--Forward primer (SEQ ID NO: 21) designed for
mitochondrial Control region d-Loop of Stenobrachius leucopsarus
(slmb primer pro-L) with 5' to 3' end sequences (SENSE) and
summaries of oligonucleotide and structural analyses.
[0116] Table 6--Backward primer (SEQ ID NO: 22) designed for
mitochondrial Control region d-Loop Stenobrachius leucopsarus (slmb
primer D loop-H) with 5' to 3' end sequences (ANTISENSE) and
summaries of oligonucleotide and structural analyses.
[0117] Table 7--Forward primer (SEQ ID NO: 23) designed for
Rhodopsin gene region of Stenobrachius leucopsarus (slmb primer
ROD-L) with 5' to 3' end sequences (SENSE) and summaries of
oligonucleotide and structural analyses.
[0118] Table 8--Backward primer (SEQ ID NO: 24) designed for
Rhodopsin gene region of Stenobrachius leucopsarus (slmb primer ROD
-H) with 5' to 3' end sequences (ANTISENSE) and summaries of
oligonucleotide and structural analyses.
[0119] Table 9--Forward primer (SEQ ID NO: 26) designed for
mitochondrial 16S ribosomal RNA region of Lampanyctus regalis (LRMB
primer 16 S-L) with 5' to 3' end sequences (SENSE) and summaries of
oligonucleotide and structural analyses.
[0120] Table 10--Backward primer (SEQ ID NO: 25) designed for
mitochondrial 16S ribosomal RNA region of Lampanyctus regalis (LRMB
primer 16 S -H) with 5' to 3' end sequences (ANTISENSE) and
summaries of oligonucleotide and structural analyses.
[0121] Table 11--Forward primer (SEQ ID NO: 28) designed for
mitochondrial 12 S ribosomal RNA region of Lampanyctus regalis
(LRMB primer 12 S-L) with 5' to 3' end sequences (SENSE) and
summaries of oligonucleotide and structural analyses.
[0122] Table 12--Backward primer (SEQ ID NO: 27) designed for
mitochondrial 12 S ribosomal RNA region of Lampanyctus regalis
(LRMB primer 12 S -H) with 5' to 3' end sequences (ANTISENSE) and
summaries of oligonucleotide and structural analyses.
[0123] Table 13--Backward primer (SEQ ID NO: 29) designed for
mitochondrial 16 S ribosomal RNA region of Diaphus theta (DTMB
primer 16 S -H) with 5' to 3' end sequences (ANTISENSE) and
summaries of oligonucleotide and structural analyses.
[0124] Table 14--Forward primer (SEQ ID NO: 30) designed for
mitochondrial 16 S ribosomal RNA region of Diaphus theta (DTMB
primer 16 S -L) with 5' to 3' end sequences (SENSE) and summaries
of oligonucleotide and structural analyses.
[0125] Table 15--Backward primer (SEQ ID NO: 31) designed for
mitochondrial 12 S ribosomal RNA region of Diaphus theta (DTMB
primer 12 S -H) with 5' to 3' end sequences (ANTISENSE) and
summaries of oligonucleotide and structural analyses.
[0126] Table 16--Forward primer (SEQ ID NO: 32) designed for
mitochondrial 12 S ribosomal RNA region of Diaphus theta (DTMB
primer 12 S -L) with 5' to 3' end sequences (SENSE) and summaries
of oligonucleotide and structural analyses.
[0127] Table 17--Backward primer (SEQ ID NO: 33) designed for
mitochondrial 16 S ribosomal RNA region of Tarletonbenia crenularis
(TCMB primer 16 S -H) with 5' to 3' end sequences (ANTISENSE) and
summaries of oligonucleotide and structural analyses.
[0128] Table 18--Forward primer (SEQ ID NO: 24) designed for
mitochondrial 16 S ribosomal RNA region of Tarletonbenia crenularis
(TCMB primer 16 S -L) with 5' to 3' end sequences (SENSE) and
summaries of oligonucleotide and structural analyses.
[0129] Table 19--Backward primer (SEQ ID NO: 35) designed for
mitochondrial 12 S ribosomal RNA region of Tarletonbenia crenularis
(TCMB primer 12 S -H) with 5' to 3' end sequences (ANTISENSE) and
summaries of oligonucleotide and structural analyses.
[0130] Table 20--Forward primer (SEQ ID NO: 36) designed for
mitochondrial 12 S ribosomal RNA region of Tarletonbenia crenularis
(TCMB primer 12 S -L) with 5' to 3' end sequences (SENSE) and
summaries of oligonucleotide and structural analyses.
[0131] Table 21--Backward primer (SEQ ID NO: 37) designed for
mitochondrial 16 S ribosomal RNA region of Protomyctophum crockeri
(PCMB primer 16 S -H) with 5' to 3' end sequences (ANTISENSE) and
summaries of oligonucleotide and structural analyses.
[0132] Table 22--Forward primer (SEQ ID NO: 38) designed for
mitochondrial 16 S ribosomal RNA region of Protomyctophum crockeri
(PCMB primer 16 S -L) with 5' to 3' end sequences (SENSE) and
summaries of oligonucleotide and structural analyses.
[0133] Table 23--Backward primer (SEQ ID NO: 39) designed for
mitochondrial 12 S ribosomal RNA region of Protomyctophum crockeri
(PCMB primer 12 S -H) with 5' to 3' end sequences (ANTISENSE) and
summaries of oligonucleotide and structural analyses.
[0134] Table 24--Forward primer (SEQ ID NO: 40) designed for
mitochondrial 12 S ribosomal RNA region of Protomyctophum crockeri
(PCMB primer 12 S -L) with 5' to 3' end sequences (SENSE) and
summaries of oligonucleotide and structural analyses.
[0135] Table 25--Backward primer (SEQ ID NO: 15) designed for
mitochondrial 16 S ribosomal RNA region of Stenobrachius
leucopsarus (SLMB primer 16 S -H) with 5' to 3' end sequences
(ANTISENSE) and summaries of oligonucleotide and structural
analyses.
[0136] Table 26--Forward primer (SEQ ID NO: 16) designed for
mitochondrial 16 S ribosomal RNA region of Stenobrachius
leucopsarus (SLMB primer 16 S -L) with 5' to 3' end sequences
(SENSE) and summaries of oligonucleotide and structural
analyses.
[0137] Table 27--Backward primer (SEQ ID NO: 13) designed for
mitochondrial 12 S ribosomal RNA region of Stenobrachius
leucopsarus (SLMB primer 12 S -H) with 5' to 3' end sequences
(ANTISENSE) and summaries of oligonucleotide and structural
analyses.
[0138] Table 28--Forward primer (SEQ ID NO: 14) designed for
mitochondrial 12 S ribosomal RNA region of Stenobrachius
leucopsarus (SLMB primer 12 S -L) with 5' to 3' end sequences
(SENSE) and summaries of oligonucleotide and structural
analyses.
DETAILS OF THE INVENTION
[0139] This invention of DNA probes developed can be utilized for
chromosomal mapping of the myctophid species.
[0140] The present invention also contemplates the use of lantern
fish primer sets and specific probes in kit form.
[0141] Thus in a preferred mode of use, the species specific
primers will be employed to conveniently amplify a selected gene
region to produce DNA probe directed for use as genetic
markers.
[0142] The invention would be advantageous for identification of
myctophid larvae and hence can facilitate the assessment of water
bodies, estimation of genetic resources and genetic variability
between myctophid population.
[0143] Cyt b probe sequenmces of myctophids will be advantageous to
be used for study of comparative human genomics of the genes
related to acclimitisation to low oxygen at high altitudes and deep
oceanic explorations.
[0144] Rod gene probe sequences will be advantageous to be used for
study of comparative human genomics of the genes related to
acclimitisation to low light like night blindness.
EXAMPLE 1
Chemicals, Reagents, Apparatus Used and Their Sources
[0145]
22 LIST OF CHEMICALS Name Company Catalogue Number
8-Hydroxyquinoline HIMEDIA RM1061 Acrylamide 3Xcryst SRL 0144139
free from DNase, RNase, Protease Agar powder HIMEDIA RM 026 Agarose
SRL 014011 Agarose HIMEDIA RM 187 Benzimidazole SRL 024727
(1,3Benzodiazole) Extrapure Bromophenol blue BDH 20015 Buffer
Tablets QUALIGENS 17301 pH 7.0 Butan-1-ol GLAXO 12045 A.R
Chloroform A.R S.D.FINE 20077 CHEM Chloroform for HPLC SRL 0322123
E.D.T.A. Disodium salt HIMEDIA RM 1195 (Hydroxy Methyl) free from
DNase, RNase, protease EDTA HIMEDIA RM 678 EDTA Disodium salt
S.D.FINE 38025 L.R CHEM Ethanol MERCK UN 1170 Ethanol (Absolute)
FARCO 971109 CHEMICALS Ethidium Bromide HIMEDIA RM 813 Hydrochloric
acid Isoamyl alcohol MERCK 8.18969 For synthesis Magnesium Chloride
HIMEDIA RM 728 A.R Methanol SRL 132977 Extrapure A.R N,N-Dimethyl
Formamide SRL 042825 Phenol RANBAXY P0130 A.R Potassium Acetate
HIMEDIA RM 1091 Potassium Chloride MERCK 17533 Purified Potassium
Dihydrogen HIMEDIA RM 249 Orthophosphate A.R Propan-2-ol QUALIGENS
73827 A.R Sodium Acetate HIMEDIA RM 410 Anhydrous Sodium Chloride
HIMEDIA RM 853 A.R Sodium dihydrogen Phosphate MERCK 17845
2-hydrate Cryst.Pure Sodium Hydroxide HIMEDIA RM 467 A.R Sodium
Lauryl Sulphate HIMEDIA RM 205 Sodium Nitrite QUALIGENS 15935
Excela R Sodium Phosphate dibasic LOBA 35986 Anhydrous purified
CHEMIE Sucrose SRL 194957 From sugar cane A.R Tris Aminomethane SRL
2044122 (Tris Buffer) Tris Aminomethane SRL 204982 Hydroxy Methyl
(Tris buffer) Extrapure A.R Tris buffer HIMEDIA RM 262 A.R Tris
Hydrochloride HIMEDIA RM 613 Tri-Sodium Citrate HIMEDIA RM 255 A.R
Trypsin 3X Cryst. SRL 204013 Trypsin 1: 250 SRL 2040106 Vitamin
B.sub.12 (Cyanocobalamin) HIMEDIA RM184 Yeast Extract powder SRL
254011
[0146] DNA Extractions and Quality and Quantity Check:
[0147] Total DNA of Stenobrachius leucopsarus was extracted from
the muscle tissue by putting in the cocktail of
[0148] 613.0 .mu.l Lysis Buffer (Lysis Buffer recipe: 10 mM Tris,
400 mM NaCl, 2 mM Na.sub.2 EDTA);
[0149] 30.0 .mu.l 20% SDS (40g/200 ml water);
[0150] 7.0 .mu.l Proteinase K (20 .mu.g/.mu.l) which was stored at
-20.degree. C. and about 0.1-0.5 grams of the muscle tissue.
[0151] Incubated the eppendorf at 55.degree. C. for 12-14 hrs.
Added 375 .mu.l of 6 M NaCl and left for 30 min at room
temperature.
[0152] Centrifuged for 30 min. @14000 rpm. Removed the supernatant
and added Chloroform (800 .mu.l )& Centrifuged for 10 min@12000
rpm. Removed the supernatant and added 1 .mu.l RNAase (10 mg/ml,
stored at -20.degree. C.) and kept at 37.degree. C. for 10 min.
Added 750 .mu.l volume of isopropanol.
[0153] Left the tube at -20.degree. C. for 30 min for DNA
precipitation followed by Centrifigation for 30 min.@14000 rpm.
[0154] Removed the liquid and dried the pellet under vacuum.
[0155] After 1-2 hrs dissolved DNA pellet in 100 .mu.l of
sterilized ultrapure water. Stored at 4.degree. C.
[0156] The DNA extracted was checked for its quality and quantity
using UV Spectrophotometer (OD.sub.260-OD.sub.280 range of
wavelengths of UV. 10.D=50 .mu.g DNA/ml. 1.80-2.00 range of A1:A2
ratios.
[0157] Agarose gel electrophoresis was done to check the molecular
weight of DNA.
[0158] Agarose gel (0.8%) was prepared in 1.times. TBE buffer and 2
.mu.l etBr was added to it.
[0159] The samples were loaded and 1 Kb DNA Ladder was used as the
Marker. The samples were run for 40-45 min. at 72 volts.
[0160] The results were viewed in Biorad Gel Doc. 1000.
[0161] The computer program used was Molecular analyst.
EXAMPLE 2
[0162] The DNA preparation and quality & quantity of
Tarletonbenia crenularis was done as given in example 1.
EXAMPLE 3
[0163] The DNA preparation and quality & quantity of
Protomyctophum crockeri was done as given in example 2.
EXAMPLE 4
[0164] The DNA preparation and quality & quantity of
Lampanyctus regalis was done as given in example 3.
EXAMPLE 5
[0165] The DNA preparation and quality & quantity of Diaphus
theta was done as given in example 4.
EXAMPLE 6
[0166] PCR amplification using forward and backward D-Loop primers
of Stenobrachius leucopsarus.
[0167] The PCR master mix (100 .mu.l) comprised of Taq Buffer
MgCl.sub.2 free (10.0 .mu.l), dNTP all the four nucleotides in the
ratio of 1:1:1:1 (08.0 .mu.l); D-Loop forward primer 01.0 .mu.l
with sequences (PRO-L: 5' CTA CC 3'), D-Loop backward 01.0 .mu.l,
with sequences (D-Loop H: 5' CCT GAA GTA GGA ACC AGA TG 3') (SEQ ID
NO: 4); MgCl.sub.2 (01.0 .mu.l); Taq Polymerase (0.5 .mu.l); and
ultrapure water (78.2 .mu.l).
[0168] This master mix was divided into 4 equal parts and put in
separate eppendorf tubes. To each tube 0.3 .mu.l of DNA of
Stenobrachius leucopsarus was added and run for 35 cycles in DNA
thermo cycler. (Each cycle consisted of 94 degree C. for 45
Seconds, 48 degree for 45 seconds, and 72 degree C. for 1 minute)
and hold at 4 degree Centigrade.
[0169] Thermal Cycler used was DNA Thermal Cycler, Perkin Elmer
480.
EXAMPLE 7
[0170] As given in example 6, PCR amplification master mix was
prepared using forward and backward 12 S RNA primers; 16 S RNA
primers, Cyt b primers; ROD, ITS2 primers and DNA 0.3 .mu.l of
Stenobrachius leucopsarus was added individually to all tubes and
amplified. The primers used were ROD-F: (SEQ ID NO: 8) 5' CAT ATG
AAT ACC CTC AGT ACT ACC 3' and ROD-R: (SEQ ID NO: 7) 5' TCT TTC CGC
AGC ACA ACG TGG 3' for Rhodopsin DNA probe; 16SBR-H (SEQ ID NO: 12)
5' CCG GTC TGA ACT CAG ATC ACG T 3' and 16SAR-L (SEQ ID NO: 11) 5'
CGC CTG TTT ATC AAA AAC AT 3' 16S for 16 S RNA gene probe; 12SA-L:
(SEQ ID NO: 9) 5' AAA CTG GGA TTA GAT ACC CCA CTA T 3' and 12SB-L:
(SEQ ID NO: 10) 5' AGA GTG ACG GGC GGT GTG T 3' for 12S RNA gene
probe and run for 35 cycles in DNA thermo cycler. (Each cycle
consisted of 94 degree C. for 45 Seconds, 48 degree for 45 seconds,
and 72 degree C. for 1 minute) and hold at 4 degree Centigrade.
EXAMPLE 8
[0171] Cytochrome b DNA probe was amplified by using Cyt 1: (SEQ ID
NO: 1) 5' TGA YTT GAA RAA CCA YCG TTG 3' and Cyt 2: (SEQ ID NO: 2)
5' CTC CAR TCT TCG RYT TAC AAG 3' primers followed by
reamplification by using CBI-L: (SEQ ID NO: 3) 5' CCA TCC AAC ATC
TCA GCA TGA TGA AA 3' and Cyt 2: (SEQ ID NO: 2) 5' CTC CAR TCT TCG
RYT TAC AAG 3' primers. The DNA template was of Stenobrachius
leucopsarus and run for 35 cycles in DNA thermo cycler. (Each cycle
consisted of 94.degree. C. for 45 Seconds, 48 degree for 45
seconds, and 72 degree C. for 1 minute) and hold at 4 degree
Centigrade.
EXAMPLE 9
[0172] Similarly, primer ITS1F-ITS2R of Internal transcribed
spacers was used for the nested PCR's (ITS1-F: (SEQ ID NO: 41) 5'
TTG TAC ACA CCG CCC GTC GC 3' and ITS2-R: (SEQ ID NO: 6) 5' ATA TGC
TTA AAT TCA GCG GG 3') and amplified by PCR. Later the ITS2 was
reamplified using primers ITS2-F: (SEQ ID NO: 5) 5' CTA CGC CTG TCT
GAG TGT C 3' and
[0173] ITS2-R: (SEQ ID NO: 6) 5' ATA TGC TTA AAT TCA GCG GG 3'. The
DNA template was of Stenobrachius leucopsarus myctophid fish and
run for 35 cycles in DNA thermo cycler. (Each cycle consisted of
94.degree. C. for 45 Seconds, 48.degree. C. for 45 seconds, and
72.degree. C. for 1 minute) and hold at 4 degree Centigrade.
EXAMPLE 10
[0174] The PCR amplified and PCR reamplified DNA's were cleaned
using QIAquick.TM. PCR purification Kit which is a product of
QIAGEN Inc following their protocols.
EXAMPLE 11
[0175] Cycle sequencing was done after following protocols of
Perkin Elmer's ABI PRISM.TM. Dye terminator Cycle sequencing ready
reaction kit with AmpliTaq.sup.R DNA polymerase. Single primer for
each gene was used and PCR amplification was done following 25
cycles (each cycle comprised of 96.degree. C. for 30 seconds; Rapid
thermal ramp to 46 degree C.; 46.degree. C. for 15 seconds; rapid
Thermal ramp to 60 degree C. and 60 degree C. for 4 minutes).
EXAMPLE 12
[0176] Purified extension products was done after the same protocol
as given on page 8. The DNA pellet was dried under vacuum and
stored the dry pellet at -20.degree. C.
EXAMPLE 13
[0177] Sequencing on acrylamide gel was done in automated
sequencer.
[0178] Model 373, DNA sequencing System, Applied Biosystems. A
Division of Perkin Elmer. The protocols were used after Sambrook,
Fritsch, Maniatis, Molecular Cloning a laboratory manual, second
edition, Vol. 2, DNA sequencing 13.47.
[0179] 40% acrylamide solution was made by acrylamide DNA
sequencing grade 380 g N,N'-methylenebisacrylamide (20 g) and
distilled water 600 ml. The TBE buffer (5.times.) was used. It was
made up with Tris Base (54.00 g); Boric acid (27.50 g) Na2 EDTA
(4.65 g) and remaining ultra pure autoclaved water to make the
volume 1 litre.
EXAMPLE 14
[0180] For computation of sequence data program used was "Sequence
Navigator, DNA, Applied Systems. A division of Perkin Elmer
Corporation". The sequences were sent to Blast email and confirmed
by checking for the respective gene segments.
EXAMPLE 15
[0181] Ligating the eluted PCR product in to a suitable vector:
[0182] Eluted DNA of Stenobrachius leucopsarus of cyt b gene was
ligated. The total mixture of 20 .mu.l comprised of Water (9
.mu.l); T 4 DNA Ligase Buffer (4 .mu.l); 1 .mu.l Vectors (KS.sup.-,
cut with ECOR V); 5 .mu.l eluted DNA; 1 .mu.l T 4 DNA Ligase.
[0183] The vector and the host cells used for cloning was
Bluescript KS.sup.- phagemid with the ampicillin resistance gene
for selection.
[0184] Lac Z gene for blue white colony selection. ColE1 origin for
replication of phagemid in the absence of helper phage.
[0185] F 1 (-) origin for recovery of antisense strand of lac Z
gene when a host strain containing the bluescript II phagemid
[0186] The host cells used for transformation were E. coli blue
bacteria (Bacteria Strain XL 1 blue) XL1-Blue:--F'::Tn10, pro
A.sup.+B.sup.+lacl.sup.q(lacZ)M15/recA1endA1gyrA96(Nal.sup.r)thi
hsdR.sup.17(r.sub.k.sup.-m.sub.k.sup.+)supE44relA1lac.
[0187] All these were put in a PCR tube and left for 24 hrs at 4
degree Centigrade cold room for legation. It was labeled as "#SLMB
1, Cytb, DNA Lig."
EXAMPLE 16
[0188] Similarly the DNA ligase for genes ROD, ITS2, 12 S RNA and
16 S RNA was prepared.
EXAMPLE 17
[0189] Preparation of electro-competent cells for use in
electro-transformation.
[0190] Prepared 3 litre of LB broth media (10 g Bacto-Tryptone per
litre, 5 g Bacto- Yeast per litre and 5 g NaCl per litre ) and
divided into 4 parts.
[0191] From this 2 litres were used for E. coli culture (E. coli
strain XL 1 blue was not ampicillin resistant & so no
antibiotic was added ). It was poured in 4 no. of 1 Litre glass
bottles with 500 ml in each.
[0192] 500 ml was put in a 1 litre bottle to make plates for
growing transformed bacteria after electroporation. Prior to
autoclaving added only 10 gms of granulated agar (2% agar solution)
and later added antibiotic.
[0193] 250 ml LB was distributed in 20 ml KIMAKAP NO. 73660 tubes@5
ml in each (no antibiotic was added). These tubes were meant for
initial growing of electro-competent cells i.e. bacteria.
[0194] Last 250 ml was put in a 250 ml glass bottle for use while
electroporating to put into the cuvettes.
EXAMPLE 18
[0195] Making agar plates to grow transformed bacteria after
Electroporation:
[0196] a) After autoclaving the LB broth bottle with 500 ml LB+10 g
granulated Agar for bacteriological work was allowed to cool down
on the bench to about 50.degree. C.
[0197] Then added, X gal-500 .mu.l, 500 .mu.l* Ampicillin sodium
salt prepared by dissolving 75 mg (0.075 g) in 1.5 ml water, 200
.mu.l* IPTG under sterile conditions. Poured in petridishes. The
procedures followed were after Sambrook, Fritsch, Maniatis,
Molecular Cloning a laboratory manual, second edition, Vol. 1.
EXAMPLE 19
[0198] Electro-transformation of E. coli:
[0199] Apparatus used was: E. coli Pulser.TM.
[0200] Transformation Apparatus
[0201] Bio Rad
[0202] Took E. coli and DNA ligase in 0.2 cm cuvettes. Set the
E.Coli Pulser apparatus to 2.50 kV and followed the protocols given
in Bio Rad catalogue. The white colonies were plated and replated
for 3 consective times.
EXAMPLE 20
[0203] PCR for confirmation that transformed bacteria has the
plasmids with the D-Loop gene inserts.:
[0204] PCR amplification using forward and backward D-Loop primers
of Stenobrachius leucopsarus.
[0205] The PCR master mix (100 .mu.l ) comprised of Taq Buffer
MgCl.sub.2 free (10.0 .mu.l ), dNTP all the four nucleotides in the
ratio of 1:1:1:1 (08.0 .mu.l); D-Loop forward primer 01.0 .mu.l
with sequences (PRO-L: 5' CTA CC 3'), D-Loop backward 01.0 .mu.l,
with sequences (D-Loop H: 5' CCT GAA GTA GGA ACC AGA TG 3') (SEQ ID
NO: 4); MgCl.sub.2 (01.0 .mu.l); Taq Polymerase (0.5 .mu.l); and
ultrapure water (78.2 .mu.l).
[0206] This master mix was divided into 4 equal parts and put in
separate eppendorf tubes. To each tube DNA was added by getting a
pick from the replated last set of white colonies and run for 35
cycles in DNA thermo cycler. (Each cycle consisted of 94.degree. C.
for 45 Seconds, 48 degree for 45 seconds, and 72.degree. C. for 1
minute) and hold at 4 degree Centigrade.
[0207] Thermal Cycler used was: DNA Thermal Cycler, Perkin Elmer
480.
EXAMPLE 21
[0208] Similarly using DNA from the colonies did PCR for gene Cytb,
ITS2, 12 S RNA and 16 S RNA by using the respective primers as
mentioned above in example 6-9. The amplified DNA was checked on
the gel with the 1 Kb DNA marker and checked for the size of the
insert.
EXAMPLE 22
[0209] The colonies showing the brightest band for the particular
gene insert were inoculated in LB broth under sterile conditions
with flame, added 5 .mu.l of Ampicillin and grown at 37 degree
Centigrade.
EXAMPLE 23
[0210] Plasmid DNA purification was followed from Wizard.TM.
minipreps DNA Purification system Technical bulletin, revised
1/94.part #TB 117 and cloned gene probes were thus developed for
D-Loop, ITS2, Cyt b, 12 S and 16 s of Stenobrachius leucosparus
myctophid fish.
EXAMPLE 24
Checking Purity and Specificity of the Probe
[0211] The plasmid with insert was first cut with ECOR I
restriction enzyme and following were added in a PCR tube (1 .mu.l
DNA (plasmid), 2 .mu.l ECOR Buffer, 1 .mu.l ECOR Enzyme, 16 .mu.l
MQ water to make the volume 20 .mu.l).
[0212] Run at 0.7% agarose gel on 72 volts with 1 kb DNA ladder.
And keeping negative control of plasmids from the blue
colonies.
[0213] The band shown will be of the size of plasmid+gene. In this
case the 12 S gene is 411 bp and plasmid is 3000 bp. So the
position of the band was between the 3054 and 4072 bp bands. The
DNA probe of 12 S with us is the pure cloned gene from myctophid
Stenobrachius leucopsarus.
EXAMPLE 25
[0214] Similarly we have developed pure DNA probes of the gene
segment from 16 S RNA, D-Loop, Cytb, and ITS2 genes.
EXAMPLE 26
[0215] Sequencing of Probe:
[0216] Once the Species specific probe was ready it was sequenced
to see the sequences to ascertain that these were the same
sequences that we started with. The steps of example 6-14 were
followed in the same manner. Only the DNA template is of the probes
from example 23.
EXAMPLE 27
[0217] The blast operation was performed by sending pasta format of
all the gene sequences to the Blast@NCBI and asking for related
sequences. The results confirmed that the genes probed belonged to
the Cytb, D-Loop, Rod and ITS2, 12 S rRNA and 16 S rRNA genes.
EXAMPLE 28
[0218] Article II. The species specific primers of cyt b, D-Loop,
ROD, ITS2, 12S rRNA and 16S rRNA gene regions of the myctophid
Stenobrachius leucopsarus (SLMB) and 12S rRNA and 16S rRNA gene
regions of four more myctophids like Lampanyctus regalis, Diaphus
theta; Tarletonbenia crenularis & Protomyctophum crockeri are
designed using the "Generunner".
EXAMPLE 29
[0219] The sequences of the species specific primers of cyt b,
D-Loop, ROD, ITS2, 12S rRNA and 16S rRNA gene regions of the
myctophid Stenobrachius leucopsarus (SLMB) and 12S rRNA and 16S
rRNA gene regions of four more myctophids like Lampanyctus regalis,
Diaphus theta; Tarletonbenia crenularis & Protomyctophum
crockeri are designed using the "Generunner" program for the first
time.
EXAMPLE 30
[0220] Both the Forward (also written as "L" and "Sense") and
Backward primers (also written as "H" and "Antisense") sequences
for all the above said gene regions for all the species are
designed.
EXAMPLE 31
[0221] All the primers designed had no Hairpin Loops, Dimers, Bulge
Loops and Internal Loops. There are no palindromes.
23 Analysis of "table 1 (slmb primer cyt L)" a 20-mer DNA
Oligonucleotide (Sense) 5' CAA CCT CAT CTG TCG TAA AC 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6101.0 Delta G Temperature 25.0 degrees C. Tm thermodynamic 56.4
degrees C. Probe concentration 0.6 pMol Filter Tm 48.8 degrees C.
Salt concentration 1000.0 mMol % GC Tm 66.2 degrees C. Formamide
concentration 0.0% AT + GC Tm 58.0 degrees C. 3' End length 7 bases
Absorbance 5.3 nMol/A260 Run length 4 bases Absorbance 32.5 ug/A260
Palindrome length 8 bases Percent GC 45.0% Hairpin loop stem length
3 bases Delta G 28.7 kCal/Mol Delta H -140.6 kCal/Mol Delta S 368.0
eu 3' End Delta G 5.9 kCal/Mol Structural Analysis Summary Number
of base runs / palindromes 0 / 0 Number of hairpin loops 0 Number
of dimers / 2-oligo dimers 0 / 0 Number of bulge loops / 2-oligo
bulges 0 / 0 Number of internal loops / 2-oligo internals 0 / 0
[0222]
24 Analysis of "table 2 (slmb primer cyt H)" a 20-mer DNA
oligonuclectide (Antisense) 5' GCT CGG GCT GCT GGA ATC TT 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6220.1 Delta G Temperature 25.0 degrees C. Tm thermodynamic 70.8
degrees C. Probe concentration 0.6 pMol Filter Tm 63.2 degrees C.
Salt concentration 1000.0 mMol % GC Tm 72.3 degrees C. Formamide
concentration 0.0% AT + GC Tm 64.0 degrees C. 3' End length 7 bases
Absorbance 5.6 nMol/A260 Run length 4 bases Absorbance 34.8 ug/A260
Palindrome length 8 bases Percent GC 60.0% Hairpin loop stem length
3 bases Delta G -37.5 kCal/Mol Delta H -164.6 kCal/Mol Delta S
-419.9 eu 3' End Delta G -5.1 kCal/Mol Structural Analysis Summary
Number of base runs / palindromes 0 / 0 Number of hairpin loops 0
Number of dimers / 2-oligo dimers 0 / 0 Number of bulge loops /
2-oligo bulges 0 / 0 Number of internal loops / 2-oligo internals 0
/ 0
[0223]
25 Analysis of "table 3 (slmb primer ITS2 F)" a 20-mer DNA
Oligonucleotide (Sense) 5' ACT TGA CTG ACC TTC TTA CT 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6098.0 Delta G Temperature 25.0 degrees C. Tm thermodynamic 51.3
degrees C. Probe concentration 0.6 pMol Filter Tm 43.7 degrees C.
Salt concentration 1000.0 mMol % GC Tm 64.2 degrees C. Formamide
concentration 0.0% AT + GC Tm 56.0 degrees C. 3' End length 7 bases
Absorbance 5.6 nMol/A260 Run length 4 bases Absorbance 34.0 ug/A260
Palindrome length 8 bases Percent GC 40.0% Hairpin loop stem length
3 bases Delta G -26.5 kCal/Mol Delta H -137.7 kCal/Mol Delta S
-365.8 eu 3' End Delta G -3.9 kCal/Mol Structural Analysis Summary
Number of base runs / palindromes 0 / 0 Number of hairpin loops 0
Number of dimers / 2-oligo dimers 0 / 0 Number of bulge loops /
2-oligo bulges 0 / 0 Number of internal loops / 2-oligo internals 0
/ 0
[0224]
26 Analysis of "table 4 ( slmb primer ITS2-H)" a 24-mer DNA
Oligonucleotide (Antisense) 5' ATA CTC TGC GGA CAT ACT TGA CTG 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
7407.0 Delta G Temperature 25.0 degrees C. Tm thermodynamic 65.4
degrees C. Probe concentration 0.6 pMol Filter Tm 57.8 degrees C.
Salt concentration 1000.0 mMol % GC Tm 72.2 degrees C. Formamide
concentration 0.0% AT + GC Tm 70.0 degrees C. 3' End length 7 bases
Absorbance 4.4 nMol/A260 Run length 4 bases Absorbance 32.4 ug/A260
Palindrome length 8 bases Percent GC 45.8 % Hairpin loop stem
length 3 bases Delta G -35.5 kCal/Mol Delta H -169.5 kCal/Mol Delta
S -442.0 eu 3' End Delta G -5.2 kCal/Mol Structural Analysis
Summary Number of base runs / palindromes 0 / 0 Number of hairpin
loops 0 Number of dimers / 2-oligo dimers 0 / 0 Number of bulge
loops / 2-oligo bulges 0 / 0 Number of internal loops / 2-oligo
internals 0 / 0
[0225]
27 Analysis of "table 5 (slmb primer pro-L)" a 24-mer DNA
Oligonucleotide (Sense) 5' CAG TCT CGT CAA ACC AAG TCA AAC 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
7354.9 Delta G Temperature 25.0 degrees C. Tm thermodynamic 67.8
degrees C. Probe concentration 0.6 pMol Filter Tm 60.2 degrees C.
Salt concentration 1000.0 mMol % GC Tm 72.2 degrees C. Formamide
concentration 0.0% AT + GC Tm 70.0 degrees C. 3' End length 7 bases
Absorbance 4.3 nMol/A260 Run length 4 bases Absorbance 31.4 ug/A260
Palindrome length 8 bases Percent GC 45.8% Hairpin loop stem length
3 bases Delta G -36.5 kCal/Mol Delta H -169.9 kCal/Mol Delta S
-439.7 eu 3' End Delta G -4.9 kCal/Mol Structural Analysis Summary
Number of base runs / palindromes 0 / 0 Number of hairpin loops 0
Number of dimers / 2-oligo dimers 0 / 0 Number of bulge loops /
2-oligo bulges 0 / 0 Number of internal loops / 2-oligo internals 0
/ 0
[0226]
28 Analysis of "table 6 (slmb primer Dloop-H)" a 23-mer DNA
Oligonuclectide (Antisense) 5' ATA ATC ATC CAG CAT AAA CAC AC 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
7033.7 Delta G Temperature 25.0 degrees C. Tm thermodynamic 61.2
degrees C. Probe concentration 0.6 pMol Filter Tm 53.6 degrees C.
Salt concentration 1000.0 mMol % GC Tm 66.4 degrees C. Formamide
concentration 0.0% AT + GC Tm 62.0 degrees C. 3' End length 7 bases
Absorbance 4.3 nMol/A260 Run length 4 bases Absorbance 30.0 ug/A260
Palindrome length 8 bases Percent GC 34.8% Hairpin loop stem length
3 bases Delta G -32.9 kCal/Mol Delta H -163.3 kCal/Mol Delta S
-429.7 eu 3' End Delta G -4.6 kCal/Mol Structural Analysis Summary
Number of base runs / palindromes 0 / 0 Number of hairpin loops 0
Number of dimers / 2-oligo dimers 0 / 0 Number of bulge loops /
2-oligo bulges 0 / 0 Number of internal loops / 2-oligo internals 0
/ 0
[0227]
29 Analysis of "table 7 ( slmb primer ROD-L)" a 20-mer DNA
Oligonucleotide (Sense) 5' CCT GGT AGA GTT CGC CGT CA 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6189.0 Delta G Temperature 25.0 degrees C. Tm thermodynamic 67.4
degrees C. Probe concentration 0.6 pMol Filter Tm 59.8 degrees C.
Salt concentration 1000.0 mMol % GC Tm 72.3 degrees C. Formamide
concentration 0.0% AT + GC Tm 64.0 degrees C. 3' End length 7 bases
Absorbance 5.3 nMol/A260 Run length 4 bases Absorbance 33.0 ug/A260
Palindrome length 8 bases Percent GC 60.0% Hairpin loop stem length
3 bases Delta G -34.7 kCal/Mol Delta H -154.3 kCal/Mol Delta S
-394.4 eu 3' End Delta G -9.6 kCal/Mol Structural Analysis Summary
Number of base runs / palindromes 0 / 0 Number of hairpin loops 0
Number of dimers / 2-oligo dimers 0 / 0 Number of bulge loops /
2-oligo bulges 0 / 0 Number of internal loops / 2-oligo internals 0
/ 0
[0228]
30 Analysis of "table 8 (slmb primer ROD-H)" a 22-mer DNA
Oligonucleotlde (Antisense) 5' CGT GTT CCT TAT CAT TGT GCC T 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6738.4 Delta G Temperature 25.0 degrees C. Tm thermodynamic 66.4
degrees C. Probe concentration 0.6 pMol Filter Tm 58.8 degrees C.
Salt concentration 1000.0 mMol % GC Tm 69.5 degrees C. Formamide
concentration 0.0% AT + GC Tm 64.0 degrees C. 3' End length 7 bases
Absorbance 5.2 nMol/A260 Run length 4 bases Absorbance 34.9 ug/A260
Palindrome length 8 bases Percent GC 45.5% Hairpin loop stem length
3 bases Delta G -35.4 kCal/Mol Delta H -165.0 kCal/Mol Delta S
-427.3 eu 3' End Delta G -7.9 kCal/Mol Structural Analysis Summary
Number of base runs / palindromes 0 / 0 Number of hairpin loops 0
Number of dimers / 2-oligo dimers 0 / 0 Number of bulge loops /
2-oligo bulges 0 / 0 Number of internal loops / 2-oligo internals 0
/ 0
[0229]
31 Analysis of "table 9 ( LRMB primer 16S-L)" a 21-mer DNA
Oligonucleotide (Sense) 5' CAC CAG CCA AGT ATG TTT CTC 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6421.2 Delta G Temperature 25.0 degrees C. Tm thermodynamic 61.5
degrees C. Probe concentration 0.6 pMol Filter Tm 53.9 degrees C.
Salt concentration 1000.0 mMol % GC Tm 68.9 degrees C. Formamide
concentration 0.0% AT + GC Tm 62.0 degrees C. 3' End length 7 bases
Absorbance 5.1 nMol/A260 Run length 4 bases Absorbance 33.0 ug/A260
Palindrome length 8 bases Percent GC 47.6% Hairpin loop stem length
3 bases Delta G -31.9 kCal/Mol Delta H -152.3 kCal/Mol Delta S
-396.4 eu 3' End Delta G -4.9 kCal/Mol Structural Analysis Summary
Number of base runs / palindromes 0 / 0 Number of hairpin loops 0
Number of dimers / 2-oligo dimers 0 / 0 Number of bulge loops /
2-oligo bulges 0 / 0 Number of internal loops / 2-oligo internals 0
/ 0
[0230]
32 Analysis of "table 10 (LRMB primer 16S-H)" a 18-mer DNA
Oligonucleotide (Antisense) 5' TCG TAG TTC AGC AGT CAG 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
5594.7 Delta G Temperature 25.0 degrees C. Tm thermodynamic 51.2
degrees C. Probe concentration 0.6 pMol Filter Tm 43.6 degrees C.
Salt concentration 1000.0 mMol % GC Tm 64.5 degrees C. Formamide
concentration 0.0% AT + GC Tm 54.0 degrees C. 3' End length 7 bases
Absorbance 5.7 nMol/A260 Run length 4 bases Absorbance 31.8 ug/A260
Palindrome length 8 bases Percent GC 50.0% Hairpin loop stem length
3 bases Delta G -25.3 kCal/Mol Delta H -123.0 kCal/Mol Delta S
-320.5 eu 3' End Delta G -4.9 kCal/Mol Structural Analysis Summary
Number of base runs / palindromes 0 / 0 Number of hairpin loops 0
Number of dimers / 2-oligo dimers 0 / 0 Number of bulge loops /
2-oligo bulges 0 / 0 Number of internal loops / 2-oligo internals 0
/ 0
[0231]
33 Analysis of "table 11 (LRMB primer 12S-L)" a 19-mer DNA
Oligonucleotide(Sense) 5' CTA TTC GCC TCG CTC AGA C 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
5779.8 Delta G Temperature 25.0 degrees C. Tm thermodynamic 62.1
degrees C. Probe concentration 0.6 pMol Filter Tm 54.5 degrees C.
Salt concentration 1000.0 mMol % GC Tm 69.7 degrees C. Formamide
concentration 0.0% AT + GC Tm 60.0 degrees C. 3' End length 7 bases
Absorbance 6.0 nMol/A260 Run length 4 bases Absorbance 34.6 ug/A260
Palindrome length 8 bases Percent GC 57.9% Hairpin loop stem length
3 bases Delta G -31.8 kCal/Mol Delta H -146.6 kCal/Mol Delta S
-378.6 eu 3' End Delta G -4.6 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0232]
34 Analysis of "table 12 (LRMB primer 12S-H)" a 23-mer DNA
Oligonucleotide(Antisense) 5' GCC TCC ATC ATC CCT CAC CTT AC 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6895.5 Delta G Temperature 25.0 degrees C. Tm thermodynamic 70.8
degrees C. Probe concentration 0.6 pMol Filter Tm 63.2 degrees C.
Salt concentration 1000.0 mMol % GC Tm 75.3 degrees C. Formamide
concentration 0.0% AT + GC Tm 72.0 degrees C. 3' End length 7 bases
Absorbance 5.1 nMol/A260 Run length 4 bases Absorbance 34.9 ug/A260
Palindrome length 8 bases Percent GC 56.5% Hairpin loop stem length
3 bases Delta G -38.9 kCal/Mol Delta H -174.6 kCal/Mol Delta S
-448.9 eu 3' End Delta G -5.1 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0233]
35 Analysis of "table 13 (DTMB primer 16S-H)" a 20-mer DNA
Oligonucleotide(Antisense) 5' CTC CGT CCG TCT CGC CTC TG 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6052.0 Delta G Temperature 25.0 degrees C. Tm thermodynamic 71.7
degrees C. Probe concentration 0.6 pMol Filter Tm 64.1 degrees C.
Salt concentration 1000.0 mMol % GC Tm 76.4 degrees C. Formamide
concentration 0.0% AT + GC Tm 68.0 degrees C. 3' End length 7 bases
Absorbance 6.1 nMol/A260 Run length 4 bases Absorbance 37.2 ug/A260
Palindrome length 8 bases Percent GC 70.0% Hairpin loop stem length
3 bases Delta G -37.1 kCal/Mol Delta H -157.8 kCal/Mol Delta S
-398.9 eu 3' End Delta G -7.9 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0234]
36 Analysis of "table 14 (DTMB primer 16S-L)" a 22-mer DNA
Oligonucleotide(Sense) 5' AAA TCC GCC CTT ATG TGT GTT C 3'
1Oligonucleotide Analysis Analysis Parameters Molecular weight
6756.4 Delta G Temperature 25.0 degrees C. Tm thermodynamic 67.9
degrees C. Probe concentration 0.6 pMol Filter Tm 60.3 degrees C.
Salt concentration 1000.0 mMol % GC Tm 69.5 degrees C. Formamide
concentration 0.0% AT + GC Tm 64.0 degrees C. 3' End length 7 bases
Absorbance 4.9 nMol/A260 Run length 4 bases Absorbance 33.3
.mu.g/A260 Palindrome length 8 bases Percent GC 45.5% Hairpin loop
stem length 3 bases Delta G -36.9 kCal/Mol Delta H -171.5 kCal/Mol
Delta S -444.2 eu 3' End Delta G -4.9 kCal/Mol Structural Analysis
Summary Number of base runs/palindromes 0/0 Number of hairpin loops
0 Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0235]
37 Analysis of "table 15 (DTMB primer 12S-H)" a 22-mer DNA
Oligonucleotide(Antisense) 5' CAT CGG CTT GCT CTA TTC CTT G 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6723.4 Delta G Temperature 25.0 degrees C. Tm thermodynamic 68.8
degrees C. Probe concentration 0.6 pMol Filter Tm 61.2 degrees C.
Salt concentration 1000.0 mMol % GC Tm 71.3 degrees C. Formamide
concentration 0.0% AT + GC Tm 66.0 degrees C. 3' End length 7 bases
Absorbance 5.3 nMol/A260 Run length 4 bases Absorbance 35.5 ug/A260
Palindrome length 8 bases Percent GC 50.0% Hairpin loop stem length
3 bases Delta G -37.5 kCal/Mol Delta H -172.0 kCal/Mol Delta S
-444.3 eu 3' End Delta G -7.0 kCal/Mol Structural Analysis Summary
Number of base runs/palindrome 0/0 Number of hairpin loops 0 Number
of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo bulges
0/0 Number of internal loops/2-oligo internals 0/0
[0236]
38 Analysis of "table 16 (DTMB primer 12S-L)" a 19-mer DNA
Oligonucleotide(Sense) 5' TCT ATC GGC GGC GTA TCA C 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
5859.8 Delta G Temperature 25.0 degrees C. Tm thermodynamic 65.8
degrees C. Probe concentration 0.6 pMol Filter Tm 58.2 degrees C.
Salt concentration 1000.0 mMol % GC Tm 69.7 degrees C. Formamide
concentration 0.0% AT + GC Tm 60.0 degrees C. 3' End length 7 bases
Absorbance 5.7 nMol/A260 Run length 4 bases Absorbance 33.4 ug/A260
Palindrome length 8 bases Percent GC 57.9% Hairpin loop stem length
3 bases Delta G -33.9 kCal/Mol Delta H -152.5 kCal/Mol Delta S
-391.2 eu 3' End Delta G -3.5 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0237]
39 Analysis of "table 17 (TCMB primer 16S-H)" a 21-mer DNA
Oligonucleotide(Antisense) 5' GGC GAT TCT ACG GCA CGG GCG 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6568.3 Delta G Temperature 25.0 degrees C. Tm thermodynamic 80.4
degrees C. Probe concentration 0.6 pMol Filter Tm 72.8 degrees C.
Salt concentration 1000.0 mMol % GC Tm 78.6 degrees C. Formamide
concentration 0.0% AT + GC Tm 72.0 degrees C. 3' End length 7 bases
Absorbance 5.1 nMol/A260 Run length 4 bases Absorbance 33.3 ug/A260
Palindrome length 8 bases Percent GC 71.4% Hairpin loop stem length
3 bases Delta G -44.7 kCal/Mol Delta H -186.4 kCal/Mol Delta S
-468.6 eu 3' End Delta G -12.8 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0238]
40 Analysis of "table 18 (TCMB primer 16S-L)" a 22-mer DNA
Oligonucleotide(Sense) 5' AAA CTG GTC CTC AAC TAT GTC A 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6758.5 Delta G Temperature 25.0 degrees C. Tm thermodynamic 60.7
degrees C. Probe concentration 0.6 pMol Filter Tm 53.1 degrees C.
Salt concentration 1000.0 mMol % GC Tm 67.6 degrees C. Formamide
concentration 0.0% AT + GC Tm 62.0 degrees C. 3' End length 7 bases
Absorbance 4.7 nMol/A260 Run length 4 bases Absorbance 31.7 ug/A260
Palindrome length 8 bases Percent GC 40.9% Hairpin loop stem length
3 bases Delta G -31.7 kCal/Mol Delta H -153.3 kCal/Mol Delta S
-400.5 eu 3' End Delta G -4.1 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0239]
41 Analysis of "table 19 (TCMB primer 12S-H)" a 22-mer DNA
Oligonucleotide(Antisense) 5' CCG ATT CAG CCA CGA TTC CCT C 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6671.4 Delta G Temperature 25.0 degrees C. Tm thermodynamic 74.6
degrees C. Probe concentration 0.6 pMol Filter Tm 67.0 degrees C.
Salt concentration 1000.0 mMol % GC Tm 75.0 degrees C. Formamide
concentration 0.0% AT + GC Tm 70.0 degrees C. 3' End length 7 bases
Absorbance 5.1 nMol/A260 Run length 4 bases Absorbance 34.2 ug/A260
Palindrome length 8 bases Percent GC 59.1% Hairpin loop stem length
3 bases Delta G -40.8 kCal/Mol Delta H -176.0 kCal/Mol Delta S
-447.5 eu 3' End Delta G -7.9 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0240]
42 Analysis of "table 20 (TCMB primer 12S-L)" a 21-mer DNA
Oligonucleotide(Sense) 5' CCT AAA GCC CAG ATA ACT ACA 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6432.3 Delta G Temperature 25.0 degrees C. Tm thermodynamic 59.2
degrees C. Probe concentration 0.6 pMol Filter Tm 51.6 degrees C.
Salt concentration 1000.0 mMol % GC Tm 66.9 degrees C. Formamide
concentration 0.0% AT + GC Tm 60.0 degrees C. 3' End length 7 bases
Absorbance 4.8 nMol/A260 Run length 4 bases Absorbance 30.6 ug/A260
Palindrome length 8 bases Percent GC 42.9% Hairpin loop stem length
3 bases Delta G -31.7 kCal/Mol Delta H -159.4 kCal/Mol Delta S
-421.0 eu 3' End Delta G -3.9 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0241]
43 Analysis of "table 21 (PCMB primer 16S-H)" a 22-mer DNA
Oligonucleotide(Antisense) 5' CGT GTT CTG ATG ATG ATG TGC T 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6867.5 Delta G Temperature 25.0 degrees Tm thermodynamic 64.7
degrees C. Probe concentration 0.6 pMol Filter Tm 57.1 degrees C.
Salt concentration 1000.0 mMol % GC Tm 69.5 degrees C. Formamide
concentration 0.0% AT + GC Tm 64.0 degrees C. 3' End length 7 bases
Absorbance 4.9 nMol/A260 Run length 4 bases Absorbance 33.4 ug/A260
Palindrome length 8 bases Percent GC 45.5% Hairpin loop stem length
3 bases Delta G -33.0 kCal/Mol Delta H -150.2 kCal/Mol Delta S
-385.9 eu 3' End Delta G -6.3 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0242]
44 Analysis of "table 22 (PCMB primer 16S-L)" a 19-mer DNA
Oligonucleotide(Sense) 5' ATT CCT TCC TCT TAG TAT G 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
5799.8 Delta G Temperature 25.0 degrees C. Tm thermodynamic 49.5
degrees C. Probe concentration 0.6 pMol Filter Tm 41.9 degrees C.
Salt concentration 1000.0 mMol % GC Tm 61.1 degrees C. Formamide
concentration 0.0% AT + GC Tm 52.0 degrees C. 3' End length 7 bases
Absorbance 5.8 nMol/A260 Run length 4 bases Absorbance 33.6 ug/A260
Palindrome length 8 bases Percent GC 36.8% Hairpin loop stem length
3 bases Delta G -26.1 kCal/Mol Delta H -138.8 kCal/Mol Delta S
-371.5 eu 3' End Delta G -3.1 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0243]
45 Analysis of "table 23 (PCMB primer 12S-H)" a 22-mer DNA
Oligonucleotide(Antisense) 5' GCT GAA CTT ACT ATG CCC TAC T 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6725.4 Delta G Temperature 25.0 degrees C. Tm thermodynamic 60.3
degrees C. Probe concentration 0.6 pMol Filter Tm 52.7 degrees C.
Salt concentration 1000.0 mMol % GC Tm 69.5 degrees C. Formamide
concentration 0.0% AT + GC Tm 64.0 degrees C. 3' End length 7 bases
Absorbance 5.0 nMol/A260 Run length 4 bases Absorbance 33.6 ug/A260
Palindrome length 8 bases Percent GC 45.5% Hairpin loop stem length
3 bases Delta G -32.7 kCal/Mol Delta H -164.7 kCal/Mol Delta S
-435.2 eu 3' End Delta G -6.6 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0244]
46 Analysis of "table 24 (PCMB primer 12S-L)" a 20-mer DNA
Oligonucleotide(Sense) 5' CCG ATT GAC GCC GAA CTA TG 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6182.1 Delta G Temperature 25.0 degrees C. Tm thermodynamic 68.1
degrees C. Probe concentration 0.6 pMol Filter Tm 60.5 degrees C.
Salt concentration 1000.0 mMol % GC Tm 70.3 degrees C. Formamide
concentration 0.0% AT + GC Tm 62.0 degrees C. 3' End length 7 bases
Absorbance 5.3 nMol/A260 Run length 4 bases Absorbance 32.5 ug/A260
Palindrome length 8 bases Percent GC 55.0% Hairpin loop stem length
3 bases Delta G -35.6 kCal/Mol Delta H -159.4 kCal/Mol Delta S
-408.5 eu 3' End Delta G -4.1 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0245]
47 Analysis of "table 25 (SLMB primer 16S-H)" a 18-mer DNA
Oligonucleotide(Antisense) 5' TAC GCA TAA CGG CTC TGG 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
5579.7 Delta G Temperature 25.0 degrees C. Tm thermodynamic 61.4
degrees C. Probe concentration 0.6 pMol Filter Tm 53.8 degrees C.
Salt concentration 1000.0 mMol % GC Tm 66.8 degrees C. Formamide
concentration 0.0% AT + GC Tm 56.0 degrees C. 3' End length 7 bases
Absorbance 5.9 nMol/A260 Run length 4 bases Absorbance 32.8 ug/A260
Palindrome length 8 bases Percent GC 55.6% Hairpin loop stem length
3 bases Delta G -31.0 kCal/Mol Delta H -143.5 kCal/Mol Delta S
-370.2 eu 3' End Delta G -7.9 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0246]
48 Analysis of "table 26 (SLMB primer 16S-L)" a 22-mer DNA
Oligonucleotide(Sense) 5' CTA CTA CAC CTC AAC TAC ATC T 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6638.4 Delta G Temperature 25.0 degrees C. Tm thermodynamic 52.4
degrees C. Probe concentration 0.6 pMol Filter Tm 44.8 degrees C.
Salt concentration 1000.0 mMol % GC Tm 67.6 degrees C. Formamide
concentration 0.0% AT + GC Tm 62.0 degrees C. 3' End length 7 bases
Absorbance 4.9 nMol/A260 Run length 4 bases Absorbance 32.8 ug/A260
Palindrome length 8 bases Percent GC 40.9% Hairpin loop stem length
3 bases Delta G -27.6 kCal/Mol Delta H -146.8 kCal/Mol Delta S
-392.2 eu 3' End Delta G -3.8 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0247]
49 Analysis of "table 27 (SLMB primer 12S-H)" a 19-mer DNA
Oligonucleotide(Antisense) 5' CCC ACT CAC TGC TAA CTC C 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
5708.8 Delta G Temperature 25.0 degrees C. Tm thermodynamic 58.4
degrees C. Probe concentration 0.6 pMol Filter Tm 50.8 degrees C.
Salt concentration 1000.0 mMol % GC Tm 69.7 degrees C. Formamide
concentration 0.0% AT + GC Tm 60.0 degrees C. 3' End length 7 bases
Absorbance 6.1 nMol/A260 Run length 4 bases Absorbance 35.0 ug/A260
Palindrome length 8 bases Percent GC 57.9% Hairpin loop stem length
3 bases Delta G -29.4 kCal/Mol Delta H -138.5 kCal/Mol Delta S
-359.0 eu 3' End Delta G -5.4 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0248]
50 Analysis of "table 28 (SLMB primer 12S-L)" a 21-mer DNA
Oligonucleotide(Sense) 5' GGC TAA CTA CAA TCA TCT GCT 3'
Oligonucleotide Analysis Analysis Parameters Molecular weight
6445.2 Delta G Temperature 25.0 degrees C. Tm thermodynamic 58.5
degrees C. Probe concentration 0.6 pMol Filter Tm 50.9 degrees C.
Salt concentration 1000.0 mMol % GC Tm 66.9 degrees C. Formamide
concentration 0.0% AT + GC Tm 60.0 degrees C. 3' End length 7 bases
Absorbance 5.1 nMol/A260 Run length 4 bases Absorbance 32.6 ug/A260
Palindrome length 8 bases Percent GC 42.9% Hairpin loop stem length
3 bases Delta G -30.8 kCal/Mol Delta H -153.4 kCal/Mol Delta S
-403.9 eu 3' End Delta G -6.3 kCal/Mol Structural Analysis Summary
Number of base runs/palindromes 0/0 Number of hairpin loops 0
Number of dimers/2-oligo dimers 0/0 Number of bulge loops/2-oligo
bulges 0/0 Number of internal loops/2-oligo internals 0/0
[0249]
Sequence CWU 1
1
45 1 21 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 tgayttgaar aaccaycgtt g 21 2 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 2 ctccartctt cgryttacaa g
21 3 26 DNA Artificial Sequence Description of Artificial Sequence
Primer 3 ccatccaaca tctcagcatg atgaaa 26 4 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 4 cctgaagtag
gaaccagatg 20 5 19 DNA Artificial Sequence Description of
Artificial Sequence Primer 5 ctacgcctgt ctgagtgtc 19 6 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 6
atatgcttaa attcagcggg 20 7 21 DNA Artificial Sequence Description
of Artificial Sequence Primer 7 tctttccgca gcacaacgtg g 21 8 24 DNA
Artificial Sequence Description of Artificial Sequence Primer 8
catatgaata ccctcagtac tacc 24 9 25 DNA Artificial Sequence
Description of Artificial Sequence Primer 9 aaactgggat tagatacccc
actat 25 10 19 DNA Artificial Sequence Description of Artificial
Sequence Primer 10 agagtgacgg gcggtgtgt 19 11 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 11 cgcctgttta
tcaaaaacat 20 12 22 DNA Artificial Sequence Description of
Artificial Sequence Primer 12 ccggtctgaa ctcagatcac gt 22 13 19 DNA
Artificial Sequence Description of Artificial Sequence Primer 13
cccactcact gctaactcc 19 14 21 DNA Artificial Sequence Description
of Artificial Sequence Primer 14 ggctaactac aatcatctgc t 21 15 18
DNA Artificial Sequence Description of Artificial Sequence Primer
15 tacgcataac ggctctgg 18 16 22 DNA Artificial Sequence Description
of Artificial Sequence Primer 16 ctactacacc tcaactacat ct 22 17 20
DNA Artificial Sequence Description of Artificial Sequence Primer
17 gctcgggctg ctggaatctt 20 18 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 18 caacctcatc tgtcgtaaac
20 19 24 DNA Artificial Sequence Description of Artificial Sequence
Primer 19 atactctgcg gacatacttg actg 24 20 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 20 acttgactga
ccttcttact 20 21 23 DNA Artificial Sequence Description of
Artificial Sequence Primer 21 cagtctcgtc aaaccaagtc aaa 23 22 23
DNA Artificial Sequence Description of Artificial Sequence Primer
22 ataatcatcc agcataaaca cac 23 23 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 23 cctggtagag ttcgccgtca
20 24 22 DNA Artificial Sequence Description of Artificial Sequence
Primer 24 cgtgttcctt atcattgtgc ct 22 25 18 DNA Artificial Sequence
Description of Artificial Sequence Primer 25 tcgtagttca gcagtcag 18
26 21 DNA Artificial Sequence Description of Artificial Sequence
Primer 26 caccagccaa gtatgtttct c 21 27 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 27 gcctccatca tccctcacct
tac 23 28 19 DNA Artificial Sequence Description of Artificial
Sequence Primer 28 ctattcgcct cgctcagac 19 29 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 29 ctccgtccgt
ctcgcctctg 20 30 22 DNA Artificial Sequence Description of
Artificial Sequence Primer 30 aaatccgccc ttatgtgtgt tc 22 31 22 DNA
Artificial Sequence Description of Artificial Sequence Primer 31
catcggcttg ctctattcct tg 22 32 19 DNA Artificial Sequence
Description of Artificial Sequence Primer 32 tctatcggcg gcgtatcac
19 33 21 DNA Artificial Sequence Description of Artificial Sequence
Primer 33 ggcgattcta cggcacgggc g 21 34 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 34 aaactggtcc tcaactatgt
ca 22 35 22 DNA Artificial Sequence Description of Artificial
Sequence Primer 35 ccgattcagc cacgattccc tc 22 36 21 DNA Artificial
Sequence Description of Artificial Sequence Primer 36 cctaaagccc
agataactac a 21 37 22 DNA Artificial Sequence Description of
Artificial Sequence Primer 37 cgtgttctga tgatgatgtg ct 22 38 19 DNA
Artificial Sequence Description of Artificial Sequence Primer 38
attccttcct cttagtatg 19 39 22 DNA Artificial Sequence Description
of Artificial Sequence Primer 39 gctgaactta ctatgcccta ct 22 40 20
DNA Artificial Sequence Description of Artificial Sequence Primer
40 ccgattgacg ccgaactatg 20 41 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 41 ttgtacacac cgcccgtcgc
20 42 747 DNA Unknown Organism Description of Unknown Organism
Nucleotide sequence of PSL CYTL 42 cttncccatt ttgggcgctt nggcncgctn
ctccncgaga ctctgcgtan taatccaant 60 cnctncgggc cnctccctac
cantncncta caccncaaat tncaacccng tttcctcatc 120 antcaaccac
atctgtcgaa aacntcaact acggctgact aatccgaaaa catgcacgct 180
aacggtgcct ctttcttctt catctgtatt tatctncncn ttggangagg actatnctac
240 ggatcctacc tctacgaaga gacgtgaggt gttggtgtta ttcttctcct
tctaataatg 300 atgactgcnt ttgttggcta tgtgctnccc ngaggacaaa
tgtccttttg aggtgctact 360 gtcattacaa ncctactctc tgctgtnccg
tntgttngcg gcnctctant tcaatgaatt 420 tgaggtggct tctccgtaaa
cacgcaacgc tcactcgttt cttcgcnttc cacttcttgt 480 tcccatttgt
tgtcgcngct ataaccnngg ttcaccngat ttnccgacat caaacaggct 540
ctaaancccc cccggnttga ctccatacaa caaaaccctc caccctattc nctataaaac
600 tctaggttcg tgcccgtatt ggcttacttc atgnctattt cccngncgga
gggacnaaaa 660 ttcctgcacc ccctccccnc aaaataaana atgtgtctnt
cctaccanaa aacaacnnan 720 acggggtntg cncttccatc atccacn 747 43 225
DNA Unknown Organism Description of Unknown Organism Nucleotide
sequence of PSLITS2F 43 tctacgatct accggcnttt nntgtggaaa gacgatcatg
catttatgtg tgtctttcta 60 tggatttgaa ccgtgtggta cgtctttgcg
tactgcttgg aaggctcaac ttgcttctgt 120 ccttctcttg cagtctcgca
ctgtctatgc aacgtgttct acttcgactt ctgtcgaaaa 180 atcttacttt
tgacctcaga tcagacaaga ctacccgctg aattt 225 44 750 DNA Unknown
Organism Description of Unknown Organism Nucleotide sequence of PSL
PROL 44 ccttttcggn ataggcccan ctcaaatgaa ttccttctct cctggtccaa
gcccaaactg 60 tggacggcag gttgacaatg gttacaaatc gtgacaaatc
ggctacataa ttgccgatag 120 cgatgtcgtc aaaccaagtc aaacaatggc
cgatgtatat cggccaaacc catatatggg 180 tctggctgta gtttgtgttg
agcaacgtca caccagtgtc tggtcagcat ataagatgtt 240 gacatcttgc
aacatcttac ccacagacag acagttacgg ctgcttacga anggcgctag 300
tgttgtggtg agaaacgaag atacatacgt caaacagacg ccggtgcact tgaagacact
360 gtttgaaggt gccgcactac ttgacagaca gcccatgatg cgctggacag
tgaccaaagc 420 tacnggagga ccanatggaa atcctgttgg cgttgccgtg
ggactcaagt tgtacacttt 480 tggatggttg atcactanan ccgctgccgg
gagaagcact cgctcctggt tcactaatca 540 gattgaggtt aaccanattg
angtaaacat cttcaacaca gtgtctttat gctggatgaa 600 attnagccca
cnggacacca naaaagaatt nccnctggtt ctnncggggg nccccnnnaa 660
cgnntnttcc ccttntctcn nnngcggnga agttnccccc ccccactnan ntcttccttc
720 aananntttc cnccnnnaga ggttttcccn 750 45 748 DNA Unknown
Organism Description of Unknown Organism Nucleotide sequence of ROD
PSL SLMB 45 cctggtaggg ttccccgtca acttcctcac actgtacctc acnttcgagc
acaagaagct 60 actaaccccc ttaaactaca tcctgctcaa cctggcggtc
ggagacctcc tgatggtgta 120 aggagggttc accaccacca tctacacctc
catgcacggc tacttcgtcc tagggaaact 180 gggctgcgcc atcgaaggtt
tcatggccac ccatggtggt caggtcgccc tttggtccct 240 ggtggttttg
gccgtggaaa ggtggctggt cgtctgcaan cccatctcca gcttccgctt 300
ccaggagtcc cactccctca tgggcctggc cgtgacctgg gtgatggcga cggcttgttc
360 tgtgcccccc ctgggtcggc tggtctcgct acatcccaga aggcatgcag
tgctcatgcg 420 gaatggacta ctacactccc gcgccgggcg tcaacaatga
atcctacgtn gtgtacatgt 480 tcntcanaaa aanaatngga ccncngggcg
atcatnttgn tangnnaagg ccagntgntg 540 ngagcagtca aggcggccgc
cgccgcccag caagagtccg agaccaccca gagggccgag 600 agggaagtca
cccgnatggt natnangatg gtnatntcnt tcntggtaag nagggngcca 660
nacgccagcg tggcctggtg gatcttnngn aaccagggng cagaattagg cccngtnttc
720 atgaccctgc cggcnttctt tgccaaga 748
* * * * *