Amplification and detection of chlamydia trachomatis nucleic acids

Amplification primers useful in assays for species-specific detection of a target sequence in the cryptic plasmid of C. trachomatis are described. The primers of the invention amplify a target in the region of nucleotides 2219-2366 of the cryptic plasmid sequence, and the target binding sequences disclosed may be adapted for use in amplification primers for a variety of amplification reactions.

FIELD OF THE INVENTION 
The invention relates to nucleic acid amplification, including detection 
and/or identification of microorganisms using nucleic acid amplification. 
BACKGROUND OF THE INVENTION 
Chlamydia trachomatis is a major cause of urogenital infections in both 
males and females in the United States. The in vivo diagnosis of Chlamydia 
requires culture on McCoy cell monolayers, which is labor intensive and 
requires approximately 48-72 hours. Several more rapid tests have been 
developed based on antigen detection by direct fluorescent antibody 
staining (DFA), enzyme immunoassays (EIA) and enzyme-linked immunosorbent 
assays (ELISA). A probe hybridization assay for direct detection of 
Chlamydia (E2, GenProbe, see Warren, et al. 1993. J. Clin. Microbiol. 
31:1663-1666.) has also been developed. Most of these currently available 
tests require endocervical swabs in females and urethral swabs in males. 
However, DNA amplification technologies such as PCR, LCR and SDA have the 
potential to provide highly sensitive alternatives for Chlamydia detection 
using less invasive clinical specimens such as urine. Domeika et al. 
(1994. J. Clin. Microbiol. 32:2350-2352), Bauwens, et al. (1993. J. Clin. 
Microbiol. 31:3013-3106), and U.S. Pat. No. 5,232,829 report amplification 
of Chlamydia trachomatis DNA using the PCR, followed by microtiter plate 
hybridization detection. Amplification tests which use the LCR followed by 
microparticle sandwich immunoassay detection have also been reported 
(Chernesky et al. 1994. J. Clin. Microbiol. 32:2682-2685, Lee et al. 1995. 
Lancet 345:213-216, Bassiri et al. 1995 J. Clin. Microbiol. 33:898-900). 
Currently, these tests take 4-6 hours to complete. 
The cryptic plasmid of Chlamydia trachomatis is a 7.4 kb plasmid which is 
specific to this organism. It is present in about 10 copies per genome 
equivalent and detects all 200 clinical strains of C. trachomatis when 
used as a hybridization probe (Palmer and Falkow. 1986. Plasmid 16:52-62). 
Hatt, et al. (1988. Nucl. Acids Res. 16:4053-4067) reported the sequence 
of the cryptic plasmid of the L1 serovar, and EP 0 499 681 describes the 
sequence of the cryptic plasmid of serotype D. The sequence of the cryptic 
plasmid of C. trachomatis L2/434/Bu is shown in EP 0 336 412, which also 
describes oligonucleotide probes derived from this cryptic plasmid 
sequence. One embodiment of the plate capture assay described in U.S. Pat. 
No. 5,232,829 is directed to detection of cryptic plasmid target 
sequences. Two amplification primer sets are described: one which produces 
a 208 base pair amplicon using primers which hybridize at positions 
195-219 and 377-402, and a second which produces a 173 base pair amplicon 
using primers which hybridize to positions 678-700 and 827-850. In one 
example, WO 93/00447 describes gap-filling LCR for detection of Chlamydia 
target sequences. The oligonucleotides employed are based on map positions 
6693.1, 6693.2, 6693.3 and 6694.4 of Hatt, et al., supra. 
The following terms are defined herein as follows: 
An amplification primer is a primer for amplification of a target sequence 
by extension of the primer after hybridization to the target sequence. The 
3' end of an SDA amplification primer (the target binding sequence) 
hybridizes at the 3' end of the target sequence. The target binding 
sequence is about 10-25 nucleotides in length and confers hybridization 
specificity on the amplification primer. The SDA amplification primer 
further comprises a recognition site for a restriction endonuclease 5' to 
the target binding sequence. The recognition site is for a restriction 
endonuclease which will nick one strand of a DNA duplex when the 
recognition site is hemimodified, as described by G. Walker, et al. (1992. 
PNAS 89:392-396 and 1992 Nucl. Acids Res. 20:1691-1696). The about 10-25 
nucleotides 5' to the restriction endonuclease recognition site (the 
"tail") function as a polymerase repriming site when the remainder of the 
amplification primer is nicked and displaced during SDA. The repriming 
function of the tail nucleotides sustains the SDA reaction and allows 
synthesis of multiple amplicons from a single target molecule. The 
sequence of the tail is generally not critical and can be routinely 
selected and modified to obtain the desired T.sub.m for hybridization. As 
the target binding sequence is the portion of a primer which determines 
its target-specificity, for amplification methods which do not require 
specialized sequences at the ends of the target the amplification primer 
generally consists essentially of only the target binding sequence. For 
amplification methods which require that specialized sequences other than 
the nickable restriction site and primer tail of SDA be appended to the 
target (e.g., an RNA polymerase promoter for 3SR, NASBA or transcription 
based amplification), the required specialized sequence may be linked to 
the target binding sequence using routine methods for preparation of 
oligonucletoides without altering the hybridization specificity of the 
primer. 
A bumper primer or external primer is a primer used to displace primer 
extension products in isothermal amplification reactions. The bumper 
primer anneals to a target sequence upstream of the amplification primer 
such that extension of the bumper primer displaces the downstream 
amplification primer and its extension product. 
The terms target or target sequence refer to nucleic acid sequences to be 
amplified. These include the original nucleic acid sequence to be 
amplified and its complementary second strand and either strand of a copy 
of the original sequence which is produced by the amplification reaction. 
Copies of the target sequence which are generated during the amplification 
reaction are referred to as amplification products, amplimers or 
amplicons. 
The term extension product refers to the copy of a target sequence produced 
by hybridization of a primer and extension of the primer by polymerase 
using the target sequence as a template. 
The term species-specific refers to detection, amplification or 
oligonucleotide hybridization in a species of organism or a group of 
related species without substantial detection, amplification or 
oligonucleotide hybridization in other species of the same genus or 
species of a different genus. C. trachomatis-specific detection, 
amplification or hybridization refers to species-specificity for C. 
trachomatis. 
The term assay probe refers to any oligonucleotide used to facilitate 
detection or identification of a nucleic acid. For example, in the present 
invention, assay probes are used for detection or identification of C. 
trachomatis nucleic acids, either directly or after amplification. 
Detector probes, capture probes and signal primers are examples of assay 
probes. 
SUMMARY OF THE INVENTION 
Primers for species-specific detection of a target sequence in the cryptic 
plasmid of C. trachomatis are described. The amplification primers of the 
invention amplify a target in the region of nucleotides 2219-2366 of the 
cryptic plasmid sequence. Preferred amplification primers comprise the 
specialized sequences required for SDA, however, amplification primers 
with similar specificity may be constructed using the disclosed target 
binding sequences and, optionally, other sequences required for a selected 
amplification reaction.

DETAILED DESCRIPTION OF THE INVENTION 
To identify potential target binding sequences for use in amplification 
primers, the nucleotide sequence of the cryptic plasmid of C. trachomatis 
(GenBank Accession #X06707) was analyzed for open reading frames with low 
GC content and no runs of three consecutive Cs or Gs. A potential target 
in the region of nucleotides 2219-2366 (bumper to bumper in the 
exemplified SDA reactions) was identified by these criteria (GC content 
about 34%). Target binding sequences for three upstream amplification 
primers (SEQ ID NO:1-3) and two downstream amplification primers (SEQ ID 
NO:4-5) were selected, and amplification primers for SDA comprising these 
target binding sequences were synthesized by conventional methods. The 
amplification primers are shown below, with the target binding sequences 
italicized and the nickable restriction endonuclease recognition site 
bolded: 
##STR1## 
For testing in SDA, a pair of bumper primers useful with all pairwise 
combinations of upstream and downstream amplification primers was also 
designed: 
##STR2## 
For detection of the amplification products, a detector probe which 
hybridized to the amplification products of all pairwise combinations of 
upstream and downstream amplification primers was selected: 
##STR3## 
The target binding sequences of the amplification primers confer C. 
trachomatis hybridization specificity and therefore provide the 
species-specificity of the assay. By way of example, the amplification 
primers listed above contain a recognition site for the restriction 
endonuclease BsoBI which is nicked during the SDA reaction. It will be 
apparent to one skilled in the art that other nickable restriction 
endonuclease recognition sites may be substituted for the BsoBI 
recognition site, including but not limited to those recognition sites 
disclosed in EP 0 684 315. Preferably, the recognition site is for a 
thermophilic restriction endonuclease so that the amplification reaction 
may be performed under optimum temperature conditions. Similarly, the tail 
sequence of the amplification primer (5' to the restriction endonuclease 
recognition site) is generally not critical, although it is important to 
avoid including the restriction site used for SDA and to avoid sequences 
which will hybridize either to their own target binding sequence or to 
other primers. Amplification primers for SDA according to the invention 
therefore consist of the 3' target binding sequences indicated above, a 
nickable restriction endonuclease recognition site 5' to the target 
binding sequence and a tail sequence about 10-25 nucleotides in length 5' 
to the restriction endonuclease recognition site. The length of the tail 
depends on the T.sub.m of the selected sequence, and can be easily 
adjusted by one skilled in the art to obtain sufficient hybridization. The 
tail sequence and the restriction endonuclease recognition site are 
sequences required for amplification by SDA. Other embodiments of 
amplification primers according to the invention consist of 1) the target 
binding sequence alone when no additional specialized sequence is required 
by the selected amplification reaction (e.g., the PCR), or 2) the target 
binding sequence and the additional sequences required for the 
amplification reaction (e.g., the RNA polymerase promoter required for 
amplification by 3SR or NASBA). 
Amplification products may be detected by hybridization to an assay probe. 
The assay probe may be tagged with a detectable label to facilitate its 
detection when hybridized to the amplification product. The detectable 
label may be conjugated to the probe after it is synthesized or it may be 
incorporated into the probe during synthesis, for example in the form of a 
label-derivatized nucleotide. Such labels are known in the art and include 
directly and indirectly detectable labels. Directly detectable labels 
produce a signal without further chemical reaction and include such labels 
as fluorochromes, radioisotopes and dyes. Indirectly detectable labels 
require further chemical reaction or addition of reagents to produce the 
detectable signal. These include, for example, enzymes such as horseradish 
peroxidase and alkaline phosphatase, ligands such as biotin which are 
detected by binding to label-conjugated avidin, and chemiluminescent 
molecules. The assay probes may be hybridized to their respective 
amplification products in solution, on gels, or on solid supports. 
Following hybridization, the signals from the associated labels are 
developed, detected and optionally quantitated using methods appropriate 
for the selected label and hybridization protocol. The amount of signal 
detected for each amplification product may be used to indicate the 
relative amount of amplification product present. Ligand labels may also 
be used on assay probes to facilitate capture of the hybrid on a solid 
phase (capture probe). 
An alternative method for detecting amplification products is by polymerase 
extension of a primer specifically hybridized to the target sequence. The 
primer is labeled as described above, for example with a radioisotope, so 
that the label of the primer is incorporated into the extended reaction 
product. This method is described by Walker, et al. (1992) Nuc. Acids Res. 
and PNAS, supra. Another method for detecting amplified target and control 
sequences is a chemiluminescent method in which amplified products are 
detected using a biotinylated capture probe and an enzyme-conjugated 
detector probe as described in U.S. Pat. No. 5,470,723. After 
hybridization of these two assay probes to different sites in the assay 
region of the target sequence, the complex is captured on a 
streptavidin-coated microtiter plate, and the chemiluminescent signal is 
developed and read in a luminometer. As another alternative for detection 
of amplification products, a signal primer (essentially a detector probe 
which is extended by polymerase, displaced and rendered double-stranded in 
a target amplification-dependent manner) as described in EP 0 678 582 may 
be included in an SDA reaction. In this embodiment, labeled secondary 
amplification products are generated during SDA in a target 
amplification-dependent manner and may be detected as an indication of 
target amplification. 
SDA reactions employing the primers of the invention may incorporate 
thymine as taught by Walker, et al., supra, or they may wholly or 
partially substitute 2'-deoxyuridine 5'-triphosphate (dUTP) for TTP in the 
reaction, as taught in EP 0 624 643, to reduce cross-contamination of 
subsequent SDA reactions. dU (uridine) is incorporated into amplification 
products of both target and control sequences and can be excised by 
treatment with uracil DNA glycosylase (UDG). These abasic sites render the 
amplification product unamplifiable in subsequent SDA reactions. UDG may 
be inactivated by uracil DNA glycosylase inhibitor (Ugi) prior to 
performing the subsequent amplification to prevent excision of dU in 
newly-formed amplification products. 
The primers or probes for performing the assay methods of the invention may 
be packaged in the form of a diagnostic kit for species-specific 
amplification or detection of C. trachomatis DNA. Kits for amplification 
may comprise amplification primers comprising the described target binding 
sequences for amplification of the C. trachomatis target sequence and, 
optionally, the reagents required for performing the selected 
amplification reaction (e.g., deoxynucleoside triphosphates, enzymes, 
buffers, polymerase, additional primers, etc.). The kits for target 
sequence amplification may further optionally include assay probes useful 
for detecting or identifying the amplified C. trachomatis target sequence, 
and/or an internal control sequence to be co-amplified with the target 
sequences as described in U.S. Pat. No. 5,457,027. Assay probes for 
detection of the amplified target may comprise a detectable label as 
described herein, and, optionally, the kits may include reagents for 
hybridization and/or detection of the assay probe. 
EXAMPLE 1 
The six pairwise combinations of the upstream and downstream amplification 
primers described above were tested in tSDA reactions essentially as 
described in EP 0 684 315. The 50 .mu.l amplification reactions contained 
5 mM MgCl.sub.2, 0.2 mM each dGTP, dATP and TTP, 1.4 mM .alpha.-thio-dCTP, 
10 ng/.mu.l human placental DNA, 35 mM K.sub.i PO.sub.4 pH 7.6, 10% (v/v) 
glycerol, 0.5 .mu.M SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, 0.5 .mu.M 
SEQ ID NO:4 or SEQ ID NO:5, 0.05 .mu.M SEQ ID NO:6 and SEQ ID NO:7, 3.2 
units/.mu.l BsoBI (New England BioLabs), 0.125 unit/.mu.l exonuclease 
deficient Bst DNA polymerase (Molecular Biology Resources) and 10.sup.5 or 
10.sup.3 C. trachomatis elementary bodies (EBs). Prior to addition of 
BsoBI, Bst polymerase and MgCl.sub.2, the reactions were heated at 
95.degree. C. for 5 min. to denature the target DNA. After denaturing the 
target, the samples were allowed to equilibrate at 53.5.degree. C. for 5 
min. Amplification was initiated by addition of 10 .mu.l of enzyme mix (5 
.mu.l 50 mM MgCl.sub.2, 0.5 .mu.l of 25 units/.mu.l Bst polymerase, 1 
.mu.l of 160 units/.mu.l BsoBI and 3.5 .mu.l 1X NEB2 buffer). After 30 
min. of amplification at 53.5.degree. C., 5 .mu.l of each reaction was 
assayed for amplification products by hybridization of .sup.32 P-labeled 
SEQ ID NO:8 and primer extension as described by Walker, et al (1992. 
Nucl. Acids Res., supra). The products were separated by 8% polyacrylamide 
gel electrophoresis and analyzed by autoradiography. 
All amplification primer pairs containing SEQ ID NO:1 or SEQ ID NO:3 as the 
upstream primer produced readily detectable amplification products. In 
contrast, primer pairs containing SEQ ID NO:2 as the upstream primer 
performed poorly. Quantitation of the bands of the autoradiographs showed 
that amplification of 10.sup.5 targets using the SEQ ID NO:2/SEQ ID NO:4 
primer pair was reduced 159-fold as compared to amplification using primer 
pairs which did not contain SEQ ID NO:2, and amplification of 10.sup.3 
targets was reduced 31-fold. Amplification using SEQ ID NO:2/SEQ ID NO:5 
was reduced 75-fold for 10.sup.5 targets and reduced about 14-fold for 
10.sup.3 targets. This results was unexpected, as the sequence of SEQ ID 
NO:2 overlaps the other upstream amplification primers, all of which 
performed well in the amplification reaction. Based on these results, 
however, it was concluded that amplification using SEQ ID NO:2 was too 
insensitive to be useful for clinical diagnosis. 
EXAMPLE 2 
SEQ ID NO:1 and SEQ ID NO:4 were selected as the primer pair for further 
optimization in SDA reactions employing simultaneous amplification and 
detection, as described in EP 0 678 582. SDA was performed as in Example 1 
except that 10 nM of .sup.32 P-labeled SEQ ID NO:8 was added prior to 
amplification as a signal primer. Secondary amplification products were 
detected on polyacrylamide gels as in Example 1. For comparison of 
amplification efficiency, SDA was also performed with post-amplification 
detection as in Example 1. Amplification levels were about equivalent for 
simultaneous amplification/detection and post-amplification detection, 
with an amplification factor of about 1.times.10.sup.10. 
SEQ ID NO:1 and SEQ ID NO:4 were then tested in simultaneous 
amplification/detection SDA reactions for specificity and cross 
reactivity: Target DNA from the following species was tested: C. 
trachomatis serovars A, B, Ba, C, D, E, F, G, I, J, K and L3; C. psittaci, 
N. gonorrhea, N. meningititis, C. albicans and E. coli. All C. trachomatis 
serovars tested (10.sup.4 EBs/reaction) produced detectable amplification 
products. No signal above background was detected in any of the reactions 
containing DNA from non-C. trachomatis bacteria (10.sup.6 EBs/reaction). 
EXAMPLE 3 
The sensitivity of detection of the C. trachomatis target sequence was 
tested using SEQ ID NO:1 and SEQ ID NO:4 in an assay employing a signal 
primer with post-amplification detection by fluorescence polarization as 
described by Walker, et al. (1996. Clin. Chem. 42, 9-13). SEQ ID NO:8 was 
labeled with 5-(4,6-dichlorotriazin-2-yl)amino fluorescein (5-DTAF) for 
use as a signal primer and added to the SDA reaction prior to 
amplification. The 50 .mu.l amplification reactions contained 5 mM 
MgCl.sub.2, 0.2 mM each dGTP, dATP and TTP, 1.4 mM .alpha.-thio-dCTP, 20 
.mu.g/ml non-acetylated bovine serum albumin, 1 ng/.mu.l human placental 
DNA, 40 mM K.sub.i PO.sub.4 pH 7.6, 5% (v/v) glycerol, 3% (v/v) DMSO, 0.75 
.mu.M SEQ ID NO:1, 0.1875 .mu.M SEQ ID NO:4, 10 nM 5-DTAF labeled SEQ ID 
NO:8, 0.075 .mu.M SEQ ID NO:6 and SEQ ID NO:7, 3.2 units/.mu.l BsoBI, 0.25 
units/.mu.l exonuclease deficient Bst DNA polymerase and varying amounts 
of Chlamydia EBs (0, 5, 10, 50, 500). An amplification reaction containing 
EDTA was used to inhibit amplification, representing a sample with no 
amplicon contamination and no target DNA Prior to addition of BsoBI, Bst 
polymerase, BSA and MgCl.sub.2, the reactions (40 .mu.l) were heated at 
95.degree. C. for 5 min. to denature the target DNA. After denaturing the 
target, the samples were allowed to equilibrate at 53.5.degree. C. for 5 
min. Amplification was initiated by addition of 10 .mu.l of enzyme mix (5 
.mu.l 50 mM MgCl.sub.2, 1 .mu.l 1 mg/ml BSA, 1 .mu.l of 25 units/.mu.l Bst 
polymerase, 1 .mu.l of 160 units/.mu.l BsoBI and 2 .mu.l 1X NEB2). After 
30 min. of amplification, 45 .mu.l of the reaction was diluted into 1 ml 
of fluorescence polarization buffer in a 12.times.75 borosilicate glass 
tube (Fisher) at 37.degree. C. and fluorescence polarization (FP) was 
measured on an FPM-1 fluorometer (Jolley Research and Consultants) blanked 
against FP buffer. On the FPM-1, FP is measured by exciting the label in 
the vertical plane at 488 nm and measuring emission intensity in the 
vertical and horizontal planes at 520 nm. FP is expressed as 
millipolarization units (mP). 
FP increased as the number of targets (EBs) in the amplification reaction 
increased (FIG. 1). Comparison of the 0 target sample with the EDTA sample 
shows slight contamination with about 10-20 amplicons. As few as five 
initial EBs could be detected over background. 
EXAMPLE 4 
The SEQ ID NO:1/SEQ ID NO:4 amplification primer pair was tested in a 
real-time SDA/fluorescence polarization detection system as described by 
Walker, et al. (1996) using an SLM 8100 fluorometer. This instrument has 
four cuvette chambers which can be read sequentially. Four 1 ml SDA 
reactions were performed with varying numbers of Chlamydia EBs (10.sup.6, 
10.sup.4, 10.sup.2 or 0) essentially as in Example 3. The 0 EB reaction 
also contained 10 .mu.l of 0.5M EDTA to inhibit amplification, 
representing a sample lacking Chlamydia EBs and free of amplicon 
contamination. Amplification was detected in real-time at 53.5.degree. C. 
The results are shown in FIG. 2. The reactions containing 10.sup.6 and 
10.sup.4 EBs plateaued by 18 min. at an asymptotic FP value of 
approximately 215 mP. However, a quantitative difference between the 
samples could be seen, as the reactions containing higher initial target 
levels showed an increase in FP more quickly than reactions containing 
lower initial target levels. That is, the sample containing 10.sup.6 EBs 
began to show an increase in FP at 8 min., while the samples containing 
10.sup.4 and 10.sup.2 EBs did not begin to show an increase in FP until 10 
and 16 min., respectively. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 8 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ACCGCATCGAATCGATGTCTCGGGTAGAAAATCGCATGCAAGATA45 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ACCGCATCGAATCGATGTCTCGGGCGCATGCAAGATATCGAGTAT45 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ACGCCATCGAATCGATGTCTCGGGATGCAAGATATCGAGTATGCG45 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 46 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CGATTCCGCTCCAGACTTCTCGGGAGCTGCCTCAGAATATACTCAG46 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CGATTCCGCTCCAGACTTCTCGGGGCAAGCTGCCTCAGAATATAC45 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TAAACATGAAAACTCGTTCCG21 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
TTTTATGATGAGAACACTTAAACTCA26 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TAGAGTCTTCAAATATCAGAGCTTTACCTAACAA34 
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