Oligonucleotide repeat arrays

A solid support based hybridization assay is provided which allows for the systematic and reproducible analysis of repeat and tandem repeat oligonucleotide sequences of DNA and RNA by hybridization to a reverse dot blot array comprising strings of such repeats complementary to those found in particular nucleic acid targets (e.g., analyte PCR product). An addressable library (i.e., an indexed set) of complementary repeats is synthesized on a suitable support. Preferably, the support comprises a low fluorescent background support, thereby facilitating the use of non-radioisotopic modes of detection (such as fluorescence or chemiluminescence); particularly suitable in this regard is an aminated polypropylene support or similar material. Preferred arrays permit screening of DNA and RNA samples for complete sets of particular types of nucleotide repeat sequences (e.g., all nucleotide doublet or triplet repeats).

BACKGROUND OF THE INVENTION 
The present invention relates generally to the fields of biochemistry and 
medicine. In particular, the invention is directed to materials and 
methods useful in the diagnosis of genetic mutations of clinical 
relevance. 
Short tandem repeats (STR) have been identified in a number of genes. It 
has been proposed that particular unstable triplet repeat oligonucleotides 
are correlated with a number of genetic diseases in humans, including 
Kennedy's disease [La Spada, A. et al., Nature, 352, 77-79 (1991)], 
fragile-X syndrome [Verkerk, A. J. M. H. et al., Cell 65, 905-914 (1991)], 
myotonic dystrophy [Fu, Y. H. et al, Science 255, 1256-1258 (1992)], 
Huntington disease [The Huntington's Disease Collaborative Research Group, 
Cell 72, 971-983 (1993)] and spinocerebellar ataxia type 1 [Orr, H. T. et 
al., Nature Genet. 4, 221-226 (1993)]. Similarly, doublet repeats have 
also been reported to be associated with particular disease states; for 
example, correlations have been proposed with cystic fibrosis [Chu, C.-S. 
et al., Nature Genetics 3, 151-156 (1993)] and colorectal cancer 
[Thibodeau, S. N. et al., Science 260, 816-819 (1993)]. Higher-order 
repeats, such as tetramers [see, e.g., Gen, M. W. et al., Genomics 17, 
770-772 (1993)], have also been identified. 
One gene which has been subject of intense scrutiny is the Huntington's 
disease gene. The trinucleotide hybridization approach was recently 
utilized to map out tandem repeats across a section of the gene. In this 
section, 51 triplet repeats spanning a 1.86 Mbp DNA segment were 
identified by Southern transfer of restriction enzyme digests of a 
specific cosmid and probing with .sup.32 P-labelled oligonucleotide probes 
[Hummerich, et al., "Distribution of trinucleotide repeat sequences across 
a 2 Mbp region containing the Huntington's disease gene," Human Molecular 
Genetics 3, 73 (1994)]. 
DNA polymorphisms which arise from allelic differences in the number of 
repeats have been identified by such terminology as short tandem repeats 
(STR), variable number of tandem repeats (VNTR), minisatellites (tandem 
repeats of a short sequence, originally defined as 9-60 bp) and 
microsatellites (originally defined as 1-5 bp) [McBride, L. J. & O'Neill, 
M. D., American Laboratory, pp. 52-54 (November 1991)]; minisatellites and 
microsatellites would be considered subclasses of the VNTR. It is 
estimated that there are up to 500,000 microsatellite repeats distributed 
throughout the human genome, at an average spacing of 7000 bp. Therefore, 
it is apparent that most genes will contain VNTR regions and that these 
regions can be used as genetic markers. For example, VNTRs are currently 
being used as markers in studies concerned with the inheritance of certain 
mutations leading to various forms of cancer. Recently, it has been 
discovered that certain triplet repeat expansions are associated with a 
predisposition towards certain diseases; a large expansion is typically 
associated with the onset of the disease. For example, the (CGG) triplet 
repeat region associated with Fragile X occurs at a frequency of 10-50 
repeat units in the normal population, while in those afflicted with the 
disease the expansion is between 200-2000 repeats. 
As it becomes possible to determine whether a particular genotype comprises 
an unstable repeat and/or is associated with a particular disease state, 
there is a considerable incentive to develop useful methods to 
characterize STRs. The heretofore available methods for initial scanning 
for STRs have generally required time-consuming sequential oligonucleotide 
hybridizations to filter-bound target DNAs to identify specific STRs [see, 
e.g., Litt, M. and Luty, J. A., Am. J. Hum. Genet. 44, 397-401 (1989); 
Weber, J. L. and May, P. E., Am. J. Hum. Genet. 44, 388-396 (1989); Fu et 
al., supra]. In particular, the analysis of oligonucleotide repeats is 
typically carried out at the present time by Southern blotting of 
restriction fragments followed by hybridization analysis using a specified 
repetitive sequence probe. Alternatively, it is possible to probe dot 
blots of the target DNA [Iizuka, et al., GATA 10:2-5 (1993)]. 
Both of these heretofore-known techniques are time-consuming and tedious 
for large sample populations. Moreover, multiple probings may be required 
to identify which repeat might be present. Further, it is often difficult 
to reproducibly spot or transfer equivalent amounts of DNA to these 
supports; thus, conventional dot blots and transfers show variation in 
signal intensity from batch to batch. In addition, any regions of DNA that 
might become cross-linked to the support (e.g., through UV light) would be 
inaccessible to probes. 
It would be highly useful for clinical investigators to be able to screen 
large sample populations of patients DNAs in an effective manner. As 
additional STRs are identified and associated with particular conditions, 
the need for simple and effective screening methods becomes greater. 
PCT published application No. WO 89/10977 describes methods and apparatus 
for analyzing polynucleotide sequences in which an array of the whole or a 
chosen part of a complete set of oligonucleotides are bound to a solid 
support. The different oligonucleotides occupy separate cells of the array 
and are capable of taking part in hybridization reactions. For studying 
differences between polynucleotide sequences, the array may comprise the 
whole or a chosen part of a complete set of oligonucleotides comprising 
the polynucleotide sequences. While it is suggested that a small array may 
be useful for many applications, such as the analysis of a gene for 
mutations, there is no teaching or suggestion of a specific array or 
method for using same which would permit the rapid and accurate screening 
of a wide range of biological materials for tandem repeats. Moreover, the 
arrays described in WO 89/10977 are designed specifically for use in 
sequencing by hybridization; the presence of long tandem nucleotide 
repeats can present a significant problem in attempts to sequence a sample 
using the methods described in WO 89/10977. 
It is an object of the present invention to provide methods and apparatus 
for rapid and accurate identification of nucleotide tandem repeats in DNA 
and RNA sequences from a wide variety of sources. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, a solid support based 
hybridization assay is provided which allows for the systematic and 
reproducible analysis of repeat and tandem repeat oligonucleotide 
sequences of DNA and RNA by hybridization to a reverse dot blot array 
comprising strings of such repeats complementary to those found in 
particular nucleic acid targets (e.g., analyte PCR product). An 
addressable library (i.e., an indexed set) of complementary repeats is 
synthesized on a suitable support. Preferably, the support comprises a low 
fluorescent background support, thereby facilitating the use of 
non-radioisotopic modes of detection (such as fluorescence or 
chemiluminescence); particularly suitable in this regard is an aminated 
polypropylene support or similar material. Pursuant to a preferred 
embodiment of the invention, arrays are provided which permit screening of 
DNA and RNA samples for complete sets of particular types of nucleotide 
repeat sequences (e.g., all nucleotide doublet or triplet repeats).

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, defined repeat and tandem repeat 
arrays for use in screening nucleic acid targets for the presence of 
genetic markers generally known as variable number [of] tandem repeats 
(VNTRs) are synthesized on a suitable support. After hybridization of the 
target materials with the array, the identity of any tandem repeat 
sequence(s) in the target materials may be readily ascertained by 
observing the location(s) at which binding occurs. Pursuant to the present 
invention, probes are reproducibly synthesized on the surface, freely 
accessible to target DNA. Moreover, all hybridizations can be rapidly 
identified under a limited number of stringency conditions. 
The arrays of the present invention comprise a predetermined set of 
oligonucleotides attached to the surface of the solid support. One 
particularly useful class of tandem repeats for arrays in accordance with 
the present invention comprises the complete class of 60 tandem triplet 
repeats (i.e., all possible triplet combinations minus the four 
homopolymer combinations). Another useful class of tandem repeats is the 
complete class of 6 tandem doublet repeats (i.e., the 10 possible doublet 
combinations of the nucleic acids A, C, G and T minus the four homopolymer 
combinations). Of course, those skilled in the art would readily 
appreciate that a wide range of different combinations of repeat elements 
could also be employed in accordance with the present invention. For 
example, repeats of a higher order (i.e., repeats of four or more 
nucleotides) may be useful in some instances. In addition, particular 
subclasses of any complete class of all possible tandem repeats of a given 
size may be suitable for carrying out particular types of screenings. For 
purposes of the present invention, all predetermined sets of tandem 
repeats are contemplated as within the scope of the present invention. 
The sequences forming the array may be directly linked to the support. In 
other embodiments of the arrays of the present invention, the repeat units 
may be attached to the support by non-repetitive sequences of 
oligonucleotides or other molecules serving as spacers or linkers to the 
solid support. In preferred examples of this embodiment, specific leader 
sequences are encoded on either side of the tandem repeat region in an 
array format. Depending upon the relative position of the leader sequence 
a PCR or sequencing primer may be designed. Such primers may then be used 
to aid in the characterization of the length of the tandem repeat and/or 
the specific flanking sequences, respectively. In general, a triplet 
tandem repeat sequence of sufficient length effectively defines two 
additional tandem repeat sequences; for example, a 21 mer complementary to 
(ACG).sub.n also hybridizes to (GAC).sub.n and (CGA).sub.n. By 
systematically including a degenerate set of leader sequences while 
reducing the size of the tandem repeat region, hybridization stringency is 
increased to allow for identification of the combination of leader plus 
the tandem repeat; in the example, selectivity of CCC ACG ACG ACG [SEQ ID 
NO:1] would be observed over, e.g., CCC GAC GAC GAC [SEQ ID NO:2]. FIG. 1 
illustrates a set of instructions for synthesis of suitable leader 
sequences for triplet tandem repeats. Such leader arrays are particularly 
advantageous for the purpose of identifying leader sequences for use as 
PCR primers to tandem repeat regions. 
The method of the present invention is generally applicable to a wide range 
of tandem repeat patterns, including higher order tandem repeats. As by 
definition a tandem repeat consists of at least 2 units of a given 
oligomer (for example, a dimer or 2 mer), then a (2 mer).sub.n wherein 
n=2, 3, 4 . . . would represent a dinucleotide repeat forming a 4 mer, 6 
mer, 8 mer, etc. (e.g., ACAC, ACACAC, ACACACAC, etc.). Similarly, a 
triplet repeat would be defined as a (3 mer).sub.n and a tetramer repeat 
as (4 mer).sub.n, wherein n represents the number of repeats present. 
Contemplated as within the scope of the present invention are all tandem 
repeats of the general formula (Nmer).sub.n wherein N is an integer 
greater than 1 representing the number of nucleotides in the repeat 
pattern and n is an integer representing the number of times the pattern 
is repeated; in general, the product of N and n is in the range of 4 to 
about 100, and preferably 6 to about 60. 
Higher order tandem repeat combinations representing combinations of two or 
more individual tandem repeats are also contemplated as within the scope 
of the present invention; for example, such higher order tandem repeat 
combinations may include two dimer patterns, a dimer and a triplet, two 
triplets, etc. In general terms, such repeat combinations may be described 
as 
EQU (Nmer).sub.n (Mmer).sub.m 
in which N and M are independently selected integers greater than 1 and 
represent the number of nucleotides in the respective repeat pattern and n 
and m are independently selected integers and represent the number of 
times the respective pattern is repeated. In the case of three tandem 
patterns, the structure may be represented as 
EQU (Nmer).sub.n (Mmer).sub.m (Pmer).sub.p 
in which P is defined in the same manner as N and M, and p in the same 
manner as n and m. Moreover, these higher order tandem repeat combinations 
may also be found in a repeat pattern; such a complex higher order tandem 
repeat combination may be described as 
EQU [(Nmer).sub.n (Mmer).sub.m)].sub.x 
or 
EQU [(Nmer).sub.n (Mmer).sub.m (Pmer).sub.p ].sub.x 
in which N, M, P, n, m and p are as previously defined and x is an integer 
which represents the number of times the [(Nmer).sub.n (Mmer).sub.m)] or 
[(Nmer).sub.n (Mmer).sub.m (Pmer).sub.p ] pattern is repeated. 
Contemplated as within the scope of the present invention are those 
combinations wherein x(Nn+Mm) or x(Nn+Mm+Pp) is in the range of 4 to about 
100, preferably in the range of 6 to about 60. In the simplest case 
comprising two repeat patterns and wherein x is 1, N and M are both 2 and 
n and m are both 1; 1.times.2+1.times.2=4. Table 1 illustrates the 
construction of higher order repeats in which n and m are both 2. 
TABLE 1 
______________________________________ 
Tandem 
Repeat (2 mer).sub.n (3 mer).sub.n (4 mer).sub.n (5 mer).sub.n (6 
mer).sub.n (7 mer).sub.n 
______________________________________ 
(2 mer).sub.m 
8 mer 10 mer 12 mer 
14 mer 16 mer 
18 mer 
(3 mer).sub.m 10 mer 12 mer 14 mer 16 mer 18 mer 20 mer 
(4 mer).sub.m 12 mer 14 mer 16 mer 18 mer 20 mer 22 mer 
(5 mer).sub.m 14 mer 16 mer 18 mer 20 mer 22 mer 24 mer 
(6 mer).sub.m 16 mer 18 mer 20 mer 22 mer 24 mer 26 mer 
(7 mer).sub.m 18 mer 20 mer 22 mer 24 mer 26 mer 28 mer 
______________________________________ 
Thus, for example, the 8 mer comprising a (2 mer).sub.n =(AC).sub.2 and a 
(2 mer).sub.m =(AT).sub.2 would have the formula ACACATAT; similarly, the 
12 mer comprising a (3 mer).sub.n =(ACG).sub.2 and a (3 mer).sub.m 
=(ATC).sub.2 would have the formula ACGACGATCATC [SEQ ID NO:3]. A complex 
higher order repeat [(AC).sub.n (AT).sub.m ].sub.x in which n and m are 1 
and x is 2 would have the formula ACATACAT; where x is 3, the formula 
would be ACATACATACAT [SEQ ID NO:4]. Those skilled in the art would 
readily appreciate the variety of simple and higher-order tandem repeat 
patterns falling within the scope of the present invention. 
The methods and apparatus in accordance with the present invention take 
advantage of the fact that under appropriate conditions oligonucleotides 
form base paired duplexes with oligonucleotides which have a complementary 
base sequence. The stability of the duplex is dependent on a number of 
factors, including the length of the oligonucleotides, the base 
composition, and the composition of the solution in which hybridization is 
effected. The effects of base composition on duplex stability may be 
reduced by carrying out the hybridization in particular solutions, for 
example in the presence of high concentrations of tertiary or quaternary 
amines. 
The thermal stability of the duplex is also dependent on the degree of 
sequence similarity between the sequences. By carrying out the 
hybridization at temperatures close to the anticipated Tm's of the type of 
duplexes expected to be formed between the target material(s) and the 
oligonucleotides bound to the array, the rate of formation of mismatched 
duplexes may be substantially reduced. 
The number of repeats in the tandem sequences attached to the array may 
vary over a broad range from the minimum of two necessary to constitute a 
repeat to a maximum on the order of about 50. Of course, the optimum range 
for the number of tandem repeats in any given instance is dependent upon a 
number of factors, including in particular the composition and the length 
of the repeat. In general, the Tm of the complex formed between a given 
sequence in the target material and the complementary sequence in the 
array increases as the length of the sequences increase; however, only 
minor increases in Tm are observed once the sequences have reached a 
length of about 50-60 bases. The sequences on the array contain at least 
four bases (the minimum for a repeat of a doublet pattern). It is 
generally preferred that the sequences on the array comprise at least 
about 6 bases, more preferably at least about 10 bases, and most 
preferably on the order of about 15 to about 60 bases. As previously 
noted, there is little practical advantage in using sequences 
substantially longer than about 60 bases; nonetheless, extended sequences 
of up to about 100 bases in length (corresponding to, e.g., 50 repeats of 
a doublet sequence) and longer are also contemplated as within the scope 
of the present invention. 
In addition, in accordance with preferred embodiments of the invention the 
length of each sequence employed in the array may be selected to as to 
optimize binding of target materials to the array. For any given tandem 
repeat sequence, an optimum length for use with any particular target 
material under specified screening conditions may be determined 
empirically. Thus, the length for each individual element of the set of 
tandem repeats comprising the array may be optimized for the screening of 
particular target materials under specific conditions (e.g., at a given 
temperature). 
A wide variety of array formats may be employed in accordance with the 
present invention. One particularly useful format is a linear array of 
oligonucleotide bands, generally referred to in the art as a dipstick. 
Another suitable format comprises a two-dimensional pattern of discrete 
cells (e.g., 4096 squares in a 64 by 64 array). Of course, as would be 
readily appreciated by those skilled in the art, other array formats 
(e.g., circular) would be equally suitable for use in accordance with the 
present invention. While arrays may be prepared on a variety of materials 
including glass plates, it is presently preferred to use an organic 
polymer medium. As used herein, the term "organic polymer" is intended to 
mean a support material which is most preferably chemically inert under 
conditions appropriate for biopolymer synthesis and which comprises a 
backbone comprising various elemental substituents including, but not 
limited to, hydrogen, carbon, oxygen, fluorine, chlorine, bromine, sulfur 
and nitrogen. Representative polymers include, but are not limited to, the 
following: polypropylene, polyethylene, polybutylene, polyisobutylene, 
polybutadiene, polyisoprene, polyvinylpyrrolidone, 
polytetrafluoroethylene, polyvinylidene difluoride, 
polyfluoroethylene-propylene, polyethylene-vinyl alcohol, 
polymethylpentene, polychlorotrifluoroethylene, polysulfones, and blends 
and copolymers thereof. As used herein, the term "medium" is intended to 
mean the physical structural shape of the polymer. Thus, medium can be 
generally defined as polymer films (i.e., polymers having a substantially 
non-porous surface); polymer membranes (i.e., polymers having a porous 
surface); polymer filaments (e.g., mesh and fabrics); polymer beads; 
polymer foams; polymer frits; and polymer threads. Preferably, the polymer 
medium is a thread, membrane or film; most preferably, the polymer medium 
is a film. An exemplary organic polymer medium is a polypropylene sheet 
having a thickness on the order of about 1 mil (0.001 inch), although the 
thickness of the film is not critical and may be varied over a fairly 
broad range. Particularly preferred for preparation of arrays at this time 
are biaxially oriented polypropylene (BOPP) films; in addition to their 
durability, BOPP films exhibit a low background fluorescence. 
The array formats of the present invention may be included in a variety of 
different types of device. As used herein, the term "device" is intended 
to mean any device to which the solid support can be affixed, such as 
microtiter plates, test tubes, inorganic sheets, dipsticks, etc. For 
example, when the solid support is a polypropylene thread, one or more 
polypropylene threads can be affixed to a plastic dipstick-type device; 
polypropylene membranes can be affixed to glass slides. The particular 
device is, in and of itself, unimportant. All that is necessary is that 
the solid support can be affixed thereto without affecting the functional 
behavior of the solid support or any biopolymer adsorbed thereon, and that 
the device is stable to any materials into which the device is introduced 
(e.g., clinical samples, etc.). 
The arrays of the present invention may be prepared by a variety of 
approaches which are known to those working in the field. Pursuant to one 
type of approach, the complete sequences are synthesized separately and 
then attached to a solid support. However, it is presently considered 
particularly advantageous to synthesize the sequences directly onto the 
support to provide the desired array. Suitable methods for covalently 
coupling oligonucleotides to a solid support and for directly synthesizing 
the oligonucleotides onto the support would be readily apparent to those 
working in the field; a summary of suitable methods may be found in, e.g., 
Matson, R. S. et al., Analytical Biochem. 217, 306-310 (1994), hereby 
incorporated by reference. Advantageously, the oligonucleotides are 
synthesized onto the support using conventional chemical techniques as 
heretofore employed for preparing oligonucleotides on solid supports 
comprising, e.g., controlled pore size glass (CPG), as described for 
example in PCT applications WO 85/01051 and WO 89/10977, or polypropylene, 
as described in copending U.S. patent application Ser. No. 07/091,100, 
which has been assigned to the assignee of the present application and is 
herein incorporated by reference. Pursuant to one preferred approach, a 
polypropylene support (for example, a biaxially-oriented polypropylene) is 
first surface aminated by exposure to an ammonia plasma generated by 
radiofrequency plasma discharge. The reaction of a 
phosphoramidite-activated nucleotide with the aminated membrane followed 
by oxidation with, e.g., iodine provides a base stable amidate bond to the 
support. 
As described in U.S. patent application Ser. No. 08/144,954 filed Oct. 28, 
1993, which has been commonly assigned to the assignee of the present 
invention and is incorporated by reference herein, a suitable array may 
advantageously be produced using automated means to synthesize 
oligonucleotides in the cells of the array by laying down the precursors 
for the four bases in a predetermined pattern. Briefly, a multiple-channel 
automated chemical delivery system is employed to create oligonucleotide 
probe populations in parallel rows (corresponding in number to the number 
of channels in the delivery system) across the substrate. Following 
completion of oligonucleotide synthesis in a first (10 ) direction, the 
substrate may then be rotated by 90.degree. to permit synthesis to proceed 
within a second (2.degree.) set of rows that are now perpendicular to the 
first set. This process creates a multiple-channel array whose 
intersection generates a plurality of discrete cells. Table 2 describes an 
exemplary vertical array of 64 oligonucleotides consisting of 60 triplet 
tandem repeat sequences (21 mers) and dinucleotide tandem repeat sequences 
(20 mers). 
For the example of the preferred array comprising specific leader sequences 
as described in FIG. 1 (and as more fully described in the above-noted 
U.S. patent application Ser. No. 08/144,954), all of the degenerate 
triplet repeats (n=64, including homopolymers) are synthesized in a first 
direction (1.degree. synthesis) in the 64 channels in Cycles 1-3 as 3' 
Leader Sequences (LLL). For example, lane 1 is AAA, lane 2 AAC, lane 5 ACA 
and lane 64 TTT. Then, the film is rotated 90.degree. to perform synthesis 
in the second direction (20 synthesis or cross-synthesis) in Cycles 4-15. 
All 64 triplet tandem repeat sequences of (NNN).sub.4 are then produced. 
The 2-dimensional array created thereby is the product of a bidirectional 
synthesis and comprises 4096 discrete cells containing 15 mer 
oligonucleotide products (LLL)(NNN).sub.4, in which the Leader Sequence is 
placed at the 3'-end of each completed oligonucleotide. Thus, the 
following sequences would be found at the indicated cell positions: 
Cell 1,1' (AAA)(AAA).sub.4 
[SEQ ID NO:5] 
Cell 1,2' (AAA)(AAC).sub.4 [SEQ ID NO:6] 
Cell 5,1' (ACA)(AAA).sub.4 [SEQ ID NO:7] 
Cell 64,1' (TTT)(AAA).sub.4 [SEQ ID NO:8] 
Cell 64,64' (TTT)(TTT).sub.4 [SEQ ID NO:9] 
This type of array comprising leader sequences (at either the 5' or 3' end) 
is particularly preferred in accordance with the present invention. 
In order to accommodate a suitably large array, the pixel size should be as 
small as possible. Cells having a width on the order of about 10 .mu.m to 
about 1 mm would be particularly suitable. In one preferred embodiment of 
the invention, arrays with a pixel width of about 500 .mu.m are prepared 
on biaxially-oriented polypropylene. 
Pursuant to the present invention, there are also provided methods for 
screening DNA and RNA samples comprising labelling the samples to form 
labelled material, applying the labelled material under suitable 
hybridization conditions to an array as described herein, and observing 
the location of the label on the surface associated with particular 
members of the set of oligonucleotides. Identification of the cell(s) in 
which binding occurs permits a rapid and accurate identification of any 
nucleotide repeats present in the sample from which the probes are 
derived. 
In a hybridization reaction in accordance with the present invention, the 
array is explored by the labelled target material in essentially the same 
manner as a labelled probe is employed to screen, e.g., a DNA library 
containing a gene complementary to the probe. The target material may 
suitably comprise labelled sequences amplified from genomic DNA by the 
polymerase chain reaction (PCR), a mRNA population, or a partial or 
complete set of oligonucleotides from one or more chromosomes or an entire 
genome. To prepare the target material, the sample may be degraded to form 
fragments; where appropriate, the degraded material may then be sorted 
(for example, by electrophoresis on a sequencing gel) to provide a set of 
oligomers having a specific length. 
The target material is then labelled to facilitate detection of duplex 
formation Suitably, conventional methods for end-labelling of oligomers 
are employed. Both radioactive and fluorescent labelling methods would be 
suitable for use in accordance with the present invention. 
Commonly-employed techniques routinely permit the introduction of label 
into a significant fraction of the target materials. Using conventional 
methods for labelling oligomers with .sup.32 P, for example, the 
radioactive yield of any individual oligomer even from a total human 
genome could be more than 10.sup.4 dpm/mg of total DNA. For detection, 
only a small fraction of the labelled material would be necessary for 
hybridization to a pixel of a size within the preferred range specified 
herein. Hybridization conditions for a given combination of array and 
target material can routinely be optimized in an empirical manner to be 
close to the Tm of the expected duplexes, thereby maximizing the 
discriminating power of the method. Autoradiography (in particular, with 
32P) may cause image degradation which may be a limiting factor 
determining resolution; the limit for silver halide films is about 25 
.mu.m. Accordingly, the use of fluorescent probes (in particular, in 
conjunction with an array prepared on a low-fluorescence solid support) is 
presently preferred. In view of the low background fluorescence of the 
preferred biaxially-oriented polypropylene substrate, fluorescence-based 
labelling techniques may advantageously be employed with arrays on such a 
substrate. With either type of labeled target material, the substantial 
excess in bound oligonucleotides of the array makes it possible to operate 
at conditions close to equilibrium with most types of target materials 
contemplated herein. 
As would be readily understood by those skilled in the art, the chosen 
conditions of hybridization must be such as to permit discrimination 
between exactly matched and mismatched oligonucleotides. Hybridization 
conditions may be initially chosen to correspond to those known to be 
suitable in standard procedures for hybridization to filters and then 
optimized for use with the arrays of the present invention; moreover, 
conditions suitable for hybridization of one type of target material would 
appropriately be adjusted for use with other target materials for the same 
array. In particular, it is appropriate to control temperature closely 
(preferably, to better than about .+-.1.degree. C.) to substantially 
eliminate formation of duplexes between sequences other than identical 
sequences. Particularly when the length of the oligonucleotide in the 
target materials is small, it is necessary to be able to distinguish 
between slight differences in the rate and/or extent of hybridization. 
A variety of heretofore known hybridization solvents may suitably be 
employed, the choice of solvent for particular hybridizations being 
dependent on a number of considerations. For example, G:C base pairs are 
more stable than A:T base pairs in 1 M NaCl; thus, the Tm of 
double-stranded oligonucleotides with a high G+C content will be higher 
than corresponding oligonucleotides with a high A+T content. These effects 
are of course particularly pronounced in sequences comprising tandem 
nucleotide repeats. In order to compensate for this discrepancy, a variety 
of approaches may be employed. For example, the amount of oligonucleotide 
applied to the surface of the support may be varied in dependence on the 
nucleotide composition of the bound oligomer. Further, computer means 
employed to analyze data from hybridization experiments may be programmed 
to make compensations for variations in nucleotide compositions. Another 
expedient (which may be employed instead of or in addition to those 
already mentioned) is the use of a chaotropic hybridization solvent, such 
as a ternary or quaternary amine. In this regard, tetramethylammonium 
chloride (TMACl) at concentrations in the range of about 2 M to about 5.5 
M is particularly suitable; at TMACl concentrations around 3.5 to 4 M, the 
Tm dependence on nucleotide composition is substantially reduced. In 
addition, the choice of hybridization salt has a major effect on overall 
hybridization yield; for example, TMACl at concentrations up to 5 M can 
increase the overall hybridization yield by a factor of up to 30 or more 
(depending to some extent on the nucleotide composition) compared to 1 M 
NaCl. Finally, as previously noted, the length of the oligonucleotides 
attached to the array may be varied so as to optimize hybridization under 
the particular conditions employed. As previously noted, it would be a 
routine matter for those working in the field to optimize hybridization 
conditions for any given combination of target materials and array. 
Hybridization is typically carried out with a very large excess of the 
bound oligonucleotides over what is found in the target. In preferred 
embodiments of the invention, it is possible in some cases to distinguish 
between hybridization involving single and multiple occurrences of the 
target sequence, as yield is proportional to concentration at all stages 
in the reaction. 
In accordance with another embodiment of the present invention, an array as 
described herein may be employed to selectively isolate and size variable 
number of tandem repeats (VNTRs). This is accomplished by preparing a 
sample comprising VNTRs in a manner known per se [see, e.g., McBride & 
O'Neill, supra], capturing the VNTRs on the array, selectively 
dissociating the hybrid and eluting the VNTR from the support. A selected 
lane from the array may be cut out of the support, the ssDNA eluted 
therefrom, the number of copies thereof increased by PCR amplification and 
size analysis conducted by a conventional technique (e.g., gel 
electrophoresis against DNA size markers). The presence of large molecular 
weight strands would indicate an increase in mutational frequency (i.e., 
higher orders of tandem repeat regions). 
The invention may be better understood with reference to the accompanying 
example that is intended for purposes of illustration only and should not 
be construed as in any sense limiting the scope of the present invention 
as defined in the claims appended hereto. 
EXAMPLE 
Oligonucleotides were synthesized directly from monomers onto a 
6.6.times.6.6 cm sheet of aminated polypropylene substrate using standard 
CED-phosphoramidite chemistries. A specially designed 64 channel chemical 
delivery system (Southern Array Maker.TM., Beckman Instruments) as 
described in co-pending U.S. patent application Ser. No. 08/144,954 was 
utilized to prepare the discrete oligonucleotide sequences in parallel 
rows across the polypropylene substrate. Polypropylene film was surface 
aminated by a radiofrequency discharge into ammonia gas as described in 
Matson et al., supra. The plasma-aminated film was then placed in the 
synthesizer. Standard phosphoramidite chemistry was performed in each of 
the 64 channels to create 64 different oligonucleotide sequences on the 
film. The substrate was then cut into 0.5 cm widths perpendicular to the 
oligonucleotide rows to produce a panel of 64 tandem repeat sequences. For 
the present study 60 trinucleotide (21 mers) and 4 dinucleotide tandem (20 
mers) repeat sequences were arrayed on a vertical order shown in Table 2. 
The arrayed trinucleotide repeat set represents all triplet frames except 
(AAA).sub.7 [SEQ ID NO:10], (CCC).sub.7 [SEQ ID NO:11], (GGG).sub.7 [SEQ 
ID NO:12], and (TTT).sub.7 [SEQ ID NO:13] in 3'.fwdarw.5' direction as 
well as minus strand orientation. 
In order to confirm that all sequences were fully represented on the panel 
a series of complementary probes were prepared that would verify each 
sequence by row position on the strip. As each triplet repeat (n) of a 
sufficient length in fact reads the n-1 and n-2 frames as well, only 20 
probe sequences were required to identify the 60 triplet repeats on the 
panel. For example, row 2 containing the sequence (AAC).sub.7 [SEQ ID 
NO:14] also represents (ACA).sub.6 [SEQ ID NO:15] and (CAA).sub.6 [SEQ ID 
NO:16] (sequences found at row 5 and row 17, respectively); thus, a single 
tandem repeat probe which has been labeled will identify the sequences at 
row positions 2, 5 and 17. Four additional probe sequences were required 
to identify the 4 doublet tandem repeats synthesized on the strip. It is 
also possible to combine the sequences of non-complementary probes (i.e., 
those that will not self-hybridize or form hairpin loops) to reduce the 
total number of probes necessary to read all row positions. The following 
.sup.32 P 5'-end labeled probes were prepared and used to identify all row 
positions 1-64 (listed in Table 2) on the panel: 
(TGC).sub.6 ; [SEQ ID NO:17] 
(TGG).sub.4 (TG).sub.6 (TTA).sub.4 ; [SEQ ID NO:18] 
(TGG).sub.4 (TGA).sub.4 ; [SEQ ID NO:19] 
(TCG).sub.6 ; [SEQ ID NO:20] 
(GGC).sub.5 ; [SEQ ID NO:21] 
(CG).sub.4 (GAC).sub.4 ; [SEQ ID NO:22] 
(TCA).sub.4 (TAA).sub.4 (TAC).sub.4 ; [SEQ ID NO:23] 
(CCA).sub.4 (CAA).sub.4 ; [SEQ ID NO:24] 
(TTC).sub.4 (TC).sub.6 (TCC).sub.4 ; and [SEQ ID NO:25] 
(GAA).sub.4 (GA).sub.6 (GGA).sub.4 ; [SEQ ID NO:26] 
(GCC).sub.4 (GCA).sub.4 [SEQ ID NO:27]. 
A number of different test DNAs were employed. This included a 
5'-(CAG).sub.7 -3' oligonucleotide [SEQ ID NO:28] and 200 bp PCR fragments 
containing a trinucleotide short tandem repeat of (CTG).sub.11 [SEQ ID 
NO:29] [Fu, Y. H., et al, Science 255, 1256-1258 (1992)] generated from 
human genomic DNA of a wild type individual. An 800 bp PCR fragment 
containing a (CAG).sub.10 [SEQ ID NO:30] repeat and a 3.0 kb PCR fragment 
containing a tandemly combined (GCA).sub.8 +(GCG).sub.4 repeat [SEQ ID 
NO:31] were generated from cDNA clones G13 and A12, respectively, recently 
isolated in a new cDNA identification system [Lee, C. C., et al., Am. J. 
Hum. Genet. 53 (Suppl.), 1321 (1993)]. DNA samples of the cosmid MDY2 [Fu, 
Y. H., et al, Science 255, 1256-1258 (1992)] containing the entire 
myotonin protein kinase gene and cosmid 22.3 containing the FMR-1 gene 
[Verkerk, A. J. M. H., et al., Cell 65, 905-914 (1991)] have also been 
utilized as target materials to evaluate the test strips. 
STRs were identified using the GCG sequence analysis software package 
(Genetics Computer Group, Inc. 1991). 11,613 bp of cosmid MDY2 (GenBank 
accession L00727) and 61,612 bp of a contiguous sequence containing the 
complete 34,977 bp sequence of cosmid 22.3 were searched for STRs. 
TABLE 2 
__________________________________________________________________________ 
Vertical array of 64 oligonucleotides consisting of 60 triplet tandem 
repeat sequences (2lmers) and dinucleotide tandem repeat sequences 
(2Omers) on a polypropylene substrate 
Seq Seq 
Oligonucteotides ID 
Oligonucleotides ID 
# 3' - - - &gt; 5' No. # 3' - - - &gt; 5' No. 
__________________________________________________________________________ 
1 AC AC AC AC AC AC AC AC AC AC 
32 33 
GAA GAA GAA GAA GAA GAA GAA 
62 
2 AAC AAC AAC AAC AAC AAC AAC 14 34 GAC GAC GAC GAC GAC GAC GAC 63 
3 AAG AAG AAG AAG AAG AAG AAG 33 35 GAG GAG GAG GAG GAG GAG GAG 64 
4 AAT AAT AAT AAT AAT AAT AAT 34 36 GAT GAT GAT GAT GAT GAT GAT 65 
5 ACA ACA ACA ACA ACA ACA ACA 35 37 GCA GCA GCA GCA GCA GCA GCA 66 
6 ACC ACC ACC ACC ACC ACC ACC 36 38 GCC GCC GCC GCC GCC GCC GCC 67 
7 ACG ACG ACG ACG ACG ACG ACG 37 39 GCG GCG GCG GCG GCG GCG GCG 68 
8 ACT ACT ACT ACT ACT ACT ACT 38 40 GCT GCT GCT GCT GCT GCT GCT 69 
9 AGA AGA AGA AGA AGA AGA AGA 39 41 GGA GGA GGA GGA GGA GGA GGA 70 
10 AGC AGC AGC AGC AGC AGC AGC 40 42 GGC GGC GGC GGC GGC GGC GGC 71 
11 AGG AGG AGG AGG AGG AGG AGG 41 43 AG AG AG AG AG AG AG AG AG AG 72 
12 AGT AGT AGT AGT AGT AGT AGT 42 44 GGT GGT GGT GGT GGT GGT GGT 73 
13 ATA ATA ATA ATA ATA ATA ATA 43 45 GTA GTA GTA GTA GTA GTA GTA 74 
14 ATC ATC ATC ATC ATC ATC ATC 44 46 GTC GTC GTC GTC GTC GTC GTC 75 
15 ATG ATG ATG ATG ATG ATG ATG 45 47 GTG GTG GTG GTG GTG GTG GTG 76 
16 ATT ATT ATT ATT ATT ATT ATT 46 48 GTT GTT GTT GTT GTT GTT GTT 77 
17 CAA CAA CAA CAA CAA CAA CAA 47 49 TAA TAA TAA TAA TAA TAA TAA 78 
18 CAC CAC CAC CAC CAC CAC CAC 48 50 TAC TAC TAC TAC TAC TAC TAC 79 
19 CAG CAG CAG CAG CAG CAG CAG 28 51 TAG TAG TAG TAG TAG TAG TAG 80 
20 CAT CAT CAT CAT CAT CAT CAT 49 52 TAT TAT TAT TAT TAT TAT TAT 81 
21 CCA CCA CCA CCA CCA CCA CCA 50 53 TCA TCA TCA TCA TCA TCA TCA 82 
22 CG CG CG CG CG CG CG CG CG CG 51 54 TCC TCC TCC TCC TCC TCC TCC 83 
23 CCG CCG CCG CCG CCG CCG CCG 52 55 TCG TCG TCG TCG TCG TCG TCG 84 
24 CCT CCT CCT CCT CCT CCT CCT 53 56 TCT TCT TCT TCT TCT TCT TCT 85 
25 CGA CGA CGA CGA CGA CGA CGA 54 57 TGA TGA TGA TGA TGA TGA TGA 86 
26 CGC CGC CGC CGC CGC CGC CGC 55 58 TGC TGC TGC TGC TGC TGC TGC 87 
27 CGG CGG CGG CGG CGG CGG CGG 56 59 TGG TGG TGG TGG TGG TGG TGG 88 
28 CGT CGT CGT CGT CGT CGT CGT 57 60 TGT TGT TGT TGT TGT TGT TGT 89 
29 CTA CTA CTA CTA CTA CTA CTA 58 61 TTA TTA TTA TTA TTA TTA TTA 90 
30 CTC CTC CTC CTC CTC CTC CTC 59 62 TTC TTC TTC TTC TTC TTC TTC 91 
31 CTG CTG CTG CTG CTG CTG CTG 60 63 TTG TTG TTG TTG TTG TTG TTG 92 
32 CTT CTT CTT CTT CTT CTT CTT 61 64 CT CT CT CT CT CT CT CT CT CT 
__________________________________________________________________________ 
93 
Oligonucleotide probes were end-labelled with .sup.32 P-Gamma-dATP under 
standard conditions [Sambrook, J., et al., Molecular Cloning: A Laboratory 
Manual, Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring 
Harbor (1989)]. Double stranded DNA was radiolabelled with .sup.3 
P-Alpha-dCTP using a Pharmacia random priming labelling kit according to 
the manufacturer's instructions. To improve the labelling reaction cosmid 
DNA was digested with EcoRI prior to radiolabelling. The test strips were 
hybridized without prehybridization in plastic bags containing 6.times. 
SSCP (saline, sodium citrate-phosphate buffer) and 0.01% sodium dodecyl 
sulfate (SDS) for 16 hrs. Only target-specific binding to the 
polypropylene membranes was observed, eliminating the need for a 
prehybridization step. The specific activity of the radiolabelled probes 
was adjusted to 5.times.10.sup.6 cpm/ml hybridization solution. After 
hybridization the test strips were washed in 2.times.SSCP, 0.01% SDS for 
20 min. A variety of hybridization and wash temperatures was employed, as 
hereinafter described. Autoradiograms were developed after 5 minutes to 6 
hours exposure at -70.degree. C. The resulting signals on the test strips 
were evaluated visually. 
As expected, the synthetic (CAG).sub.7 [SEQ ID NO:28] oligonucleotide probe 
hybridized specifically at 60.degree. C. to three rows of the array. These 
corresponded to the oligonucleotide repeats (CGT).sub.7 [SEQ ID NO:57], 
(GTC).sub.7 [SEQ ID NO:69], and (TCG).sub.7 [SEQ ID NO:84], respectively. 
Using double-stranded DNA of 200 bp and 800 bp containing a (CTG).sub.11 
[SEQ ID NO:29] and a (CAG).sub.10 [SEQ ID NO:30] repeat resulted in a 
pattern of 6 bands corresponding to (ACG).sub.7 [SEQ ID NO:37], 
(CGA).sub.7 [SEQ ID NO:54], (CGT).sub.7 [SEQ ID NO:57], (GAC).sub.7 [SEQ 
ID NO:63], (GTC).sub.7 [SEQ ID NO:75], and (TCG).sub.7 [SEQ ID 
NO:84]--i.e., the sense and antisense orientations. Differences in the 
signal intensity were observed between the various triplet-representing 
lanes. 
Using the 3 kb PCR fragment containing a combined repeat (GCA).sub.8 
+(GCG).sub.4 [SEQ ID NO:31] to probe the test strips resulted in a 
complete set of six oligonucleotides--(ACG).sub.7 [SEQ ID NO:37], 
(CGA).sub.7 [SEQ ID NO:54], (CGT).sub.7 [SEQ ID NO:57], (GAC).sub.7 [SEQ 
ID NO:63], (GTC).sub.7 [SEQ ID NO:75], and (TCG).sub.7 [SEQ ID NO:84]. 
This set represents the six different frame shifts for the (GCA).sub.8 
[SEQ ID NO:94] repeat. Additionally, the signals found with (CCG).sub.7 
[SEQ ID NO:52], (CGC).sub.7 [SEQ ID NO:55], and (GCC).sub.7 [SEQ ID 
NO:67]were evident for the 3'.fwdarw.5' directed frame of the (GCG).sub.4 
[SEQ ID NO:95] repeat. No signal was detected under these conditions for 
the reversed direction indicated by (GGC).sub.7 [SEQ ID NO:71], 
(GCG).sub.7 [SEQ ID NO:68], and (CGC).sub.7 [SEQ ID NO:55]. 
Using cosmid MDY2 with an insert size of 31 kb as a probe, a band pattern 
was observed indicative of the presence of (CAG).sub.n, (GCC).sub.n, and 
(CCT).sub.n repeats, respectively. For the (CCT).sub.n, only one direction 
of the oligonucleotide frame as represented by (CCT).sub.7 [SEQ ID NO:53], 
(CTC).sub.7 [SEQ ID NO:59], and (TCC).sub.7 [SEQ ID NO: 83] was found to 
hybridize. A search of 11,613 nt available sequence information (GenBank 
accession L00727) of cosmid MDY2 revealed the presence of all types of 
triplet repeats identified by the test strip (Table 3). The repeated 
triplet numbers vary from 3 for the CCT and GCG type repeats to 11 for the 
CTG repeat. 
TABLE 3 
______________________________________ 
Position 
(Nucleotide #) STR 
______________________________________ 
809-817 (CCT).sub.3 
8,172-8,180 (CCT).sub.3 
9,093-9,101 (GGA).sub.3 
10,364-10,372 (GCC).sub.3 
10,677-10,709 (CTG).sub.11 
[SEQ ID NO:29] 
______________________________________ 
The influence of temperature on the STR detection was evaluated by 
hybridizing cosmid 22.3 at 40.degree. C., 50.degree. C., and 60.degree. C. 
to the test strips. The strips were then washed at the temperatures used 
for the respective hybridizations. At 40.degree. C., a band pattern was 
obtained indicative of (CA).sub.n, (ACC).sub.n, (CCT).sub.n and 
(GCC).sub.n type repeats, respectively (FIG. 2A); for the triplet repeats, 
only one set of signals representing one direction of the oligonucleotide 
frame was observed. The pattern at 50.degree. C. was also specific for 
(CA).sub.n, (ACC).sub.n, and (GCC).sub.n type repeats (FIG. 2B); however, 
unlike the pattern at 40.degree. C. the signals representing a (TCC).sub.n 
type repeat disappeared and additional bands indicative of a (CGT).sub.n 
type repeat occurred; again, for the trinucleotide repeats only one set of 
signals representing one direction of the respective oligonucleotide frame 
was found. At 60.degree. C. only signals representing (CA).sub.n and 
(CCG).sub.n type repeats persisted (FIG. 2C). Under these hybridization 
conditions a full set of the expected 6 bands evident for a (CCG).sub.n 
type repeat was observed. 
All types of STRs indicated by the test strips were found to be present in 
34,977 bp sequence available from cosmid 22.3 (FIG. 3). They range from a 
single repeat unit of (CCT).sub.3 and (AGC).sub.3 up to 18 repeat units 
for a (TG)-dinucleotide repeat. There was no unspecific hybridization 
signal observed. AT-rich repeats also occurring in the sequence in three 
or less repeat units were not detected by the test strips under the 
hybridization conditions used. 
The array was designed to represent trinucleotide repeats by all three 
possible frames in 3'.fwdarw.5' direction, as well as in the reverse 
direction. Thus, using single stranded DNA a complementary sequence to a 
given trinucleotide repeat should result in three signals on the test 
strips; using double stranded DNA six respective bands for a given repeat 
should occur. For the four dinucleotide repeats only one frame was used 
for each type. Using this reverse blotting system, the obtained band 
pattern provided qualitatively the precise identification of previously 
known STRs in DNA samples of various complexities between 21 bp-34,977 bp. 
Moreover, there was no random or cross hybridization to unspecific 
sequences observed. Based on the Tm and size of the STRs as well as 
possible influences by flanking sequences, varying the hybridization 
stringency can enhance the specificity. 
From the foregoing description, one skilled in the art can readily 
ascertain the essential characteristics of the invention and, without 
departing from the spirit and scope thereof, can adapt the invention to 
various usages and conditions. Changes in form and substitution of 
equivalents are contemplated as circumstances may suggest or render 
expedient, and although specific terms have been employed herein, they are 
intended in a descriptive sense and not for purposes of limitation. 
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- - GCCGCCGCCG CCGCAGCAGC AGCA - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO:28: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
- - CAGCAGCAGC AGCAGCAGCA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:29: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
- - CTGCTGCTGC TGCTGCTGCT GCTGCTGCTG CTG - # - # 
33 
- - - - (2) INFORMATION FOR SEQ ID NO:30: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
- - CAGCAGCAGC AGCAGCAGCA GCAGCAGCAG - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO:31: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
- - GCAGCAGCAG CAGCAGCAGC AGCAGCGGCG GCGGCG - # - 
# 36 
- - - - (2) INFORMATION FOR SEQ ID NO:32: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
- - ACACACACAC ACACACACAC - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO:33: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
- - AAGAAGAAGA AGAAGAAGAA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:34: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
- - AATAATAATA ATAATAATAA T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:35: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
- - ACAACAACAA CAACAACAAC A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:36: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
- - ACCACCACCA CCACCACCAC C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:37: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
- - ACGACGACGA CGACGACGAC G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:38: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
- - ACTACTACTA CTACTACTAC T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:39: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
- - AGAAGAAGAA GAAGAAGAAG A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:40: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
- - AGCAGCAGCA GCAGCAGCAG C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:41: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
- - AGGAGGAGGA GGAGGAGGAG G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:42: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
- - AGTAGTAGTA GTAGTAGTAG T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:43: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
- - ATAATAATAA TAATAATAAT A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:44: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
- - ATCATCATCA TCATCATCAT C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:45: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
- - ATGATGATGA TGATGATGAT G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:46: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
- - ATTATTATTA TTATTATTAT T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:47: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
- - CAACAACAAC AACAACAACA A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:48: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
- - CACCACCACC ACCACCACCA C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:49: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
- - CATCATCATC ATCATCATCA T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:50: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
- - CCACCACCAC CACCACCACC A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:51: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
- - CGCGCGCGCG CGCGCGCGCG - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO:52: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 
- - CCGCCGCCGC CGCCGCCGCC G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:53: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: 
- - CCTCCTCCTC CTCCTCCTCC T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:54: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: 
- - CGACGACGAC GACGACGACG A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:55: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: 
- - CGCCGCCGCC GCCGCCGCCG C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:56: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: 
- - CGGCGGCGGC GGCGGCGGCG G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:57: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: 
- - CGTCGTCGTC GTCGTCGTCG T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:58: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: 
- - CTACTACTAC TACTACTACT A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:59: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: 
- - CTCCTCCTCC TCCTCCTCCT C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:60: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: 
- - CTGCTGCTGC TGCTGCTGCT G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:61: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 
- - CTTCTTCTTC TTCTTCTTCT T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:62: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: 
- - GAAGAAGAAG AAGAAGAAGA A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:63: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: 
- - GACGACGACG ACGACGACGA C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:64: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: 
- - GAGGAGGAGG AGGAGGAGGA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:65: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: 
- - GATGATGATG ATGATGATGA T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:66: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: 
- - GCAGCAGCAG CAGCAGCAGC A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:67: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: 
- - GCCGCCGCCG CCGCCGCCGC C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:68: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 
- - GCGGCGGCGG CGGCGGCGGC G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:69: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 
- - GCTGCTGCTG CTGCTGCTGC T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:70: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: 
- - GGAGGAGGAG GAGGAGGAGG A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:71: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 
- - GGCGGCGGCG GCGGCGGCGG C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:72: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: 
- - AGAGAGAGAG AGAGAGAGAG - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO:73: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: 
- - GGTGGTGGTG GTGGTGGTGG T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:74: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: 
- - GTAGTAGTAG TAGTAGTAGT A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:75: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: 
- - GTCGTCGTCG TCGTCGTCGT C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:76: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: 
- - GTGGTGGTGG TGGTGGTGGT G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:77: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: 
- - GTTGTTGTTG TTGTTGTTGT T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:78: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: 
- - TAATAATAAT AATAATAATA A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:79: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: 
- - TACTACTACT ACTACTACTA C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:80: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: 
- - TAGTAGTAGT AGTAGTAGTA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:81: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: 
- - TATTATTATT ATTATTATTA T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:82: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: 
- - TCATCATCAT CATCATCATC A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:83: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: 
- - TCCTCCTCCT CCTCCTCCTC C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:84: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: 
- - TCGTCGTCGT CGTCGTCGTC G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:85: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: 
- - TCTTCTTCTT CTTCTTCTTC T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:86: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: 
- - TGATGATGAT GATGATGATG A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:87: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: 
- - TGCTGCTGCT GCTGCTGCTG C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:88: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: 
- - TGGTGGTGGT GGTGGTGGTG G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:89: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:89: 
- - TGTTGTTGTT GTTGTTGTTG T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:90: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: 
- - TTATTATTAT TATTATTATT A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:91: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: 
- - TTCTTCTTCT TCTTCTTCTT C - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:92: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: 
- - TTGTTGTTGT TGTTGTTGTT G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:93: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: 
- - CTCTCTCTCT CTCTCTCTCT - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO:94: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: 
- - GCAGCAGCAG CAGCAGCAGC AGCA - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO:95: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other nucleic acid 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95: 
- - GCGGCGGCGG CG - # - # 
- # 12 
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