U.S. patent application number 09/984842 was filed with the patent office on 2003-04-24 for methods for analyzing ltc4 synthase polymorphisms and diagnostic use.
This patent application is currently assigned to ZENECA LIMITED. Invention is credited to Norris Morten, John Edward.
Application Number | 20030077592 09/984842 |
Document ID | / |
Family ID | 10817852 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030077592 |
Kind Code |
A1 |
Norris Morten, John Edward |
April 24, 2003 |
Methods for analyzing LTC4 synthase polymorphisms and diagnostic
use
Abstract
This invention relates to single nucleotide polymorphisms in the
LTC.sub.4 synthase gene, EMBL accession no. U50136, particularly at
one or more of positions 375, 815, 1003, 2169 and 2742. The
invention also relates to methods and materials for analysing
allelic variation in the LTC.sub.4 synthase gene, and to the use of
LTC.sub.4 synthase polymorphism in the diagnosis and treatment of
leukotriene mediated diseases such as asthma and allergic
rhinitis.
Inventors: |
Norris Morten, John Edward;
(Macclesfield, GB) |
Correspondence
Address: |
PILLSBURY WINTHROP, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Assignee: |
ZENECA LIMITED
|
Family ID: |
10817852 |
Appl. No.: |
09/984842 |
Filed: |
October 31, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09984842 |
Oct 31, 2001 |
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09485636 |
Feb 15, 2000 |
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6316196 |
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Current U.S.
Class: |
435/6.14 ;
536/23.2; 536/24.3 |
Current CPC
Class: |
Y10S 435/81 20130101;
C12Q 2600/156 20130101; C12Q 1/6883 20130101 |
Class at
Publication: |
435/6 ; 536/23.2;
536/24.3 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 22, 1997 |
GB |
9717766.1 |
Aug 18, 1998 |
GB |
PCT/GB98/02468 |
Claims
1. A method for the diagnosis of a single nucleotide polymorphism
in LTC.sub.4 synthase in a human, which method comprises
determining the sequence of the nucleic acid of the human at one or
more of positions 375, 815, 1003, 2169 and 2742 in the LTC.sub.4
synthase gene as defined by the positions in SEQ ID NO: 1, and
determining the status of the human by reference to polymorphism in
the LTC.sub.4 synthase gene.
2. A method according to claim 1 in which the single nucleotide
polymorphism at position 375 is presence of G and/or A.
3. A method according to claim 1 in which the single nucleotide
polymorphism at position 815 is presence of C and/or A.
4. A method according to claim 1 in which the single nucleotide
polymorphism at position 1003 is presence of A and/or C.
5. A method according to claim 1 in which the single nucleotide
polymorphism at position 2169 is presence of C and/or T.
6. A method according to claim 1 in which the single nucleotide
polymorphism at position 2742 is presence of C and/or T.
7. A method according to any one of claims 1-6 in which the
sequence is determined by a method selected from amplification
refractory mutation system and restriction fragment length
polymorphism.
8. A nucleic acid comprising the 5' untranslated region of
LTC.sub.4 synthase comprising a polymorphism corresponding with one
or more of positions 375, 815 and 1003 as defined by the positions
in SEQ ID NO: 1 and in which there is an A at position 375, an A at
position 815 and a C at position 1003.
9. A nucleic acid comprising the first intron of the LTC.sub.4
synthase gene comprising a polymorphism at one or more of positions
2169 and 2742 as defined by the position in SEQ ID NO: 1 and in
which there is a T at position 2169 and there is a T at position
2742.
10. A diagnostic nucleic acid primer capable of detecting a
LTC.sub.4 synthase gene polymorphism at one or more of positions
375, 815, 1003, 2169 and 2742 in the LTC.sub.4 synthase gene as
defined by the positions in SEQ ID NO: 1.
11. An allele-specific oligonucleotide probe capable of detecting a
LTC.sub.4 synthase gene polymorphism at one or more of positions
375, 815, 1003, 2169 and 2742 in the LTC.sub.4 synthase gene as
defined by the positions in SEQ ID NO: 1.
12. A diagnostic kit comprising a diagnostic primer as defined in
claim 10 and/or an allele-specific oligonucleotide primer as
defined in claim 11.
13. A method of treating a human in need of treatment with an
antileukotriene drug in which the method comprises: i) diagnosis of
a single nucleotide polymorphism in LTC.sub.4 synthase in the
human, which diagnosis comprises determining the sequence of the
nucleic acid at one or more of positions 375, 815, 1003, 2169 and
2742 in the LTC.sub.4 synthase gene as defined by the positions in
SEQ ID NO: 1, and determining the status of the human by reference
to polymorphism in the LTC.sub.4 synthase gene; and ii)
administering an effective amount of an antileukotriene drug.
14. Use of an antileukotriene drug in preparation of a medicament
for treating a leukotriene mediated disease in a human diagnosed as
having a single nucleotide polymorphism at one or more of positions
375, 815, 1003, 2169 and 2742 in LTC.sub.4 synthase gene as defined
by the positions in SEQ ID NO: 1.
Description
[0001] This invention relates to polymorphisms in the LTC.sub.4
synthase gene. The invention also relates to methods and materials
for analysing allelic variation in the LTC.sub.4 synthase gene, and
to the use of LTC.sub.4 synthase polymorphism in the diagnosis and
treatment of leukotriene mediated diseases such as asthma and
allergic rhinitis.
[0002] The cysteinyl-leukotrienes, LTC.sub.4, LTD.sub.4 and
LTE.sub.4, are potent bronchoconstrictors, increase vascular
permeability and increase mucus production in airways. They are
implicated in the pathophysiology of asthma and allergic rhinitis
and are found at elevated levels in bronchoalveolar lavage from
asthma patients, particularly after allergen challenge. LTD.sub.4
and LTE.sub.4 may also enhance the neurogenic inflammatory response
in airways. Compounds which inhibit leukotriene synthesis e.g. the
5-lipoxygenase inhibitor, zileuton, or the leukotriene receptor
antagonist, zafirlukast, have been shown to be effective against
asthma and rhinitis (Busse W. W, Clin. Exp. Allergy, 26, 868-879,
1996; see particularly FIG. 1 therein which shows the arachidonic
acid cascade, indicating the role of LTC.sub.4 synthase in
catalysing the formation of LTC.sub.4).
[0003] Leukotrienes are derived from membrane phospholipids.
Arachidonic acid is released from the phospholipid by cytosolic
phospholipase A2 and converted to LTA.sub.4 by 5-lipoxygenase in
the presence of 5-lipoxygenase activating protein, FLAP.
Polymorphisms in 5-LO have been reported in international patent
application WO 97/42347, Brigham & Women's Hospital. LTA.sub.4
is conjugated with reduced glutathione by LTC.sub.4 synthase to
form LTC.sub.4. The biologically active metabolites, LTD.sub.4 and
LTE.sub.4 are formed, following carrier mediated export of
LTC.sub.4, by the sequential action of gamma-glutamyl
transpeptidase and dipeptidases.
[0004] The LTC.sub.4 synthase gene has been cloned and published as
a 4,465 nucleotide genomic sequence comprising 1,446 nucleotides of
sequence 5' to the coding sequence, the 5 exons and intervening
introns and 3' sequence extending 398 nucleotide beyond the polyA
signal (Penrose et al., J. Biol. Chem., 271, 11356-11361, 1996;
EMBL accession no. U50136).
[0005] One approach is to use knowledge of polymorphisms to help
identify patients most suited to therapy with particular
pharmaceutical agents (this is often termed "pharmacogenetics").
Pharmacogenetics can also be used in pharmaceutical research to
assist the drug selection process. Polymorphisms are used in
mapping the human genome and to elucidate the genetic component of
diseases. The reader is directed to the following references for
background details on pharmacogenetics and other uses of
polymorphism detection: Linder et al. (1997), Clinical Chemistry,
43, 254; Marshall (1997), Nature Biotechnology, 15, 1249;
International Patent Application WO 97/40462, Spectra Biomedical;
and Schafer et al. (1998), Nature Biotechnology, 16, 33.
[0006] Clinical trials have shown that patient response to
treatment with leukotriene antagonists is heterogeneous. Thus there
is a need for improved approaches to pharmaceutical agent design
and therapy with leukotriene antagonists.
[0007] The present invention is based on the discovery of five
single nucleotide polymorphisms (SNPs) in the LTC.sub.4 synthase
gene. Three SNPs have been found in the 5' untranslated region of
the gene and two in the first intron, located at positions 375,
815, 1003, 2169 and 2742 respectively, based on the numbering of
U50136. Before our first filing date, we believe there has been no
disclosure of polymorphism/allelic variation in the LTC.sub.4
synthase gene.
[0008] According to one aspect of the present invention there is
provided a method for the diagnosis of a single nucleotide
polymorphism in LTC.sub.4 synthase in a human, which method
comprises determining the sequence of the nucleic acid of the human
at one or more of positions 375, 815, 1003, 2169 and 2742 in the
LTC.sub.4 synthase gene as defined by the positions in SEQ ID NO:
1, and determining the status of the human by reference to
polymorphism in the LTC.sub.4 synthase gene.
[0009] The term human includes both a human having or suspected of
having a leukotriene mediated disease and an asymptomatic human who
may be tested for predisposition or susceptibility to leukotriene
mediated disease. At each position the human may be homozygous for
an allele or the human may be a heterozygote.
[0010] In one embodiment of the invention preferably the method for
diagnosis described herein is one in which the single nucleotide
polymorphism at position 375 is presence of G and/or A.
[0011] In another embodiment of the invention preferably the method
for diagnosis described herein is one in which the single
nucleotide polymorphism at position 815 is presence of C and/or
A.
[0012] In another embodiment of the invention preferably the method
for diagnosis described herein is one in which the single
nucleotide polymorphism at position 1003 is presence of A and/or C.
Testing for the presence of the C allele at this position is
especially preferred because, without wishing to be bound by
theoretical considerations, of its association with increased
levels of LTC.sub.4 synthase (as explained herein).
[0013] In another embodiment of the invention preferably the method
for diagnosis described herein is one in which the single
nucleotide polymorphism at position 2169 is presence of C and/or
T.
[0014] In another embodiment of the invention preferably the method
for diagnosis described herein is one in which the single
nucleotide polymorphism at position 2742 is presence of C and/or
T.
[0015] The method for diagnosis is preferably one in which the
sequence is determined by a method selected from amplification
refractory mutation system and restriction fragment length
polymorphism.
[0016] In another aspect of the invention we provide a method for
the diagnosis of leukotriene mediated disease, which method
comprises:
[0017] i) obtaining sample nucleic acid from an individual,
[0018] ii) detecting the presence or absence of a variant
nucleotide at one or more of positions 375, 815, 1003 and 2169 in
the LTC.sub.4 synthase gene and
[0019] iii) determining the status of the individual by reference
to polymorphism in the LTC.sub.4 synthase gene.
[0020] The published sequence of the LTC.sub.4 synthase gene, EMBL
accession number U50136, is shown in SEQ ID NO: 1 in which the
variant sites discovered in the present invention are at positions
375, 815, 1003, 2169 and 2742.
[0021] Allelic variation at position 375 consists of a single base
substitution from G (the published base), for example to A. Allelic
variation at position 815 consists of a single base substitution
from C (the published base), for example to A. Allelic variation at
position 1003 consists of a single base substitution from A (the
published base), for example to C. Allelic variation at position
2169 consists of a single base substitution from C (the published
base), for example to T. Allelic variation at position 2742
consists of a single base substitution from C (the published base),
for example to T. The status of the individual may be determined by
reference to allelic variation at one, two, three, four or all five
of the above loci.
[0022] Sanak et al. (1998), Lancet, 350, 1599, have reported an
increased risk of aspirin induced asthma (AIA) being associated
with the polymorphism at position 1003. This work suggests that the
presence of the C allele at position 1003 leads to increased levels
of LTC.sub.4 synthase (see also Cowburn et al. (1998), J. Clin.
Invest., 101, 834). AIA affects about 10% of adult asthmatics.
Aspirin and other cyclo-oxygenase inhibitors cause release of LTs
into airways, leading to an asthma attack in susceptible
individuals. Clinical approaches to deal with AIA include
pretreatment with anti-leukotriene drugs (Szczeklik (1997),
Allergy, 52, 613-9). Commentators have written approvingly of the
clinical utility of detection of LTC.sub.4 polymorphisms (Holgate
(1998), Lancet, 351, 1300-1301, see last paragraph in particular).
Anti-leukotriene drugs have been reviewed in the following
publications: Horwitz et al. (1998), Am J Respir Crit Care Med,
157, 1363 (see particularly Table 1 for a list of drugs); and Tan
(1998), Current Opinion in Pulmonary Medicine, 4, 25.
[0023] The test sample of nucleic acid is conveniently a sample of
blood, bronchoalveolar lavage fluid, sputum, or other body fluid or
tissue obtained from an individual. It will be appreciated that the
test sample may equally be a nucleic acid sequence corresponding to
the sequence in the test sample, that is to say that all or a part
of the region in the sample nucleic acid may firstly be amplified
using any convenient technique e.g. PCR, before use in the analysis
of LTC.sub.4 synthase variation.
[0024] It will be apparent to the person skilled in the art that
there are a large number of analytical procedures which may be used
to detect the presence or absence of variant nucleotides at one or
more of positions 375, 815, 1003, 2169 and 2742 in the LTC.sub.4
synthase gene. In general, the detection of allelic variation
requires a mutation discrimination technique, optionally an
amplification reaction and a signal generation system. Table 1
lists a number of mutation detection techniques, some based on the
PCR. These may be used in combination with a number of signal
generation systems, a selection of which is listed in Table 2.
Further amplification techniques are listed in Table 3. Many
current methods for the detection of allelic variation are reviewed
by Nollau et al., Clin. Chem. 43, 1114-1120, 1997; and in standard
textbooks, for example "Laboratory Protocols for Mutation
Detection", Ed. by U. Landegren, Oxford University Press, 1996 and
"PCR", 2.sup.nd Edition by Newton & Graham, BIOS Scientific
Publishers Limited, 1997.
1 Abbreviations: AIA Aspirin induced asthma ALEX .TM. Amplification
refractory mutation system linear extension APEX Arrayed primer
extension ARMS .TM. Amplification refractory mutation system b-DNA
Branched DNA CMC Chemical mismatch cleavage bp base pair COPS
Competitive oligonucleotide priming system DGGE Denaturing gradient
gel electrophoresis FLAP 5-lipoxygenase activating protein FRET
Fluorescence resonance energy transfer LCR Ligase chain reaction
5-LO 5-Lipoxygenase LT Leukotriene MASDA Multiple allele specific
diagnostic assay NASBA Nucleic acid sequence based amplification
OLA Oligonucleotide ligation assay PCR Polymerase chain reaction
PTT Protein truncation test RFLP Restriction fragment length
polymorphism SDA Strand displacement amplification SNP Single
nucleotide polymorphism SSCP Single-strand conformation
polymorphism analysis SSR Self sustained replication TGGE
Temperature gradient gel electrophoresis General: DNA sequencing,
Sequencing by hybridisation Scanning: PTT*, SSCP, DGGE, TGGE,
Cleavase, Heteroduplex analysis, CMC, Enzymatic mismatch cleavage
*Note: not useful for detection of promoter polymorphisms.
[0025] Hybridisation Based
[0026] Solid phase hybridisation: Dot blots, MASDA, Reverse dot
blots, Oligonucleotide arrays (DNA Chips)
[0027] Solution phase hybridisation: Taqman.TM.--US-5210015 &
US-5487972 (Hoffmann-La Roche), Molecular Beacons--Tyagi et al
(1996), Nature Biotechnology, 14, 303; WO 95/13399 (Public Health
Inst., New York)
[0028] Extension Based: ARMS.TM., ALEX.TM.--European Patent No. EP
332435 B1 (Zeneca Limited), COPS--Gibbs et al (1989), Nucleic Acids
Research, 17, 2347.
[0029] Incorporation Based: Mini-sequencing, APEX
[0030] Restriction Enzyme Based: RFLP, Restriction site generating
PCR
[0031] Ligation Based: OLA
[0032] Other: Invader assay
[0033] Table 2--Signal Generation or Detection Systems
[0034] Fluorescence: FRET, Fluorescence quenching, Fluorescence
polarisation--United Kingdom Patent No. 2228998 (Zeneca
Limited)
[0035] Other: Chemiluminescence, Electrochemiluminescence, Raman,
Radioactivity, Colorimetric, Hybridisation protection assay, Mass
spectrometry
[0036] Table 3--Further Amplification Methods
[0037] SSR, NASBA, LCR, SDA, b-DNA
[0038] Preferred mutation detection techniques include ARMS.TM.,
ALEX.TM., COPS, Taqman, Molecular Beacons, RFLP, and restriction
site based PCR and FRET techniques.
[0039] Particularly preferred methods include ARMS.TM. and RFLP
based methods. ARMS.TM. is an especially preferred method.
[0040] In a further aspect, the diagnostic methods of the invention
are used to assess the efficacy of therapeutic compounds in the
treatment of asthma, rhinitis and other leukotriene mediated
diseases. The polymorphisms identified in the present invention
occur in the 5' untranslated region and the first intron of the
LTC.sub.4 synthase gene, regions which are of importance in the
control of gene transcription and gene translation. Furthermore,
each of the variant positions is located within a known
transcription factor binding site; it is believed that substitution
of A at variant position 375 modifies an AP-2 CS4 transcription
factor binding site, substitution of A at variant position 815
modifies an AP-2 CS5 transcription factor binding site,
substitution of C at variant position 1003 modifies the
glucocorticoid receptor binding site GGGACA and substitution of T
at variant position 2169 disrupts an MREc-(3) transcription factor
binding site.
[0041] Example 3 below describes another polymorphism which is
substitution of T for C at position 2742. This variant disrupts a
RIPE3b site (Shieh and Tsai, J. Biol. Chem. 266, 16708-16714,
1991).
[0042] Assays, for example reporter-based assays, may be devised to
detect whether one or more of the above polymorphisms affect
transcription levels and/or message stability.
[0043] Individuals who carry particular allelic variants of the
LTC.sub.4 synthase gene may therefore exhibit differences in their
ability to regulate enzyme biosynthesis under different
physiological conditions and will display altered abilities to
react to different diseases. In addition, differences in enzyme
regulation arising as a result of allelic variation may have a
direct effect on the response of an individual to drug therapy.
LTC.sub.4 synthase polymorphism may therefore have the greatest
effect on the efficacy of drugs designed to modulate the activity
of LTC.sub.4 synthase or other components of the leukotriene
pathway. However, the polymorphisms may also affect the response to
agents acting on other biochemical pathways regulated by
leukotrienes. The diagnostic methods of the invention may therefore
be useful both to predict the clinical response to such agents and
to determine therapeutic dose.
[0044] In a further aspect, the diagnostic methods of the
invention, are used to assess the predisposition and/or
susceptibility of an individual to diseases mediated by
leukotrienes. LTC.sub.4 synthase polymorphism may be particularly
relevant in the development of asthma and other inflammatory
diseases such as allergic rhinitis and the present invention may be
used to recognise individuals who are particularly at risk from
developing these conditions.
[0045] In a further aspect, the diagnostic methods of the invention
are used in the development of new drug therapies which selectively
target one or more allelic variants of the LTC.sub.4 synthase gene.
Identification of a link between a particular allelic variant and
predisposition to disease development or response to drug therapy
may have a significant impact on the design of new drugs. Drugs may
be designed to regulate the biological activity of variants
implicated in the disease process whilst minimising effects on
other variants.
[0046] In a further diagnostic aspect of the invention the presence
or absence of variant nucleotides is detected by reference to the
loss or gain of sites recognised by restriction enzymes. In the
accompanying Example 1 we provide details of convenient sites that
are lost or gained as a result of LTC.sub.4 synthase gene
polymorphisms. The person of ordinary skill will be able to design
and implement diagnostic procedures based on the detection of
restriction fragment length polymorphism due to the loss or gain of
one or more of the sites listed in Examples 1 or 3.
[0047] In yet a further aspect the invention provides a variant of
the LTC.sub.4 synthase gene comprising one or more of the specific
polymorphisms at positions 375, 815, 1003 and 2169.
[0048] Further aspects of this invention comprise the 5'
untranslated region of the LTC.sub.4 synthase gene comprising a
polymorphism at one or more of positions 375, 815 and 1003. In
particular the polymorphism at position 375 is G to A. In
particular the polymorphism at position 815 is C to A. In
particular the polymorphism at position 1003 is A to C. Another
aspect of this invention comprises the first intron of the
LTC.sub.4 synthase gene comprising a polymorphism at position 2169;
in particular this polymorphism is C to T.
[0049] According to another aspect of the present invention there
is provided a nucleic acid comprising the 5' untranslated region of
LTC.sub.4 synthase comprising a polymorphism corresponding with one
or more of positions 375, 815 and 1003 as defined by the positions
in SEQ ID NO: 1 and in which there is an A at position 375, an A at
position 815 and a C at position 1003. The 5' untranslated region
of LTC.sub.4 synthase is defined as positions 1-1446 of SEQ ID NO:
1. Fragments of the 5' untranslated region comprising at least one
of these allelic variants are also within the scope of the
invention.
[0050] Fragments are at least 17 bases, more preferably at least 20
bases, more preferably at least 30 bases. Complementary strands are
also within the scope of the invention.
[0051] According to another aspect of the present invention there
is provided a nucleic acid comprising the first intron of the
LTC.sub.4 synthase gene comprising a polymorphism at one or more of
positions 2169 and 2742 as defined by the position in SEQ ID NO: 1
and in which there is a T at position 2169 and there is a T at
position 2742. The first intron of the LTC.sub.4 synthase gene is
defined as positions 1505-2949 of SEQ ID NO: 1. Fragments of the
first intron comprising at least one of these allelic variants are
also within the scope of the invention.
[0052] The invention further provides nucleotide primers which
detect the LTC.sub.4 synthase gene polymorphisms of the
invention.
[0053] According to another aspect of the present invention there
is provided a diagnostic nucleic acid primer capable of detecting a
LTC.sub.4 synthase gene polymorphism at one or more of positions
375, 815, 1003, 2169 and 2742 in the LTC.sub.4 synthase gene as
defined by the positions in SEQ ID NO: 1.
[0054] A diagnostic nucleic acid primer is defined as an allele
specific primer, used, generally together with a constant primer,
in an amplification reaction such as a PCR reaction, which provides
the discrimination between alleles through selective amplification
of one allele at a particular sequence position e.g. as used for
ARMS.TM. assays, see Example 2 herein. The diagnostic primer is
preferably 17-50 nucleotides, more preferably about 17-35
nucleotides, more preferably about 17-30 nucleotides.
[0055] We provide diagnostic primers comprising the sequences set
out below as well as derivatives thereof wherein about 6-8 of the
nucleotides at the 3' terminus are identical to the sequences given
below and wherein up to 10, such as up to 8, 6, 4, 2, or 1 of the
remaining nucleotides may be varied without significantly affecting
the properties of the diagnostic primer. Conveniently, the sequence
of the diagnostic primer is as written below, or more preferably as
described in Example 2 below. The diagnostic primer is preferably
17-50 nucleotides, more preferably about 17-35 nucleotides, more
preferably about 17-30 nucleotides.
2 Allelic Primer variant Diagnostic (Allele Specific) number*
detected Primer sequence 1 375 A GGGGCGGCCGGGGGCGCTCCAGGCGGGGCA 2
815 A CTTGGACAGGTTTCCTCCTGGCAGGGTGGA 3 1003 C
GGGTTGCCAGGAACAGCCTGGATGGGGACC 4 2169 T
ATGGTCCGACGGGAGGTCTGGGGAGGGAGT 5 375 A
CTCCTGCCTGGAGTTCTGGGTGTCTCCCTT 6 815 A
TAGTCGTTGTAGGGGTTCCATGCACAAGGT 7 1003 C
TAACTCCTCCACCCACCTTATCTGTTCCCG 8 2169 T
GACCACACACAGACCAGTGCTGGCTGTGCA *Primers 1-8 are represented as SEQ
ID NO:2-9 respectively.
[0056] The primers may be manufactured using any convenient method
of synthesis. Examples of such methods may be found in standard
textbooks, for example "Protocols for Oligonucleotides and
Analogues; Synthesis and Properties," Methods in Molecular Biology
Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 0-89603-247-7;
1993; 1.sup.st Edition. If required the primer(s) may be labelled
to facilitate detection.
[0057] According to another aspect of the present invention there
is provided an allele-specific oligonucleotide probe capable of
detecting a LTC.sub.4 synthase gene polymorphism at one or more of
positions 375, 815, 1003, 2169 and 2742 in the LTC.sub.4 synthase
gene as defined by the positions in SEQ ID NO: 1.
[0058] The allele-specific oligonucleotide probe is preferably
17-50 nucleotides, more preferably about 17-35 nucleotides, more
preferably about 17-30 nucleotides.
[0059] The design of such probes will be apparent to the molecular
biologist of ordinary skill. Such probes are of any convenient
length such as up to 50 bases, up to 40 bases, more conveniently up
to 30 bases in length, such as for example 8-25 or 8-15 bases in
length. In general such probes will comprise base sequences
entirely complementary to the corresponding wild type or variant
locus in the LTC.sub.4 gene. However, if required one or more
mismatches may be introduced, provided that the discriminatory
power of the oligonucleotide probe is not unduly affected. The
probes of the invention may carry one or more labels to facilitate
detection.
[0060] According to another aspect of the present invention there
is provided a diagnostic kit comprising a diagnostic primer of the
invention and/or an allele-specific oligonucloetide primer of the
invention.
[0061] The diagnostic kits may comprise appropriate packaging and
instructions for use in the methods of the invention. Such kits may
further comprise appropriate buffer(s) and polymerase(s) such as
thermostable polymerases, for example taq polymerase.
[0062] The LTC.sub.4 synthase gene has been mapped to chromosome
5q35 (Penrose et al, J. Biol. Chem. 271, 11356-11361, 1996). In
another aspect of the invention, the single nucleotide
polymorphisms of this invention may be used as genetic markers for
this region in linkage studies. This particularly applies to the
polymorphism at 1003 because of its relatively high frequency,
(Krugylak, Nature Genetics, 17, 21-24, 1997).
[0063] According to another aspect of the present invention there
is provided a method of treating a human in need of treatment with
an antileukotriene drug in which the method comprises:
[0064] i) diagnosis of a single nucleotide polymorphism in
LTC.sub.4 synthase in the human, which diagnosis comprises
determining the sequence of the nucleic acid at one or more of
positions 375, 815, 1003, 2169 and 2742 in the LTC.sub.4 synthase
gene as defined by the positions in SEQ ID NO: 1, and determining
the status of the human by reference to polymorphism in the
LTC.sub.4 synthase gene; and
[0065] ii) administering an effective amount of an antileukotriene
drug.
[0066] Preferably determination of the status of the human is
clinically useful. Examples of clinical usefulness include deciding
which antileukotriene drug or drugs to administer and/or in
deciding on the effective amount of the drug or drugs.
[0067] According to another aspect of the present invention there
is provided use of an antileukotriene drug in preparation of a
medicament for treating a leukotriene mediated disease in a human
diagnosed as having a single nucleotide polymorphism at one or more
of positions 375, 815, 1003, 2169 and 2742 in LTC.sub.4 synthase
gene as defined by the positions in SEQ ID NO: 1.
[0068] According to another aspect of the present invention there
is provided a pharmaceutical pack comprising an antileukotriene
drug and instructions for administration of the drug to humans
diagnostically tested for a single nucleotide polymorphism at one
or more of positions 375, 815, 1003, 2169 and 2742 in LTC.sub.4
synthase gene as defined by the positions in SEQ ID NO: 1.
[0069] Suitable antileukotriene drugs include leukotriene D.sub.4
receptor antagonists, FLAP antagonists and 5-lipoxygenase
inhibitors (see particularly Table 1 in the following publication
for a list of drugs, Horwitz et al. (1998), Am J Respir Crit Care
Med, 157, 1363), preferably leukotriene D.sub.4 receptor
antagonists, more preferably montelukast and zafirlukast, and of
these zafirlukast is most preferred.
[0070] Testing for the presence of the C allele at position 1003 is
especially preferred because, without wishing to be bound by
theoretical considerations, of its association with increased
levels of LTC.sub.4 synthase (as explained herein).
[0071] The invention will now be illustrated but not limited by
reference to the following Examples. All temperatures are in
degrees Celsius.
[0072] In the Examples below, unless otherwise stated, the
following methodology and materials have been applied.
[0073] AMPLITAQ.TM., available from Perkin-Elmer Cetus, is used as
the source of thermostable DNA polymerase.
[0074] General molecular biology procedures can be followed from
any of the methods described in "Molecular Cloning--A Laboratory
Manual" Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring
Harbor Laboratory, 1989).
[0075] Electropherograms were obtained in a standard manner: data
was collected by ABI377 data collection software and the wave form
generated by ABI Prism sequencing analysis (2.1.2).
EXAMPLE 1
[0076] Identification of Polymorphisms
[0077] 1. Methods
[0078] DNA Preparation
[0079] DNA was prepared from frozen blood samples collected in EDTA
following protocol I (Molecular Cloning: A Laboratory Manual, p392,
Sambrook, Fritsch and Maniatis, 2.sup.nd Edition, Cold Spring
Harbor Press, 1989) with the following modifications. The thawed
blood was diluted in an equal volume of standard saline citrate
instead of phosphate buffered saline to remove lysed red blood
cells. Samples were extracted with phenol, then phenol/chloroform
and then chloroform rather than with three phenol extractions. The
DNA was dissolved in deionised water.
[0080] Template Preparation
[0081] Templates were prepared by PCR using the oligonucleotide
primers and annealing temperatures set out below. The extension
temperature was 72.degree. and denaturation temperature 94.degree..
Generally 50 ng of genomic DNA was used in each reaction and
subjected to 40 cycles of PCR.
3 Forward Reverse Oligo- Oligo- Annealing % Fragment nucleotide
nucleotide Temperature Time DMSO 62-1043 62-87 1021-1043 62.degree.
60 s 5 271-407 271-291 388-407 60.degree. 45 s 0 417-1043 417-437
1021-1043 60.degree. 45 s 10 851-1824 851-874 1801-1824 62.degree.
60 s 5 1503-2400 1503-1524 2379-2400 65.degree. 60 s 10
[0082] For dye-primer sequencing these primers were modified to
include T7 and SP6 primer sequences (ABI protocol P/N 402114,
Applied Biosystems) at the 5' end of the forward and reverse
oligonucleotides respectively.
[0083] Chemical Mismatch Cleavage (CMC)
[0084] CMC was carried out as decribed by Rowley et al. (Genomics
30, 574-592, 1995) using internal labelling of probe and target
with fluorescent dyes (RG6 or R110). 6% Acrylamide gels were run on
an automated DNA sequencer (ABI 377, Applied Biosystems) on 12 cm
plates (under module GS12-2400A) and analysed with suitable
software (ABI GeneScan.TM. 2.1).
[0085] Dye Primer Sequencing
[0086] Dye-primer sequencing using T7 and SP6 primers was as
described in the ABI protocol P/N 402114 for the ABI Prism.TM. dye
primer cycle sequencing core kit with "AmpliTaq FS".TM. DNA
polymerase, modified in that the annealing temperature was
45.degree. and DMSO was added to the cycle sequencing mix to a
final concentration of 5%.
[0087] The extension reactions for each base were pooled,
ethanol/sodium acetate precipitated, washed and resuspended in
formamide loading buffer.
[0088] 4.25% Acrylamide gels were run on an automated sequencer
(ABI 377, Applied Biosystems).
[0089] 2. Results
[0090] All positions are based on the U50136 numbering.
[0091] Variant Position 375
[0092] CMC analysis of fragment 1 (62-1043) produced cleavage
products of approximately 300 bp and 670 bp in 9/49 subjects.
Dye-primer sequence analysis of fragment 1 from 2 subjects showing
this pattern revealed a substitution of A for G at position 375.
This was confirmed by sequencing 6 clones of fragment 1 from one of
these subjects; 5/6 had A at position 375 and 1/6 had G.
[0093] Substituting A for G at position 375 modifies a Mnl I site
at position 368. PCR products from LTC.sub.4 synthase position 271
to 407 from 49 subjects were digested with Mnl I. This product
contains an invariant Mnl I site at position 335 giving an
invariant 61 bp fragment and a polymorphic fragment, 72 bp in the
absence of site 368 or 33 and 39 base pairs if the Mnl I site at
368 is present. 9/49 subjects gave both the 72 bp and 33/39 bp
products indicating that the Mnl I site at position 368 was lost
from one allele and these subjects were heterozygous at position
375. The frequency of the A allele at 375 is thus 9/98.
[0094] Additional RFLPs generated by this variant are loss of an
M.CviA IV and M.Sss I site.
[0095] This variant modifies a transcription factor binding site
AP-2 CS4.
[0096] Variant Position 815
[0097] CMC analysis of fragment 1 produced cleavage products of
approximately 230 bp and 750 bp in 2/49 subjects. Dye-primer
sequence analysis of fragment 1 from 1 subject showing this pattern
revealed a substitution of A for C at position 815. This was
confirmed by sequencing 8 clones of fragment 1 from this subject.
4/8 had A at position 815 and 4/8 had C.
[0098] Substituting A for C at position 815 generates an Ava II
site at 813.
[0099] PCR products from LTC.sub.4 synthase position 417 to 1043
from 53 subjects were digested with Ava II. In 5/53 subjects an Ava
II site at position 815 was created. These subjects were
heterozygous at position 815. The frequency of the A allele was
thus 5/106 alleles.
[0100] Additional RFLPs generated by this variant are loss of Aca
I, CviK I, M.CviA IV, CviJ I and Hae III sites and gain of Asp697
I, VpaK11A I and Sin I sites.
[0101] This variant modifies a transcription factor binding site:
AP-2 CS5.
[0102] Variant Position 1003
[0103] CMC analysis of fragment 1 produced a cleavage product of
approximately 940 bp in 22/49 subjects. CMC analysis of fragment 2
(851-1824) produced a band of approximately 800 bp. Dye-primer
sequence analysis of fragment 2 from 24 subjects showing this
pattern revealed a substitution of C for A at position 1003. This
was confirmed by sequencing 14 clones of fragment 1 from 2 subjects
with the 940 bp cleavage product. 6/14 had C at position 1003 and
8/14 had A.
[0104] Substituting C for A at position 1003 generates an Ava II
site at position 999. PCR products from LTC.sub.4 synthase position
417 to 1043 from 53 subjects were digested with Ava II. In 26/53
subjects an Ava II site at position 1003 was created. One of these
subjects was homozygous C/C at position 815 and 25 were
heterozygous C/A. The frequency of the C allele was thus 27/106
alleles.
[0105] The 1003 C variant is not on the same chromosome as the 815
A variant.
[0106] Additional RFLPs generated by this variant are gain of sites
for Bcr I, AhaB I, Asp697 I, VpaK11A I, Asu I, Fmu I, Sau96 I, Sin
I, Nla IV, Asp I, Asp748 I, BsaC I, Dsa V, Ecol831 I, Hin2 I, Hpa
II, Msp I, Bcn I, Nci I, ScrF I and M.Sss I.
[0107] This variant modifies the glucocorticoid receptor binding
site GGGACA, (Chan et al., J. Biol. Chem. 266, 22634-22644,
1991).
[0108] Sanak et al. (1998), Lancet, 350, 1599, have reported an
increased risk of aspirin induced asthma (AIA) being associated
with this polymorphism (Sanak's position -444 is equivalent to our
position 1003). AIA affects about 10% of adult asthmatics. Aspirin
and other cyclo-oxygenase inhibitors cause release of LTs into
airways, leading to an asthma attack. Clinical approaches to deal
with AIA include pretreatment with anti-leukotriene drugs
(Szczeklik (1997), Allergy, 52, 613-9). Commentators have written
approvingly of the clinical utility of detection of LTC.sub.4
polymorphisms (Holgate (1998), Lancet, 351, 1300-1301, see last
paragraph in particular).
[0109] Variant Position 2169
[0110] Dye-primer sequencing of fragment 6 (1503-2400) from 47
subjects demonstrated a substitution of T for C at position 2169 in
3 subjects.
[0111] Substituting T for C at position 2169 generates an Apa LI
site at position 647.
[0112] Fragment 6 was digested with ApaL I. In 3/54 subjects an Apa
LI site was created. These subjects were heterozygous for the RFLP,
thus the frequency of the T allele at position 2169 is 3/108
alleles.
[0113] Additional RFLPs generated by this variant are loss of
M.CviA IV, Bca I, Hinp I, Hinp1 I, Cfo I, Hha I and M.Sss I sites
and gain of Aaq I, Bka1125 I, BsaG I, CviR I, BsiHKA I, Bsp 1286 I,
Hgi A I and Nsp II sites.
[0114] This variant disrupts an MREc-(3) site (Labbe et al., Nuc.
Acid Res. 19, 4225-4231 1991).
EXAMPLE 2
[0115] Detection of Variants 375, 815, 1003 and 2169 using
ARMS.TM..
[0116] The following primers were used in ARMS.TM. PCR to
distinguish allelic variants at positions 375, 815, 1003 and 2169
of the LTC.sub.4 synthase gene.
4 Allelic Variant Allele Specific Primer Detected Sequence**
Constant Primer Sequence*** 375 G GGAGTTCTGGGTGTCTCCATC
GGTCAGTCTGGACTTTGCCAC A GGAGTTCTGGGTGTCTCCATT 815 C
TAGGGGTTCCATGCACAAGGG TTGTTACCTTGAGGCAAGAGG C TAGGGGTTCCATGCACAATGG
A TAGGGGTTCCATGCACAAGGT A TAGGGGTTCCATGCACAATGT 100 A
CACCCACCTTATCTGTTCCCT AGGCTGGCAGGCATGAGGTTT C CACCCACCTTATCTGTTCCCG
A GGAACAGCCTGGATGGGGTCA TTCGTGCCCCTTCCTTGCCTA C
GGAACAGCCTGGATGGGGTCC 2169 C CAGACCAGTGCTGGCTGTACG
CTCCAGCTGCTCCTGCACTGA T CAGACCAGTGCTGGCTGTACA **These primers are
represented by SEQ ID NO: 10-21 respectively. ***These primers are
represented by SEQ ID NO: 22-26 respectively.
[0117] Genomic DNA (50 ng) was amplified for 35 cycles with the
above pairs of primers. The annealing temperatures were 62.degree.,
60.degree., 64.degree. and 60.degree. for 375, 815, 1003 and 2169
respectively.
[0118] Homozygotes for the less common allele were only available
for position 1003. The above primers and conditions would
distinguish A/A, A/C and C/C genotypes at position 1003.
[0119] 375 A/A homozygotes were not available so it could not be
demonstrated that the G specific primer would not recognise A/A
homozygotes but the A specific primer did not recognise G/G
homozygotes. 815 A/A homozygotes were not available so it could not
be demonstrated that the C specific primer would not recognise A/A
homozygotes but the A specific primer did not recognise C/C
homozygotes. 2169 T/T homozygotes were not available so it could
not be demonstrated that the C specific primer would not recognise
T/T homozygotes but the T specific primer did not recognise C/C
homozygotes.
EXAMPLE 3
[0120] Polymorphism at Position 2742
[0121] Dye primer sequencing, as described in Example 1, of
fragment 2180-2972 from 5 subjects demonstrated a substitution of T
for C at position 2742 in 2 subjects. Template was prepared as
described in Example 1 using the conditions set out below.
5 Forward Reverse Oligo- Oligo- Annealing % Fragment nucleotide
nucleotide Temperature Time DMSO 2180-2973 2180-2200 2953-2973
65.degree. 60 s 10
[0122] This variant disrupts a RIPE3b site (Shieh and Tsai, J.
Biol. Chem. 266, 16708-16714, 1991).
Sequence CWU 1
1
26 1 4465 DNA Homo sapiens 1 gagctcacag agcccccagc tggggcatat
ctggtttccg ggggcagggg cgatacccag 60 aggaggaaga agggattctg
agagagccca acaggctccg agcctcaggc tggagctgag 120 cttggggcag
caaggaagga ccaggtgcga gggcagaacc atgcggcccg acccctgcag 180
cacggcctgt ggcctccccc agctcctgcc cgtgcttctg ggtcagtctg gactttgcca
240 cttctgacca aaagccaccg caaacccact caagccaaaa gaggaagtga
ccgttaggcc 300 caactgggaa ggctggcggc caggggcact ccaggcaggg
cgaggggggc ggccgggggc 360 gctccaggcg gggcgaggga gacacccaga
actccaggca ggagtcctcg ggtgccacct 420 ttcctctcca cctggccctg
cgtgggctct gtcctcaggg tggcccgccg tagtccccct 480 ccccactctg
agtttcctgt cccaaagtcc taaggaagtt tccagaacta catctcacca 540
tcttgagtca gccttggctc agtgtccatc tcacaggcct ggaaggggca ggagtcagca
600 ctgtccagac cacagggcct gagtgtgggg agggcagccg tctaggaagg
tggtggaggg 660 ttgttacctt gaggcaagag ggctgcgggg cagaaagaca
cagcaggtga ctgttgtggg 720 aggcccaaga gaggcctggg agagggatgg
cccacaaggg ctgaccctcc cgccacccag 780 ggggccttgg acaggtttcc
tcctggcagg gtggcccttg tgcatggaac ccctacaacg 840 actaaggctg
gcaggcatga ggtttcctga aggagaaaga gcttgtgggg cccagtgtgg 900
ctgggggggc gctgggactc cattctgaag ccaaaggcac tgggaagggc ttccgcagag
960 gagggtttgg caggggttgc caggaacagc ctggatgggg acagggaaca
gataaggtgg 1020 gtggaggagt tagccgggag cctggggctg gctccagcat
gatgtggggg tctgcaaggc 1080 cctggagaaa gtggggtggt gcagcagggg
gcacacccac agctggagct gacccagatg 1140 gacagcttgg gctctgccac
gcgggactag gcaaggaagg ggcacgaaca agcaggaagt 1200 ggtgaggcgg
tctccagcta gctgctctcc cctgcccaga ctttggtttc ctccctgctg 1260
gcttggcctg gctccctggc tctgtgtggt atggtcacac ccccgtgcac cccctccact
1320 gagatggggc ggggagagca ccgaggctgc tcttcctctc ctgggccgtc
ctctgagcag 1380 cagacggggc taagcgttcc ccagctcgcc ttcacacaca
gcccgtgcca ccacaccgac 1440 ggtaccatga aggacgaggt agctctactg
gctgctgtca ccctcctggg agtcctgctg 1500 caaggtgggc tggttcctat
ctaggaagag ggtgggcctt agatccctac agcttgccct 1560 ctgcccccta
ggcccaggtg gagggcagag gtggggactc cagcccaggc ccaagctgga 1620
agagggtggg gactttcagg gaactggggg gcacctggct gtgagagctg taggacttgg
1680 gggtggcaag ggtgccagga caaatggtag gatagccatg ggcttgggga
agctgatctc 1740 tgctctttcc agctgtcccc tctctgggcg tcccagcaag
cggcccccat tccctggctc 1800 tgcttcaaag gcacctccat actgggacca
cgtggagcag ggtagaggtg ggactccttc 1860 ctccagcccc ctaaaaagag
cctgcttaat gcctttctca gactggccct aaaggacaca 1920 ttccttggcc
agatatcctt gccacctaag agacaccact actccacagt gtgtgggcta 1980
ggataaggca cagcctgggg agggggctct gaaggggctg aacagacagg ccagcctgac
2040 ctccagctgc tcctgcactg agctggatgg ccaccctgtg acacccatct
gcagagggcc 2100 cagaaccaaa ggtgccaggg ctgcaggact cagggggaga
tggtccgacg ggaggtctgg 2160 ggagggagcg cacagccagc actggtctgt
gtgtggtctg gcctggcctc acctgaccaa 2220 gagaagggct cctgcccaca
gagaaacttt agggccagcc caccctctgc aactacccca 2280 gccctggggt
cctggggtta ggctaggaga gtcccagctg caacctcctg ggagcaggag 2340
agaaggtgtc tgtcagattt aggcctggga ccggaatgca ggaacagaga aactgaggtt
2400 tggaggcaca gggacgcagg ctttagtgat cccggcctga ggcagggtca
gagggccctg 2460 ctggtgggcg ctggtaggtg ggtgaccagg gactgttagc
tacagggagt gtgcttcctt 2520 gcacctggga ggatgcagcc agctctgccc
tcagactccc gaggcacttc ctggccaggg 2580 acctgaaagc tgcatttgcc
tgtgttttga gagtgaaatg attcagaaac aaggactcaa 2640 gtggtctctc
tcgcggagca ggtgtccctg tgcctgaatc actcaccctc ccccatacac 2700
tcacaggttg ggacagggcc tctctgcgcc ccaggcttca gccctgccct cctcgctgaa
2760 tgtcagggac acagggcagg ccagggatgg gtgagacgag aggtctcctc
gggcggggag 2820 ggggcggggt tccgccttag ggaggagagg acacggccaa
gtgaagggcc agattgcagg 2880 atccctccca ctcccatctc tggggcttcg
ggtgtccaga cctgactccc gctccccctc 2940 ctcccccagc ctacttctcc
ctgcaggtga tctcggcgcg cagggccttc cgcgtgtcgc 3000 cgccgctcac
caccggccca cccgagttcg agcgcgtcta ccgagcccag tgaggcgcgg 3060
cgggagggcg cggggcgggg agcgagcccc aggcgggtcc gggtcgcagg accatcccgg
3120 ccggcgcgct catcccaccc gcccaccgca gggtgaactg cagcgagtac
ttcccgctgt 3180 tcctcgccac gctctgggtc gccggcatct tctttcatga
aggtcggggt gtggggcagg 3240 ggcgcacgcg ctggaccccc gggacccgcg
cagggcgctc accaggcccg tgcgtacctc 3300 tcgcaggggc ggcggccctg
tgcggcctgg tctacctgtt cgcgcgcctc cgctacttcc 3360 agggctacgc
gcgctccgcg cagctcaggt gagggccggg cggggagcgg ggcggggccg 3420
gggaaagatc gcgggcgggc ggggctcctg gggagcggga ccgaagctgg gggcgggcga
3480 cgggccggag cccagcgcct ttggggattc ggtgggcgag ccctggcggc
ggccagagga 3540 agtccccgtg gggccagggt tgcggcgggg aagaagcggg
cctcctcgcg ccacctcccc 3600 gctgaccgcc gcccgcaggc tggcaccgct
gtacgcgagc gcgcgcgccc tctggctgct 3660 ggtggcgctg gctgcgctcg
gcctgctcgc ccacttcctc ccggccgcgc tgcgcgccgc 3720 gctcctcgga
cggctccgga cgctgctgcc gtgggcctga gaccaaggcc cccgggccga 3780
cggagccggg aaagaagagc cggagcctcc agctgccccg gggaggggcg ctcgcttccg
3840 catcctagtc tctatcatta aagttctagt gaccgagacc cgggctgcgt
tctctgggtc 3900 cgcgggggtg gcgcaccgcg ggctacggag cctggagggg
cccagcccga gtccgggcag 3960 cccggggcgg gcttcctagt ggcggcgtga
gagtggctgc gaaggaacga gccctccccc 4020 tggggcggga ctggatccgg
tcttcacctc ctaccccact ccctactcag cctcggggtc 4080 acaaggccgc
ccagtcctgc cggggttcac cctcctagcg ctcagcggtc tcctcaccgg 4140
tccccctcct caggggcctt ccctcgactc tcagccgccg cagtccctcg tcccctggcc
4200 ttcacagctg acactagata gagcctgtgg ctctctcccc aggtgagggc
aggggttttt 4260 cttttggtca gcactggatc cccctcgtta actgtaggtg
ttcagggcag ccctccgagg 4320 tccgcagagc tgcgggcacc atgggaacga
agtgagtcag tgacaggcgg tctcaaggaa 4380 atgtccagaa gccttgggga
tccaggggag gcccacagaa acaaagaagt gacttttagc 4440 caagtatgca
ggagaaacgg aggag 4465 2 30 DNA Artificial Sequence Description of
Artificial Sequence Primer 2 ggggcggccg ggggcgctcc aggcggggca 30 3
30 DNA Artificial Sequence Description of Artificial Sequence
Primer 3 cttggacagg tttcctcctg gcagggtgga 30 4 30 DNA Artificial
Sequence Description of Artificial Sequence Primer 4 gggttgccag
gaacagcctg gatggggacc 30 5 30 DNA Artificial Sequence Description
of Artificial Sequence Primer 5 atggtccgac gggaggtctg gggagggagt 30
6 30 DNA Artificial Sequence Description of Artificial Sequence
Primer 6 ctcctgcctg gagttctggg tgtctccctt 30 7 30 DNA Artificial
Sequence Description of Artificial Sequence Primer 7 tagtcgttgt
aggggttcca tgcacaaggt 30 8 30 DNA Artificial Sequence Description
of Artificial Sequence Primer 8 taactcctcc acccacctta tctgttcccg 30
9 30 DNA Artificial Sequence Description of Artificial Sequence
Primer 9 gaccacacac agaccagtgc tggctgtgca 30 10 21 DNA Artificial
Sequence Description of Artificial Sequence Primer 10 ggagttctgg
gtgtctccat c 21 11 21 DNA Artificial Sequence Description of
Artificial Sequence Primer 11 ggagttctgg gtgtctccat t 21 12 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 12
taggggttcc atgcacaagg g 21 13 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 13 taggggttcc atgcacaatg
g 21 14 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 14 taggggttcc atgcacaagg t 21 15 21 DNA Artificial
Sequence Description of Artificial Sequence Primer 15 taggggttcc
atgcacaatg t 21 16 21 DNA Artificial Sequence Description of
Artificial Sequence Primer 16 cacccacctt atctgttccc t 21 17 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 17
cacccacctt atctgttccc g 21 18 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 18 ggaacagcct ggatggggtc
a 21 19 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 19 ggaacagcct ggatggggtc c 21 20 21 DNA Artificial
Sequence Description of Artificial Sequence Primer 20 cagaccagtg
ctggctgtac g 21 21 21 DNA Artificial Sequence Description of
Artificial Sequence Primer 21 cagaccagtg ctggctgtac a 21 22 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 22
ggtcagtctg gactttgcca c 21 23 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 23 ttgttacctt gaggcaagag
g 21 24 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 24 aggctggcag gcatgaggtt t 21 25 21 DNA Artificial
Sequence Description of Artificial Sequence Primer 25 ttcgtgcccc
ttccttgcct a 21 26 21 DNA Artificial Sequence Description of
Artificial Sequence Primer 26 ctccagctgc tcctgcactg a 21
* * * * *