Methods and compositions for full-length cDNA Cloning using a template-switching oligonucleotide

The present invention pertains to methods for the synthesis and cloning of full-length cDNA, or cDNA fragments, that correspond to the complete sequence of 5'-ends of mRNA molecules. The method of the present invention comprises contacting RNA with a cDNA synthesis primer which can anneal to RNA, a suitable enzyme which possesses reverse transcriptase activity, and a template switching oligonucleotide under conditions sufficient to permit the template-dependent extension of the primer to generate an mRNA-cDNA hybrid. The template switching oligonucleotide hybridizes to the CAP site at the 5'-end of the RNA molecule and serves as a short, extended template for CAP-dependent extension of the 3'-end of the ss cDNA that is complementary to the template switching oligonucleotide. The resulting full-length ss cDNA includes the complete 5'-end of the RNA molecule as well as the sequence complementary to the template switching oligonucleotide, which can then serve as a universal priming site in subsequent amplification of the cDNA. The subject invention also pertains to the template switching oligonucleotides that can be used according to the subject method. Kits containing the template switching oligonucleotide are also included within the scope of the present invention.

FIELD OF THE INVENTION 
The present invention relates to improved technology for selectively 
synthesizing full-length cDNA having complete sequence information of 
full-length mRNA. 
BACKGROUND OF THE INVENTION 
A basic technology in the field of molecular biology is the conversion of 
poly(A)+RNA (mRNA) to double-stranded (ds) complementary DNA (cDNA), which 
then can be inserted into a cloning vector for generating a cDNA library 
or expression in an appropriate host cell. Advances in cDNA library 
construction technology have made possible the discovery and production of 
a wide range of biologically important proteins. 
Several procedures for generating cDNA libraries which have been used 
during the last 15 years are comprehensively reviewed in Wu, ed. Methods 
in Enzymology (1987), vol. 152. For the most part, cDNA library 
construction technologies use poly(A)+RNA as a starting material. The 
intact poly(A)+RNA is characterized by a polyadenylated "tail" at its 3' 
end and a characteristic "CAP structure or cap site" at the 5' end. A 
critical requirement for cDNA library construction is to completely copy 
poly(A)+RNA to full-length cDNA and retain the complete sequence 
information on the structure of the protein encoded by mRNA. 
One generalized and commonly used method by which the poly(A)+RNA is copied 
into cDNA employs reverse transcriptase, which starts at the 3' end of the 
mRNA from an oligo d(T) primer and proceeds towards the 5' end to generate 
a cDNA:mRNA hybrid (Gubler et al., 1983). The RNA strand is then removed 
from the hybrid by action of RNase H and a second DNA strand is then 
synthesized using DNA polymerase I. The resulting heterogeneous mixture of 
ds cDNA molecules can then be cloned into suitable recombinant DNA vector 
molecules using a variety of techniques. Unfortunately, for the majority 
of mRNAs, this method does not allow for the synthesis of "full-length" 
cDNA because reverse transcriptase can not efficiently copy them into 
full-length cDNAs. The efficiency of copying is inversely proportional to 
the length of mRNA; thus, the problem of "full-length" cDNA synthesis is 
more acute for longer mRNAs. Moreover, the current technology can generate 
deletions at the 5' and 3' ends of the cDNA. 
In an alternative approach, poly(A) tails of mRNA molecules are first 
annealed to oligo (dT) linking with linearized vector DNA (vector 
primer)(Okayama et al., 1982; Pruitt, International Patent, Appl. No. 
89110816.9). Then, the first strand of cDNA synthesized by reverse 
transcriptase is tailed at the 3' end by oligo dt which facilitates 
subsequent cloning by circularization into vector primer. This method also 
generates greater numbers of cDNA clones that contain truncated cDNAs due 
to non-full-length cDNA synthesis. 
As a result of the shortcomings using present technologies to generate 
conventional cDNA libraries, the majority of the cDNA clones lack 
sequences close to the 5' end of the mRNAs. This results in a loss of 
important information required to make functional proteins. Two selection 
procedures have been developed in efforts to enrich cDNA libraries for 
"full-length" cDNA clones. In CAP retention procedure (CAPture) 
cap-binding protein (eukaryotic initiation factor 4E) in combination with 
RNase A was used to purify full-length cDNA:mRNA hybrids (Edery et al., 
1995; Sonenberg et al., U.S. Pat. No. 5,219,989). Shorter duplexes 
corresponding to non-full-length cDNA fragments are not selected, since 
the CAP structure of mRNA is removed from the RNA moiety by nuclease 
treatment. Although the CAPture method could potentially enrich cDNA 
libraries for clones containing the authentic 5' ends, the yield of 
full-length cDNA is very low, especially for long cDNAs (1-5%). The low 
yield is a significant disadvantage for this technology. 
In the "oligo-capping" method, the CAP structure of mRNA is selectively 
replaced with an oligoribonucleotide, thus generating chimeric 
oligonucleotide/full-length mRNA intermediates which are subsequently used 
for synthesis of full-length cDNAs (Maruyama et al., 1994; Fromomt-Racine 
et al., 1993; Kato et al., International Patent, Publ. No. 0 625 572 A1, 
Appl. No. 93921061.3). However, this method is complicated, involving 
treatment of mRNA with an alkaline phosphatase, decapping mRNA with 
tobacco acid pyrophosphatase, and ligation of the oligonucleotide to the 
5' end of mRNA by T4 RNA ligase. These multiple enzymatic steps degrade 
mRNA, thereby generating incomplete cDNA fragments for subsequent cloning 
procedures. Size distribution of cDNA inserts in cDNA libraries generated 
using the "oligo-capping" method is typically less than 3 kb. This is much 
less than full-length mRNA size distribution (Kato et al., 1994) and 
indicates the low efficiency of "full-length" cDNA cloning by 
"oligo-capping" technology. 
As can be understood from the foregoing, conventional methods for 
constructing cDNA libraries containing full-length cDNA clones are 
restricted by low efficiency and the use of multiple, time-consuming 
steps. Accordingly, a simple method that would generate high quality, 
full-length cDNA from RNA is highly desirable. 
BRIEF SUMMARY OF THE INVENTION 
The present invention provides an advantageous method for synthesis and 
cloning of full-length cDNA, or cDNA fragments, that corresponds to the 
complete sequence of 5'-ends of mRNA molecules. The method of the present 
invention comprises contacting RNA with a cDNA synthesis primer which can 
anneal to RNA, a suitable enzyme which possesses reverse transcriptase 
activity, and a template switching oligonucleotide under conditions 
sufficient to permit the template-dependent extension of the primer to 
generate an mRNA-cDNA hybrid. The resulting full-length ss cDNA includes 
the complete 5'-end of the RNA molecule as well as the sequence 
complementary to the template switching oligonucleotide, which can then 
serve as a universal priming site in subsequent amplification of the cDNA. 
The subject invention also concerns novel template switching 
oligonucleotides that can be used according to the subject method. Kits 
containing the template switching oligonucleotide are also included within 
the scope of the present invention.

DETAILED DISCLOSURE OF THE INVENTION 
The subject invention concerns compositions and methods for constructing 
cDNA libraries from nanogram quantities of total or poly A+ RNA. The 
compositions and methods employ template switching oligonucleotides 
described herein. The method of the present invention comprises contacting 
RNA with a primer which can anneal to RNA, a reverse transcriptase, and a 
template switching oligonucleotide under conditions sufficient to permit 
the template-dependent extension of the annealed primer to generate an 
mRNA-cDNA hybrid. The resulting full-length ss cDNA includes the complete 
5'-end of the RNA molecule, as well as the sequence complementary to the 
template switching oligonucleotide. The template switching complementary 
sequence can then serve as a universal priming site in subsequent 
amplification of the cDNA. 
Specifically, the subject invention provides a method for synthesis of 
full-length single-stranded (ss) cDNA, or ss cDNA fragments, from RNA. The 
cDNA synthesized in the present method has an arbitrary anchor sequence at 
the 3' end, followed by a nucleotide sequence complementary to the RNA 
molecule starting from the cap site of the mRNA. In a preferred 
embodiment, the process of the subject invention comprises the following 
steps: 
1. Incubating a sample of poly(A)+RNA or total RNA in the presence of a 
cDNA synthesis primer (CDS primer) which can anneal to mRNA and an enzyme 
which possesses reverse transcriptase activity under conditions sufficient 
to permit the template-dependent extension of the primer to generate an 
mRNA-cDNA hybrid; and 
2. incubating the first-strand cDNA synthesis mixture obtained from step 1 
with a template switching oligonucleotide of the present invention (also 
referred to herein as a "CAPswitch oligonucleotide"), which can provide 
CAP-dependent extension of full-length cDNA by reverse transcriptase using 
the template switching oligonucleotide as a template, and thereby adding 
nucleotide sequence complementary to the template switching 
oligonucleotide to the 3'-end of full-length ss cDNA (referred to herein 
as "anchored cDNA:mRNA hybrid"). The template switching oligonucleotide 
has a pre-selected arbitrary nucleotide sequence at its 5'-end and at 
least one riboguanine residue at its 3'-end. 
Steps 1 and 2 of the method are separated only in time. In a preferred 
embodiment, step 1 is followed by step 2. However, it would be understood 
that the first-strand cDNA synthesis mixture from step 1 can include a 
template switching oligonucleotide which will be used at step 2. 
Alternatively, a template switching oligonucleotide can be added to the 
reaction mixture at the time of or after first-strand cDNA synthesis. In a 
preferred embodiment, the cDNA synthesis primer is a modified oligo(dT) 
primer. 
The scope of the present invention also includes a method for isolating a 
full-length cDNA fragment corresponding to a 5'-end of target mRNA(s) 
using anchored cDNA:mRNA hybrid generated at step 2 as a template. This 
method comprises the embodiment of steps 1 and 2, followed by either 
alternative step 3A or step 3B, described below: 
3A. Incubating an anchored cDNA:mRNA hybrid generated at step 2 with a 
combination of (a) an oligonucleotide primer corresponding partially or 
completely to the nucleotide sequence of the template switching 
oligonucleotide, (b) oligonucleotide primer(s) which is complementary to a 
nucleotide sequence of the target(s) mRNA, and (c) an effective amount of 
other reagents necessary to perform polymerase chain reaction (PCR). The 
incubation is conducted under conditions sufficient to perform PCR and 
thereby generate amplification product corresponding to the 5'-end 
full-length fragment of target cDNA. 
3B. Treating anchored cDNA:mRNA hybrid of step 2 under conditions in which 
a second cDNA strand is synthesized, using the first anchored cDNA strand 
as a template. 
Also within the scope of the present invention is a method for generating 
cDNA libraries containing full-length cDNAs. This method uses as a 
template anchored cDNA:mRNA hybrid generated at step 2. This method 
comprises the embodiment of steps 1 and 2, followed by either alternative 
step 3C or step 3D, described below: 
3C. Incubating anchored cDNA:mRNA hybrid generated at step 2 with a 
combination of primers corresponding partially or completely to the 
sequence of template switching oligonucleotide and cDNA synthesis primer, 
respectively, and an effective amount of other reagents necessary to 
perform PCR. The incubation is conducted under conditions sufficient to 
perform PCR to generate amplification product corresponding to the 
representative library of full-length ds cDNA. 
3D. Treating anchored cDNA mRNA hybrid of step 2 under conditions in which 
a second cDNA strand is synthesized, using the first anchored cDNA strand 
as a template. 
In one aspect of the present invention, the resulting cDNA product 
generated at step 2 or 3 can be inserted into recombinant cloning 
vehicles, and hosts can be transformed with these vehicles according to 
conventional methods well known in art (Kimmel et al., 1987). 
The subject invention enables synthesis of full-length cDNA, which has been 
difficult to synthesize by conventional methods. The present invention 
includes the novel step 2 described above, which can be utilized in 
standard cDNA preparation/cloning procedures which are well known in the 
art. The use of template switching oligonucleotides in the subject method 
advantagously allows for negative selection against cDNAs that are not 
complementary with the 5'-end of template RNA, whereas full-length cDNAs 
can be readily selected. Moreover, the subject methods can significantly 
simplify cDNA synthesis and cloning. Since the cDNA clones obtained from 
the full-length cDNA library prepared according to the present method 
contain the complete information for the primary structure of the protein, 
the invention also relates to a process for using the clones, obtained 
from said full-length cDNA library to produce the encoded proteins. 
In another aspect, the invention provides methods where the resulting cDNA 
product generated at step 3 can be used as a starting material for use 
with cDNA subtraction methods. Specifically, the method of the subject 
invention can be used in conjunction with cDNA subtraction procdures to 
prepare a cDNA population containing highly enriched representation of 
cDNA species that are present in one DNA population (the tester 
population), but that are less abundant or absent in another DNA 
population (the driver population). Preferably, tester and driver ds cDNA 
amplified by methods and materials of the present invention is used in 
combination with Suppression Subtractive Hybridization technology 
described by Chenchik et al. (U.S. Pat. No. 5,565,340). Other methods of 
subtractive hybridization, described for example, by Wigler et al. (U.S. 
Pat. No. 5,436,142); Hampson et al. (Nucl. Acids Res. 20:2899 (1992)); 
Yang et al. (Anal. Biochem. 237:109-114(1996)); Balzer et al. (Nucl. Acids 
Res. 22:2853-2854(1994)), and others, can also be employed. 
The ds cDNA prepared according to the present invention can also be used as 
a hybridization probe. As used herein, the term "hybridization probe" 
means that cDNA generated from total RNA isolated from healthy, diseased 
or infected organisms, or as subtracted as described by Chenchik et al. 
(U.S. Pat. No. 5,565,340), may be labeled by radioisotopes, fluorescent 
and other reporter groups by conventional chemical or enzymatic labeling 
procedures. Labeled cDNA can then be used in standard hybridization assays 
known in the art, i.e., the labeled cDNA is contacted with the defined 
oligonucleotide/polynucleotides corresponding to a particular set of the 
genes immobilized on a solid surface for a sufficient time to permit the 
formation of patterns of hybridization on the surfaces caused by 
hybridization between certain polynucleotide sequences in the 
hybridization probe with the certain immobilized defined 
oligonucleotide/polynucleotides. The hybridization patterns using 
available conventional techniques, such as scintillation counting, 
autoradiography, fluorescence measurement, calorimetric measurement, or 
light emission measurement. Techniques and conditions for labeling, 
hybridization and detection are well known in the art (see, e.g. Maniatis 
et al., 1989; Keller et al., 1993). 
Labeled cDNA can also be used for identifying genes which are 
differentially expressed in two different pre-determined states of an 
organism (see, e.g., Maniatis et al., 1989; International Patent WO 
95/21944). 
The ss cDNA generated after the first-strand cDNA synthesis-template 
switching procedure (step 2) or the ds cDNA generated after 5'-RACE 
amplification step (step 3) can be used as a hybridization probe for 
selectively recovering a desired complementary target nucleic acid 
molecule from a mixture or library of ss or ds nucleic acid containing 
said molecule. The method wherein the hybridization probe is haptenylated 
and used for obtaining an enriched source of target nucleic acid molecule 
based on hybridization approach as described in details in U.S. Pat. No. 
5,484,702 of Jan. 16, 1996 and U.S. Pat. No. 5,500,356 of Mar. 19, 1996 or 
RecA protein-mediated hybridization as described in details in U.S. Pat. 
No. 4,888,274 of Dec. 19, 1989. 
Also within the scope of the present invention are template switching or 
"CAPswitch" oligonucleotides useful for the preparation of cDNA libraries 
containing full-length cDNA clones. The CAPswitch oligonucleotides have at 
least two functions. One function is the ability to selectively interact 
with full-length intermediates of reverse transcriptase-mRNA-cDNA which 
are generated at the 5' end of full-length mRNA after first-strand cDNA 
synthesis. A second function of the CAPswitch oligonucleotides of the 
subject invention is as an efficient template for reverse transcriptase 
from the above-mentioned full-length intermediates which can allow 5' mRNA 
end (in most cases CAP-dependent) extension of full-length cDNA by reverse 
transcriptase using CAPswitch oligonucleotide as a template. A sequence 
complementary to CAPswitch oligonucleotide can thereby be added to the 
3'-end of full-length cDNA. 
Also within the scope of the present invention are modifications in the 
structure or sequence of CAPswitch oligonucleotide which can provide an 
advantage for selective binding to the CAP structure of mRNA. One 
modification, for example, can include covalently binding CAPswitch 
oligonucleotides with a protein capable of binding the CAP structure of 
mRNA (see U.S. Pat. No. 5,219,984). 
The invention particularly concerns the embodiments of the above methods 
wherein the CAPswitch oligonucleotide is represented by the following 
formula: 
EQU 5'-dN.sub.1 -dN.sub.2 - . . . dNm-rN.sub.1 -rN.sub.2. . . rNn-3' 
wherein dN represents a deoxyribonucleotide selected from among dAMP, dCMP, 
dGMP and dTMP; m represents an integer equal to or greater than zero, 
preferably from 10 to 50; rN represents a ribonucleotide selected from 
among AMP, CMP, GMP and UMP, preferably GMP; and n represents an integer 
of at least one or greater, preferably from 3 to 7. Some obvious and well 
known in the art modifications in the structure of the CAP switch 
oligonucleotide, such as replacement of 1-10 nucleotides with random 
nucleotides, nucleotide analogs, or nucleotides labeled with different 
hapten groups (such as, for example, biotin, digoxigenin, and 
fluorescein), incorporation of a terminator nucleotide (such as, for 
example, 3'-amino NMP, 3'-phosphate NMP, and 3'-fluoro NMP), using 
partially double-stranded DNA containing extension of a single-stranded 
CAPswitch oligonucleotide sequence 5'-dN.sub.1 -dN.sub.2 - . . . 
dNm-rN.sub.1 -rN.sub.2. . . rNn-3', incorporation of restriction sites, 
and incorporation of promoter sequences for bacteriophage RNA polymerase, 
which simplify subsequent purification and cloning of the cDNA but which 
still retain subtantially the same functional activity as an unmodified 
CAPswitch oligonucleotide, i.e., mRNA 5'-end extension of full-length cDNA 
by reverse transcriptase using CAPswitch oligonucleotide as a template are 
within the scope of present invention. 
Also included within the subject invention are cDNA library construction 
kits, for example, library construction kits which include the novel 
oligonucleotides according to the subject invention for use with PCR 
procedures. 
The subject invention also concerns novel template switching 
oligonucleotides that can be used according to the subject method. 
Also included within the scope of the present invention are kits that 
include in one or more containers template switching oligonucleotides of 
the invention. 
CAPswitch technology. The methods and materials of the present invention is 
primarily based on the use of unique CAPswitch oligonucleotides in cDNA 
synthesis. The subject cDNA synthesis can include a step of first-strand 
cDNA from polyA.sup.+ RNA using reverse transcriptase coupled with either 
second-strand cDNA synthesis or PCR amplification in a second step to 
generate a high yield of full-length ds cDNA. When included in the 
first-strand cDNA synthesis reaction mixture, the CAPswitch 
oligonucleotides create a short extended template. In the course of first 
strand cDNA synthesis, the reverse transcriptase enzyme stops at the 5' 
end of the mRNA template. The 5'-end typically includes a 
7-methylguanosine CAP structure present on the 5' ends of all eukaryotic 
mRNAs. The enzyme terminal transferase activity then adds a few additional 
nucleotides, primarily deoxycytidine, to the 3'-end of the of the newly 
synthsized cDNA strand. The CAPswitch oligonucleotide, which has an oligo 
(rG) sequence at its 3' end, base pairs with the deoxycytidine-rich 
stretch of nucleotides present on the cDNA strand, creating an extended 
template. Reverse transcriptase then switches templates and continues 
synthesis of cDNA that is complementary to the CAPswitch oligonucleotide. 
The resulting full-length ss cDNA incorporates at its 3' end a sequence 
which is complementary to both the complete 5' end of the mRNA and the 
CAPswitch oligonucleotide sequence. 
Identified herein is an oligonucleotide structure (CAPswitch 
oligonucleotide) which can provide for an efficient template switching 
reaction in the course of first-strand cDNA synthesis from poly(A)+RNA 
using, as a donor, 5'-capped full-length mRNA and, as the acceptor, a 
chemically synthesized oligonucleotide. Chimeric cDNA products having an 
oligonucleotide sequence at the 3'-end of full-length cDNA were revealed 
by subsequent amplification (e.g., 5'-RACE) using a combination of a 
gene-specific primer and a primer complementary to a portion of the 
template switching oligonucleotide. One set of oligonucleotides according 
to the present invention, having an arbitrary sequence at the 5' end and 
random sequence at the 3' end, are represented by the following 
polynucleotide sequences: 
CzR1 5'-d(TGTAGCGTGAAGACGACAGAA)r(N).sub.12 -3' (SEQ ID NO. 1) 
CzR2 5'-d(TGTAGCGTGAAGACGACAGAA(N).sub.11)r(N).sub.1 -3' (SEQ ID NO. 2) 
CzR3 5'-d(TGTAGCGTGAAGACGACAGAA(N).sub.11)-3'(SEQ ID NO. 3) 
CzR4 5'-d(TGTAGCGTGAAGACGACAGAAGGATG(N).sub.9)r(N).sub.1 -3' (SEQ ID NO. 4) 
Na21-N4 5'-d(TGTAGCGTGAAGACGACAGAA)r(N).sub.4 -3' (SEQ ID NO. 5) 
Na21-N8 5'-d(TGTAGCGTGAAGACGACAGAA)r(N).sub.8 -3' (SEQ ID NO. 6) 
NA21-N12 5'-d(TGTAGCGTGAAGACGACAGAA)r(N).sub.6 - 3' (SEQ ID NO. 7) 
wherein d() represents a deoxyribonucleotide sequence; (N).sub.11 and (N)9 
represent, respectively, a random deoxyribonucleotide sequence 11 or 9 
bases long of dAMP, dGMP, dCMP and dTTP in each base position; 
r(N).sub.1-16 represents a random sequence 1 to 16 bases long of AMP, GMP, 
CMP and UMP in each base position. 
Based on the efficiency of amplification of 5'-ends (5'-RACE) of four human 
cDNAs (smooth muscle .alpha.-actin, smooth muscle .gamma.-actin, 
cytoskeletal .gamma.-actin and transferrin receptor and model RNAs with 
and without cap structure at the 5' end), and subsequent sequence analysis 
of amplified product, conservative structures have been identified at the 
3' end of an oligonucleotide which can be used to generate a highly 
efficient CAP-dependent template switching reaction. Mutational analysis 
of conservative and non-conservative regions of the oligonucleotide 
sequence (see the sequences of additional analyzed CAPswitch 
oligonucleotides, SEQ ID NOS. 19-66) revealed that the highest efficiency 
of CAP-dependent template switching was achieved using a basic DNA-RNA 
chimeric CAPswitch oligonucleotide having an arbitrary sequence at its 5' 
end and a conservative oligo ribo(G) sequence at the 3' end. This 
oligonucleotide is represented by the general formula: 
EQU 5'-dN.sub.x -rG.sub.y -3' 
long; and rG.sub.y represents an oligo rG sequence 3-5 bases long. The 
oligo rG sequence is responsible for the main template switching function 
associated with the CAPswitch oligonucleotide. The arbitrary 
deoxyribooligonucleotide sequence can be selected so as to be useful for 
subsequent cDNA synthesis and cloning steps. 
In addition, the template switching reaction is most efficient for cDNAs 
that have sequence complementary to the 5' end of the mRNA, in comparison 
to cDNAs that were prematurely terminated during reverse transcription of 
the first strand and, therefore, do not have sequences complementary to 
the 5' end of the mRNA molecule. The presence of the CAP structure at the 
5' end of mRNA is not a necessary requirement for template switching 
reaction, but the template switching reaction is most effective for 
full-length DNA products synthesized on mRNA which has the CAP structure 
present at the 5'-end. 
Modifications in the structure of template switching oligonucleotides are 
contemplated within the scope of the present invention. Specifically, 
those modification which may affect the template switching efficiency but 
in which the template switching oligonucleotide retains the substantially 
the same functional activity as unmodifed template switching 
oligonucleotides, i.e., CAP-dependent extension of full-length cDNA by 
reverse transcriptase using the oligonucleotide as a template, is 
contemplated by the subject invention. The modified oligonucleotides can 
be used as alternatives to unmodified CAPswitch oligonucleotides. The 
following rules summarize these modifications that are useful according to 
the subject invention: 
1a. The use of shorter (1-2 bases) oligo rG 3'-end sequences, replacement 
of one or several rG residue(s) for rA, rC or rU, or replacement of oligo 
rG for oligo dG reduces efficiency of the basic structure; longer oligo rG 
sequences (7-9 bases) do not significantly influence template switching 
efficiency. 
1b. Modification of the 3' terminal G at the 3'-OH group of ribose residue 
by an amino, biotin, phosphate, fluoro or glycerol group can significantly 
reduce background in subsequent PCR amplification steps (step 3A). 
1c. Changes in the sequence of the arbitrary portion of a template 
switching oligonucleotide, replacement partially or completely 
deoxyribonucleotides for ribonucleotides, including restriction site(s), 
does not significantly influence template switching efficiency. Using a 
longer arbitrary sequence (about 22 to 42 bases) at the 3'-end of the 
template switching oligonucleotide reduces the efficiency of template 
switching, whereas shorter sequences (about 15 to 17 bases) slightly 
increase the efficiency of template switching but make subsequent PCR 
amplification steps (step 3A, 3C) less efficient. 
1d. A person skilled in this art having the benefit of the current 
disclosure would recognize that other modifications in the structure of 
template switching oligonucleotides of the present invention can be 
readily prepared. These modifications can increase the efficiency and 
specificity of the CAP-dependent template switching reaction. For example, 
using aptamer (random oligonucleotide) selection technology (Kenan et al., 
1994) it is possible to find ribonucleotide or deoxyribonucleotide 
sequences of the arbitrary portion of the template switching 
oligonucleotide which efficiently bind to the CAP structure of an mRNA 
molecule and, therefore, increase efficiency of the template switching 
reaction. The same result can be achieved by replacement of natural 
nucleotide(s) with modified nucleotides in order to increase the affinity 
of the template switching oligonucleotides binding to the CAP structure. 
Chimeric protein-template switching oligonucleotides can also be 
constructed so that the protein portion recognizes and binds the CAP 
structure, which can increase efficiency of the template switching 
reaction. These cap binding proteins or protein portions are well known in 
the art and preferably include antibodies against the CAP structure and 
eukaryotic initiation factor 4E (eIF-4E). 
Another advantage of using the methods and compositions of the present 
invention is the high flexibility of this procedure which makes it 
possible to use this new technology with conventional cDNA cloning 
procedures well known in art. Advantageously, the subject invention can 
eliminate the need for multiple enzymatic or purification procedures used 
in conventional procedures. The subject method can provide CAP-dependent 
automatic and direct addition of a template switching oligonucleotide 
sequence to the 5' end of mRNA:cDNA hybrid in the course of first-strand 
cDNA synthesis. Moreover, the methods and compositions of the subject 
invention can be combined with procedures well known in the art for cDNA 
synthesis and cloning. It will be apparent to those skilled in the art 
that the order of some of the individual steps, the exact structure of CDS 
primers, and the vectors used for cDNA library construction can be varied. 
Thus, modifications, substitutions, and optimization of the methods and 
materials disclosed herein which provide for the cloning of full-length 
cDNA or cDNA fragment(s) containing complete complementary nucleotide 
sequence of the 5'-end of mRNA molecules are within the scope of the 
present invention. The description below details only preferred steps 
which result in the efficient generation of full-length cDNA from RNA. 
First-strand cDNA synthesis. Using the subject method with conventional 
procedures, first-strand cDNA synthesis can be carried out using an 
annealed complex comprising a cDNA synthsis primer:mRNA as a template for 
reverse transcription. Primer extension can be catalyzed by reverse 
transcriptase, or by a DNA polymerase possessing reverse transcriptase 
activity, in the presence of adequate amounts of other components 
necessary to perform the reaction, for example, deoxyribonucleoside 
triphosphates ATP, CTP, GTP and TTP, Mg.sup.2+, optimal buffer. A variety 
of DNA polymerases possessing reverse transcriptase activity can be used 
for the first-strand cDNA synthesis. Examples of DNA polymerases that can 
be used in the methods of the present invention include the DNA 
polymerases derived from organisms such as thermophilic bacteria and 
archaebacteria, retroviruses, yeast, Neurospora, Drosophila, primates and 
rodents. Preferably, the DNA polymerase is isolated from Moloney murine 
leukemia virus (M-MLV) (U.S. Pat. No. 4,943,531) or M-MLV reverse 
transcriptase lacking RNaseH activity (U.S. Pat. No. 5,405,776), human 
T-cell leukemia virus type I (HTLV-I), bovine leukemia virus (BLV), Rous 
sarcoma virus (RSV), human immunodeficiency virus (HIV) or Thermus 
aquaticus (Taq) or Thermus thermophilus (Tth) (U.S. Pat. No. 5,322,770). 
These DNA polymerases may be isolated from an organism itself or, in some 
cases, obtained commercially. DNA polymerases useful with the subject 
invention can also be obtained from cells expressing cloned genes encoding 
the polymerase. As a starting material for cDNA synthesis, poly(A)+RNA or 
total RNA from yeast and higher organisms such as plants or animals can be 
used. 
The first-strand cDNA synthesis step of the subject method can include 
template switching oligonucleotides of the present invention in the 
reaction mixture, but are not a necessary component for carrying out 
first-strand cDNA synthesis. The template switching oligonucleotides can 
be added at the time of the template switching step which follows the 
first-strand synthesis step. Thus, it is understood that template 
switching oligonucleotide molecules can be included in the first-strand 
reaction composition (for example, during cDNA synthsis primer annealing 
to RNA or when contacting the RNA with an enzyme possessing reverse 
transcriptase activity) or the oligonucleotides can be added in the course 
of, or after completion of, the first-strand cDNA synthesis reaction. 
Depending on the strategy to be employed for cDNA cloning, numerous cDNA 
synthesis primers structures can be used for the first-strand cDNA 
synthesis using poly(A)+RNA as a template and catalyzed by the reverse 
transcriptase activity. The cDNA synthesis primer can be a single-stranded 
oligonucleotide, a double-stranded oligonucleotide having a 
single-stranded portion (primer-restriction-end or PRE, adapter, as 
described by Coleclough et al., 1985), or a vector primer, representing ds 
vector with a single-stranded portion (Okayama et al., 1982). In all three 
cases, a single-stranded portion of the cDNA synthesis primer is 
responsible for binding with poly(A)+RNA and initiating the first-strand 
cDNA synthesis. In a preferred embodiment of the subject method, for 
full-length cDNA library construction, a CDS primer containing an oligo dT 
tail at the 3'-end of the primer is annealed to the poly(A) portion of 
mRNA. For rapid amplification or cloning of 5' cDNA ends and for selective 
cloning of particular genes, the CDS primer can possess a random sequence 
or arbitrary sequence which may correspond to a particular sequence of a 
target gene which is to be cloned. 
The subject invention particularly concerns the embodiments of the above 
methods wherein the CDS primer can be annealed to: 
1a. The poly(A) tail of poly(A)+RNA. The cDNA synthesis primer can be 
selected from a single-stranded oligonucleotide, any partially 
double-stranded DNA fragment, or any linear vector primer. In a preferred 
embodiment, the oligonucleotide primer has the sequence: 5'-dN.sub.1 
-dN.sub.2 - . . . dNm-dTn-dN.sub.1 -dN.sub.2 - . . . dNp-3' wherein m 
represents an integer 0 and above, preferably from 0 to 20; n represents 
an integer 8 and above, preferably from 8 to 30; p is preferably from 0 to 
3; dN represents a deoxyribonucleotide selected from or represent mixture 
of dAMP, dCMP, dGMP, and dTMP; dT represents dTMP. Some modifications in 
the structure of the primer such as replacement of 1-10 nucleotides for 
nucleotides containing different hapten groups (biotin, digoxigenin, 
fluorescein, etc.), nucleotide analogs, ribonucleotides, non-natural 
nucleotides, incorporation of restriction sites, bacteriophage RNA 
polymerase promoter region which simplify subsequent purification, using 
and cloning cDNA but still retain the main function of the primer, i.e., 
priming activity from poly(A) portion of poly(A)+RNA, are within the scope 
of present invention. Using a partially double-stranded DNA primer or 
linear plasmid vectors having a single-stranded tail sequence 5'-dN.sub.1 
-dN.sub.2 - . . . dNm-dTn-dN.sub.1 -dN.sub.2 - . . . dNp-3', described 
above, and possessing priming activity for first-strand cDNA synthesis 
from poly(A) portion of poly(A)+RNA, are also considered as part of the 
subject invention. In order to simplify the cloning procedure, the 
CAPswitch oligonucleotide can be attached to the other end of vector 
primer. In this case, the vector primer will possess at one end a sequence 
corresponding to the CDS primer and, at the other end, the CAPswitch 
oligonucleotide (CAPswitch-vector primer technology). Subsequent cDNA 
synthesis and automatic template switching will generate cDNA:vector 
chimeric product, which can be easily cloned as described Okayma et al., 
1982. 
1b. Inner, non-poly(A) portion of the mRNA. These oligonucleotide primer(s) 
have the general formula dN.sub.1 -dN.sub.2 - . . . dNq, where dN 
represents a deoxyribonucleotide selected from among dAMP, dCMP, dGMP, and 
dTMP or represent a mixture of 2-4 of these bases; and q represents 
integer 6 and above, preferably from 6 to 50. These primers can have a 
random sequence, i.e., annealed to all mRNAs, an arbitrary sequence, Le., 
annealed to at least one arbitrary mRNA, or a sequence complementary to at 
least one mRNA. Also, the sequence of these primer(s) can include a 
restriction site(s) or modified bases (for example, biotinylated) to 
facilitate subsequent purification or cloning procedure. 
Second-strand cDNA synthesis and/or PCR amplification. First-strand cDNA 
synthesis based on CAPswitch technology generates a full-length (or the 
corresponding 5' end of a full-length fragment) mRNA:cDNA hybrid molecule 
intermediates flanked by a CDS primer at its 3' end, and a CAPswitch 
oligonucleotide at its 5' end. Such intermediates can be easily converted 
to a ds cDNA form suitable for subsequent cloning using conventional 
procedures. These procedures are well known and include: 
1. Direct amplification of full-length cDNA by combination of PCR primer 
corresponding CDS and CAPswitch flanking portions of mRNA:cDNA hybrid and 
effective amount of other reagents under conditions necessary to perform 
PCR. Preferably, the conditions are those developed for amplification of 
long nucleic acid sequences and described by Cheng (International Patent 
(1995)) and Barnes (U.S. Pat. No. 5,436,149 of Jul. 25, 1995). 
2. Replacement of the mRNA portion of the mRNA:cDNA hybrid with a 
second-strand cDNA essentially as described by Okayama et al. (1982) and 
Gubler et al. (1983). This process entails digestion of the RNA with a 
ribonuclease such as E. coli RNase H repair synthesis using a DNA 
polymerase having the activities of DNA polymerase I, and ligation. The 
procedure depends on the structure of the CDS primer used for the 
first-strand cDNA synthesis. Second-strand cDNA synthesis can be carried 
out using as a template mRNA:cDNA hybrid or mRNA:cDNA:vector chimeric 
product using vector primer for first-strand synthesis (CAPswitch-vector 
primer technology, see above). Alternatively, mRNA:cDNA hybrid generated 
by PRE adaptor strategy, described above, can be digested at the 5' and 3' 
flanking sequences, which correspond to PRE adaptor and CAPswitch 
oligonucleotide by at least one restriction enzyme, and then ligated into 
a conventional vector digested by the same restriction enzyme(s). Any 
restriction enzyme(s) can be used as long as it does not cut within 
mRNA:cDNA hybrid. 
Cloning into vector. In the case of using a vector primer (CAPswitch-vector 
primer) or PRE adaptor strategy, the ds cDNA generated after second-strand 
cDNA synthesis is already inserted into the vector and does not require 
this step. When an oligonucleotide CDS primer is used, the ds cDNA 
prepared in the second step by PCR or by mRNA replacement technology can 
be ligated with adaptors or digested with restriction enzyme(s) in 
sequences corresponding to CDS and CAPswitch oligonucleotide Banking 
portions, thus generating ds cDNA molecules which will be ligated to any 
conventional cloning vector (including plasmid, cosmid, phage, retroviral 
vector and so on) after digesting it with the same restriction enzyme(s). 
Then, recombinant DNA molecules comprising a full-length cDNA library can 
be introduced into prokaryotic hosts and, optionally, eukaryotic hosts, 
useful in the high frequency cloning of full-length ds cDNA and in the 
expression of recombinant proteins therefrom. 
Once cloning is completed according to the invention, the desired clone(s) 
can be detected by labeled probe, monoclonal or polyclonal antibodies 
prepared against the product in a conventional immunoassay or enriched for 
desired target by hybridization selection approach, described for example 
by Li et al. in International Patent WO 95/04745 of 9 Aug. 1994. 
Summary. Use of CAPswitch oligonucleotide in cDNA synthesis and cloning 
significantly simplify and improve technology of full-length cDNA library 
construction. The main benefits are as follows: 
1. The one-stage procedure which includes first-strand cDNA synthesis and 
addition of a defined sequence to the 3' end of cDNA which significantly 
reduces the number of steps (from 5-7 to 2-3 steps) necessary for 
conventional PCR-based standard cDNA library construction technology. A 
lower number of steps means that the novel methods and materials of the 
present invention is more efficient, easier, less labor-intensive, and 
more reproducible than conventional cDNA library construction methods. 
2. In accordance with the present invention, the CAP-dependent template 
switching mechanism provides significantly more efficient technology for 
synthesizing full-length cDNA and generating cDNA libraries mostly 
containing full-length cDNAs. The methods and materials of the present 
invention can provide a novel method for readily selecting the full-length 
cDNAs to be cloned in the cDNA library. 
As used herein, the term "full-length complementary DNA or cDNA" is defined 
as a full-length single-stranded (ss) or double-stranded (ds) cDNA(s) or 
cDNA fragment(s) which contain the complete sequence information of the 
5'-end(s) of mRNA(s). Full-length cDNA can contain the complete sequence 
information of the 5'-end of a particular (target) mRNA or of the whole 
population of polyA+RNA used as a template for the first-strand cDNA 
synthesis. 
As used herein, the term "full-length cDNA library" refers to the whole 
population of ss or ds cDNAs synthesized from polyA+RNA as a template. The 
full-length cDNA library can be used directly for different applications 
known in the art or it can be cloned into any suitable recombinant cloning 
vehicle, and host can be transformed with the cloning vehicle. 
The term "template switching" reaction refers to a process of 
template-dependent synthesis of the complementary strand by a DNA 
polymerase using two templates in consecutive order and which are not 
covalently linked to each other by phosphodiester bonds. The synthesized 
complementary strand will be a single continuous strand complementary to 
both templates. Typically, the first template is polyA+RNA and the second 
template is a template switching or "CAP switch" oligonucleotide. 
As used herein, the term "arbitrary sequence" or "anchor sequence" refers 
as any defined or pre-selected deoxyribonucleotide, ribonucleotide or 
mixed deoxyribo/ribonucleotide sequence which contains a particular 
sequence of natural or modified nucleotides. 
As used herein, the term "random sequence" is defined as 
deoxyribonucleotide, ribonucleotide or mixed deoxyribo/ribonucleotide 
sequence which contains in each nucleotide position any natural or 
modified nucleotide. 
As used herein, the term "full-length anchored cDNA" is defined as a 
full-length ss cDNA which has a defined arbitrary sequence at the 3'-end. 
As used herein, the term "mRNA:cDNA hybrid" refers to a product after 
first-strand cDNA synthesis catalyzed by reverse transcriptase using 
polyA+RNA as a template. "mRNA-cDNA hybrid" can be full-length if the cDNA 
portion includes the complete sequence of the 5'-ends of the template 
mRNA. 
As used herein, the term "reverse transcriptase" is defined as any DNA 
polymerase possessing reverse transcriptase activity which can be used for 
first-strand cDNA synthesis using polyA+RNA or total RNA as a template. 
As used herein, two sequences are said to be "complementary" to one another 
if they are capable of hybridizing to one another to form antiparallel, 
double-stranded nucleic acid structure. 
As used herein, the term "hapten" refers to a molecule that is bound to a 
nucleic acid molecule and can be recognized and bound by another molecule, 
or "binding ligand," e.g., an antibody, streptavidin and biotin, transient 
metal, and others known in the art. Examples of haptens include chelating 
group, biotin, fluorescein, digoxigenin, antigen, and others known in the 
art. 
As used herein, the term "solid support" refers to any known substrate 
which can be used for the immobilization of a binding ligand or 
oligonucleotide/polynucleotide sequences by any known method. 
As used herein, the term "reporter group" refers to any group incorporated 
into full-length cDNA by conventional chemical or enzymatic labeling 
procedures and which can be detected by use of conventional techniques, 
such as scintillation counting, autoradiography, fluorescence measurement, 
calorimetric measurement, light emission measurement, and other means 
known in the art. Examples of reporter groups include radioisotopes, 
fluorescent, chemifluorescent, chemiluminescent, hapten groups, and others 
known in the art. 
As used herein, the term "subtractive hybridization" refers to technology 
which allows for the recovery of a cDNA population containing a highly 
enriched representation of the cDNA species that are present in one cDNA 
population (tester), but that are less abundant or not found in another 
cDNA population (driver). Examples of subtractive hybridization 
technologies include Suppression Subtractive Hybridization technology 
(Chenchik et al. U.S. Pat. No. 5,565,340), representation difference 
analysis (Wigler et al., U.S. Pat. No. 5,436,142); and linker capture 
subtraction (Yang et al., Anal. Biochem. 237:109-114(1996). 
As used herein, the term "nucleotide analog" refers to a nucleotide which 
is not typically found in the in DNA or RNA and possesses some additional 
features which can improve efficiency of the template switching reaction 
or improve the usage of anchored cDNA generated. For example, suitable 
nucleotide analogs include modification in the base or sugar-phosphate 
backbone, like peptide nucleic acid, inosin, 5-nitroindole 
deoxyribofuranosyl, 5-methyldeoxycytosine, and 
5,6-dihydro-5,6-dihydroxydeoxythymidine. Other nucleotide analogs will be 
evident to those skilled in the art. 
Following are examples which illustrate procedures for practicing the 
invention. These examples should not be construed as limiting. All 
percentages are by weight and all solvent mixture proportions are by 
volume unless otherwise noted. 
EXAMPLE 1 
Preferred Method for Cloning 5'-end Sequences of Full-Length cDNA Based on 
CAPswitch Technology 
Obtaining a full-length cDNA is one of the most important, and often one of 
the most difficult, tasks in characterizing genes. Traditional methods for 
cDNA library construction usually produce only partial cDNA fragments. To 
facilitate recovery of the rest of the coding sequence, an in vitro method 
for the rapid amplification of cDNA ends (RACE) was proposed in 1988 
(Frohman et al., 1988). In spite of various modifications which have been 
developed, the current RACE technologies are complicated and inefficient. 
The methods and materials of the present invention provides a method which 
significantly simplifies and makes more efficient the 5'-RACE procedure. 
The flow chart which describes CAPswitch-based 5'-RACE procedure is shown 
in FIG. 2, and the preferred protocol is described below. Some obvious 
modifications in protocol, e.g., using other enzymes possessing similar 
enzymatic activities for first-strand synthesis step and PCR, using other 
sequences of oligo(dT) primer and CAPswitch oligonucleotide, using for 
first-strand synthesis a gene-specific primer, instead of a oligo (dT) 
primer, all fall within the scope of the present invention. 
Step. 1 First-strand cDNA synthesis-template switching procedure. 
10 pmol of cDNA synthesis primer (oligo d(T) primer) 
CDS1: 5'-d(TCTAGAATTCAGCGGCCGC(T).sub.30 VN) -3' (SEQ ID NO. 8) 
(where V=G or A or C; N=G or A or T or C) and 50 pmol of CAPswitch 
oligonucleotide (CSO1): 
CSO1: 5'-d(CTAATACGACTCACTATAGGGC)r(GGGp)-3' (SEQ ID NO. 9) 
(where p is 3'-phosphate group) were annealed to 1 .mu.g of human placenta 
poly(A).sup.+ RNA (CLONTECH Laboratories, Inc., Palo Alto, Calif.), in a 
volume of 5 .mu.l of deionized water, by heating the mixture for 2 minutes 
at 70.degree. C., followed by cooling on ice for 2 minutes. First-strand 
cDNA synthesis was then initiated by mixing the annealed primer-RNA with 
200 units of M-MLV RNase H- reverse transcriptase (SuperScript II reverse 
transcriptase, Life Technologies) in a final volume of 10 .mu.l, 
containing 50 mM Tris-HCl (pH 8.3 at 22.degree. C.); 75 mM KCl; 6 mM 
MgCl.sub.2 ; 1 mM DTT; and 1 mM each of dATP, dGTP, dCTP, and dTTP. The 
first-strand cDNA synthesis-template switching reaction was incubated at 
42.degree. C. for 1.5 hours in an air incubator and then cooled on ice. We 
also synthesized first-strand cDNA using random d(N).sub.6 primers (500 
ng) or human beta-actin antisense gene-specific primer: 
ACT1: 5'-d(ACTCGTCATACTCCTGCTTGCTGATCCACATCTGC)-3' (SEQ ID NO. 10) 
or human transferrin receptor antisense gene-specific primer: 
TFR1: 5'-d(GTCAATGTCCCAAACGTCACCAGAGA)-3' (SEQ ID NO. 11) 
instead of the oligo d(T) primer. 
The reaction mixture was then diluted 500-fold by addition of 5 ml of 10 mM 
Tricine-KOH (pH 8.5 at 22.degree. C.) and 0.1 mM EDTA, incubated at 
94.degree. C. for 1.5 min, cooled on ice, and stored at -20.degree. C. 
Step 2. 5'-RACE. 
PCR amplification was performed using the Advantage KlenTaq Polymerase Mi 
(CLONTECH Laboratories, Inc.). This kit contains a mixture of KlenTaq-1 
and Deep Vent DNA polymerases (New England Bio Labs) and TaqStart antibody 
(CLONTECH Laboratories, Inc.). The TaqStart antibody provides automatic 
hot-start PCR. Amplification was conducted in a 50-.mu.l volume containing 
5 .mu.l of diluted first-strand cDNA; 40 mM Tricine-KOH (pH 9.2 at 
22.degree. C.); 3.5 mM Mg(OAc).sub.2 ; 10 mM KOAc; 75 .mu.g/ml BSA; 200 
.mu.M each of dATP, dGTP, dCTA, and dTTP; 0.2 .mu.M each of CAPswitch 
primer (CSP1): 5'-d(CTAATACGACTCACTATAGGGC)-3' (SEQ ID NO. 12) and 
gene-specific primer (GSP 1 for beta-actin or transferrin receptor); and 1 
.mu.l of 50.times. KlenTaq Polymerase Mix. Temperature parameters of the 
PCR reactions were as follows: 1 minute at 94.degree. C. followed by 5 
cycles of 94.degree. C. for 30 seconds and 72.degree. C. for 5 minutes; 
then 5 cycles of 94.degree. C. for 30 seconds and 70.degree. C. for 5 
minutes; then 25 cycles of 94.degree. C. for 30 seconds and 68.degree. C. 
for 5 minutes; followed by a 10-minute final extension at 68.degree. C. 
PCR products were examined on 1.2% agarose/EtBr gels in 1.times. TBE 
buffer. As a DNA size marker we used a 1 kb DNA Ladder (Life 
Technologies). 
Both human beta-actin and transferrin receptor cDNA 5'-RACE reaction 
generate a single band which correspond to the expected size of 
full-length amplified 5'-RACE product. Subsequent cloning and sequence 
analysis of 18 randomly picked 5'-RACE clones confirm their identity to 
beta-actin and transferrin receptor 5'-end fragments. Moreover, 5'-end 
sequences of amplified 5'-RACE product exactly correspond to sequences of 
full-length beta-actin and transferrin receptor mRNAs followed by 
sequences corresponding to CAPswitch oligonucleotide. This example 
illustrates that CAPswitch 5'-RACE can be efficiently used not only for 
amplification of full-length 5'-end sequences of cDNAs but also for exact 
mapping of transcriptional start sites. 
EXAMPLE 2 
CAPswitch PCR-Based Technology for Full-Length cDNA Library Construction 
Methods and materials of the present invention can be effectively used for 
construction of cDNA libraries using as a template 10-100 ng of total RNA. 
Any conventional procedure well known in art can be used to purify this 
small amount of total RNA from 10-50 mg of "difficult" cells or tissues, 
like human biopsy tissues, pathogenic microorganisms, tissues at different 
developmental stages and so on. The flow chart in FIG. 3 shows the main 
step of this procedure. It will be apparent to those skilled in the art 
that some individual non-essential steps, structure of CDS primer, 
CAPswitch oligonucleotide and adaptors shown in FIG. 3 can be varied 
without changing the efficiency of the whole procedure. For example, 
instead of the adaptor ligation step (step 3), ds cDNA generated by PCR 
can be digested by rare cutting restriction endonuclease(s) in sequences 
corresponding CDS and CAPswitch oligonucleotide flanking portions and 
cloned directly into vector. Also, other conventional procedures well 
known in the art for direct cloning of PCR product, such as TA-cloning 
vector, blunt end ligation, and the like, can be used for cloning and 
generation of CAPswitch full-length cDNA libraries. Any such variations in 
the preferred protocol which are based on using methods and materials of 
the subject invention are within the scope of the invention. 
Step 1. First-strand synthesis--template switching. 
10 pmol of cDNA synthesis primer (oligo d(T) primer) CDS1: 
5'-d(TCTAGAATTCAGCGGCCGC(T).sub.30 VN)-3' (SEQ ID NO. 8) 
(where V=G or A or C; N=G or A or T or C) and 10 pmol of CAPswitch 
oligonucleotide (CSO2): 
CSO2: 5'-d(CTAATACGACTCACTATAGGGC)r(GGG)-3' (SEQ ID NO. 13) 
were annealed to 100 ng of human skeletal muscle Total RNA (CLONTECH 
Laboratories, Inc.) in a volume of 5 .mu.l of deionized water by heating 
the mixture for 2 minutes at 70.degree. C., followed by cooling on ice for 
2 minutes. First-strand cDNA synthesis was then initiated by mixing the 
annealed primer-RNA with 200 units of M-MLV RNase H- reverse transcriptase 
(SuperScript II reverse transcriptase, Life Technologies) in a final 
volume of 10 .mu.l, containing 50 mM Tris-HCl (pH 8.3 at 22.degree. C.); 
75 mM KCl; 6 mM MgCl.sub.2 ; 1 mM DTT; and 1 mM each of dATP, dGTP, dCTP, 
and dTTP. The first-strand cDNA synthesis-template switching reaction was 
incubated at 42.degree. C. for 1.5 hours in an air incubator and then 
cooled on ice. 
Step 2. Generation of full-length cDNA by PCR. 
PCR amplification of full-length cDNA was performed using the Advantage 
KlenTaq Polymerase Mix (CLONTECH Laboratories, Inc.). Amplification was 
conducted in a 100-.mu.l volume containing 2 .mu.l of first-strand cDNA; 
40 mM Tricine-KOH (pH 9.2 at 22.degree. C.); 3.5 mM Mg(OAc).sub.2, 10 mM 
KOAc; 75 .mu.g/ml BSA; 200 .mu.M each of dATP, dGTP, dCTP, and dTTP; 0.2 
.mu.M each of CAPswitch primer (CSP1) and CDS1 primer 1; and 1 ml of 
KlenTaq Polymerase mix. Temperature parameters of the PCR reactions were 
as follows: 1 minute at 95.degree. C. followed by 20-22 cycles of 
95.degree. C. for 15 seconds and 68.degree. C. for 5 minutes; followed by 
a 10-minute final extension at 68.degree. C. PCR products were examined on 
1.2% agarose/EtBr gels in 1.times. TBE buffer. As a DNA size marker we 
used a 1 kb DNA Ladder (Life Technologies). 
Step 3. Adaptor ligation. 
The 50 .mu.l of ds cDNA generated at the PCR step were combined with 2 
.mu.l of Proteinase K (2 mg/ml) and incubated at 45.degree. C. for 1 hour, 
followed by a denaturation step at 70.degree. C. for 10 minutes. Then, 3 
.mu.l (15 units) of T4 DNA polymerase were added to the reaction mixture 
and additionally incubated at 16.degree. C. for 30 minutes. ds cDNA was 
then precipitated by addition of a half volume of 4 M ammonium acetate 
(about 35 .mu.l) and 3.7 volumes of 95% ethanol (about 260 .mu.l). After 
vortexing, the tube was immediately centrifuged at 14,000 r.p.m. in a 
microcentrifuge for 20 minutes. The pellet was washed with 80% ethanol 
without vortexing, centrifuged as above for 10 minutes, air dried, and 
dissolved in 16 .mu.l of deionized water. The ds cDNA was then ligated to 
an adaptor overnight at 16.degree. C. under the following conditions: 16 
.mu.l of ds cDNA solution, 50 mM Tris-HCl (pH 7.8 at 22.degree. C.), 10 mM 
MgCl.sub.2, 1 mM DTT, 1 mM ATP, 5% polyethylene glycol (M.W. 8,000), 2 
.mu.M of adaptor (Ad1): 
Ad1: 5'-d(AATTCGCGGCCGCGTCGAC)-3' 
(SEQ ID NO. 14) 
3'-d(GCGCCGGCGCAGCTGp)-5' 
(SEQ ID NO. 15) 
(where p=a 3'-phosphate group) and 1 unit of T4 DNA ligase (Life 
Technologies) in a total volume of 30 .mu.l. The ligation mixture was then 
stopped by addition of 70 .mu.l of 10 mM EDTA. The ds cDNA was extracted 
once with phenol/chloroform/isoamyl alcohol (25:24:1, vol/vol), once with 
chloroform/isoamyl alcohol (24:1, vol/vol), and then precipitated by 
addition of 10 .mu.l of 3 M sodium acetate and 250 .mu.l of 95% ethanol. 
After vortexing, the tube was immediately centrifuged at 14,000 r.p.m. in 
a microcentrifuge for 20 minutes. The pellet was washed with 80% ethanol 
without vortexing, centrifuged as above for 10 minutes, air dried, and 
dissolved in 20 .mu.l of deionized water. The adaptor ligated ds cDNA was 
then phosphorylated at 37.degree. C. for 30 minutes under the following 
conditions: 20 .mu.l of adaptor-ligated ds cDNA solution, 50 mM Tris-HCl 
(pH 7.8 at 22.degree. C.), 10 mM MgCl.sub.2, 1 mM DTT, 1 mM ATP, 30 units 
of T4 polynucleotide kinase (Epicenter Technology) in a final volume 30 
.mu.l. Then phosphorylation reaction was terminated by adding 2 .mu.l of 
0.2 M EDTA and heat inactivated at 70.degree. C. for 15 minutes. 
Step 4. cDNA Size fractionation and cloning. 
Phosphorylated adaptor-ligated ds cDNA generated at the previous step was 
fractionated on the 1.2 ml Sephacryl S500 0 (Phamacia) gel filtration 
column equilibrated by 10 mM Tris-HCl (pH 7.4), 30 mM NaCl, 0.5 mM EDTA 
Size distribution of cDNA in the fractions was analyzed by 1.1% 
agarose/EtBr gel alongside a 1 kb DNA size marker (Life Technologies). 
Fractions corresponding to cDNA sizes longer than 0.5 kb were pooled 
together (total volume 250 .mu.l) and precipitated by adding 1/10 volume 
(25 .mu.l) of 3 M sodium acetate, 1.5 .mu.l of 20 mg/ml glycogen and 2.5 
volume (400 .mu.l) of 95% ethanol. 
After vortexing, the tube was immediately centrifuged at 14,000 r.p.m. in a 
microcentrifuge for 20 minutes. The pellet was washed with 80% ethanol 
without vortexing, centrifuged as above for 10 minutes, air dried, and 
dissolved in 15 .mu.l of deionized water. The ds cDNA was then ligated to 
the .lambda.gt11 EcoRI vector arm (CLONTECH Laboratories, Inc.) overnight 
at 16.degree. C. under the following conditions: 5 .mu.l of ds cDNA 
solution, 50 mM Tris-HCl (pH 7.8 at 22.degree. C.), 10 mM MgCl.sub.2, 1 mM 
DTT, 1 mM ATP, 5% polyethylene glycol (M.W. 8,000), 2.5 .mu.g of 
.lambda.gt11 EcoRI arms, and 2.5 units of T4 DNA ligase (Life 
Technologies) in a total volume of 25 .mu.l. The ligation mixture was then 
packaged using standard protocols as described in the laboratory manual by 
Sambrook et al. (1989). 
In order to confirm the high quality of the library generated using the 
methods and compositions of the present invention, 50 recombinant phage 
clones were selected at random for the determination of insert size. The 
size distribution of the inserts was in the range of 0.5-4.5 kb with a 
maximum of 2.0-3.0 kb that correspond to the size distribution of skeletal 
muscle poly(A)+RNA in Total RNA used for cDNA library construction. The 
same 50 inserts were sequenced using Delta Tth DNA polymerase Sequencing 
kit (CLONTECH Laboratories, Inc.). Ten of the sequences were identified in 
a search of the GenBank database. They are transferrin receptor, ribosomal 
protein L7, myosin light chain 2, LIM domain protein, ATPase factor 6, 
cytochrome C oxidase, cytoskeletal .gamma.-actin, smooth muscle 
.alpha.-actin, and smooth muscle .gamma.-actin. For three cDNAs the 
sequences of the clones were longer than published in GenBank. For seven 
cDNAs, their sequences exactly corresponded to full-length mRNA sequences 
starting from the cap site. 
These data show that methods and compositions of the present invention can 
be used for cDNA library construction to generate a high quality cDNA 
library with a very high level of full-length cDNA clones. 
EXAMPLE 3 
CAPswitch Full-Length cDNA Library Construction Using PRE Adaptor-Primer 
Strategy 
CAPswitch technology can be also effectively be combined with standard, 
conventional (non PCR-based) technologies well known in the art. As a 
result, conventional procedures can be significantly simplified, and a 
full-length cDNA rather than a cDNA fragment library will be generated. In 
this example, as a starting material for cDNA synthesis, we used 
poly(A)+RNA The flow chart in FIG. 4 illustrates the main step of 
CAPswitch full-length cDNA library construction technology mainly based on 
the conventional PRE adaptor-primer procedure described by Coleclough et 
al., (1985). It will be apparent to those skilled in the art that choice 
of enzymes possessing similar enzymatic activities, structure of CDS 
primer (PRE adaptor primer), CAPswitch oligonucleotide and vector, and 
choice of restriction sites used for cloning, as shown in FIG. 4, can be 
varied without changing the efficiency of the subject procedure. One 
modification in the procedure can be to first carry out the second-strand 
synthesis (step 3) followed by restriction digestion (step 2), and cloning 
into a vector (step 4). Another modification can include using adaptor 
ligation procedure described in Example 2 instead of restriction digestion 
(step 2). Use of the vector primer instead of the PRE adaptor-primer for 
the first-strand cDNA synthesis (step 1) can also be employed. In this 
case, the vector primer can have an oligo d(T) sequence at one end to 
initiate first-strand synthesis and a CAPswitch oligonucleotide sequence 
at the other end to provide automatic template switching after completion 
of full-length first-strand cDNA synthesis. Any such variations in the 
preferred protocol which use methods and materials of the subject 
invention are within the scope of the invention. 
Step 1. Generation of full-length mRNA: cDNA hybrid. 
10 pmol of cDNA synthesis primer (CDS3): 
5'-d(TCTAGAATTCTCGAGGCGGCCGC(T).sub.30 VN)-3' (SEQ ID NO. 16) 
3'-d(AGATCTTAAGAGCTCCGCCGGCG)-3' (SEQ ID NO. 17) 
(where V=G or A or C; N=G or A or T or C), and 10 pmol of CAPswitch 
oligonucleotide (CSO3): 
5'-d(TGCTGCGAGAAGACGACAGAATTCGG)r(GGG)-3' (SEQ ID NO. 18) 
were annealed to 5 .mu.g of human skeletal muscle poly(A)+ RNA (CLONTECH 
Laboratories, Inc.), in a volume of 12.5 .mu.l of deionized water, by 
heating the mixture for 2 minutes at 70.degree. C., followed by cooling on 
ice for 2 minutes. First-strand cDNA synthesis-template switching was then 
initiated by mixing the annealed primer-RNA with 1000 units of M-MLV RNase 
H- reverse transcriptase (SuperScript II reverse transcriptase, Life 
Technologies) in a final volume of 25 .mu.l, containing 50 mM Tris-HCl (pH 
8.3 at 22.degree. C.); 75 mM KCl; 6 mM MgCl.sub.2 ; 1 mM DTT; and 1 mM 
each of dATP, dGTP, dCTP, and dTTP. The first-strand cDNA 
synthesis-template switching reaction was incubated at 42.degree. C. for 
1.5 hours in an air incubator and stopped by addition of 75 .mu.l of 150 
.mu.g/ml glycogen, 10 mM EDTA The mRNA:cDNA hybrid was extracted once with 
phenol/chloroform/isoamyl alcohol (25:24:1, vol/vol), once with 
chloroform/isoamyl alcohol (24:1, vol/vol), and then precipitated by 
addition of a half volume of 4 M ammonium acetate (about 40 .mu.l) and 3.7 
volumes of 95% ethanol (about 300 .mu.l). After vortexing, the tube was 
immediately centrifuged at 14,000 r.p.m. in a microcentrifuge for 20 
minutes. The pellet was washed with 80% ethanol without vortexing, 
centrifuged as above for 10 minutes, air dried, and dissolved in 50 .mu.l 
of deionized water. 
Step 2. Restriction digestion. 
mRNA:cDNA hybrid generated at step 1 was digested for nondirectional 
cloning by EcoRI restriction endonuclease (EcoRI and NotI or EcoRI and 
XhoI for directional cloning) for 1 hour at 37.degree. C. in 100 ml of 
reaction mixture, containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM 
MgCl.sub.2, 1 mM DTT, and 50 units of EcoRI restriction endonuclease (New 
England BioLabs). The reaction was then stopped by addition of 5 .mu.l of 
2 mg/ml glycogen, 0.2 M EDTA. The mRNA:cDNA hybrid with EcoRI ends was 
extracted once with phenol/chloroform/isoamyl alcohol (25:24:1, vol/vol), 
once with chloroform/isoamyl alcohol (24:1, vol/vol), and then 
precipitated by addition of a half volume of 4 M ammonium acetate (about 
40 .mu.l) and 3.7 volumes of 95% ethanol (about 300 .mu.l). After 
vortexing, the tube was immediately centrifuged at 14,000 r.p.m. in a 
microcentrifuge for 20 minutes. The pellet was washed with 80% ethanol 
without vortexing, centrifuged as above for 10 minutes, air dried, and 
dissolved in 5 .mu.l of deionized water. 
Step 3. Ligation into vector. 
The EcoRI-digested mRNA:cDNA hybrid was then ligated to the .lambda.gt11 
EcoRI vector arm (CLONTECH Laboratories, Inc.) overnight at 16.degree. C. 
under the following conditions: 5 .mu.l of mRNA:cDNA hybrid solution, 50 
mM Tris-HCl (pH 7.8 at 22.degree. C.), 10 mM MgCl.sub.2, 1 mM DTT, 1 mM 
ATP, 5% polyethylene glycol (M.W. 8,000), 2.5 .mu.g of .lambda.gt11 EcoRI 
arms, and 2.5 units of T4 DNA ligase (Life Technologies) in a total volume 
of 20 .mu.l. 
Step 4. Second-strand cDNA synthesis. 
Second-strand cDNA synthesis was carried out in a total volume of 100 
.mu.l, containing 20 .mu.l of the vector-ligated mRNA:cDNA hybrid, 20 mM 
Tris-HCl (pH 7.5 at 22.degree. C.), 100 mM KCl, 10 mM (NH.sub.4).sub.2 
SO.sub.4, 5 mM MgCl.sub.2, 0.15 mM .beta.-NAD, 50 .mu.g/ml BSA, 300 
units/ml E. coli DNA polymerase I, 12 units/ml E. coli RNase H, and 60 
units/ml E. coli DNA ligase. The reaction mixture was incubated at 
16.degree. C. for 1.5 hours and stopped by addition of 4 .mu.l of 2 mg/ml 
glycogen, 0.2 M EDTA The ds cDNA was extracted once with 
phenol/chloroform/isoamyl alcohol (25:24:1, vol/vol), once with 
chloroform/isoamyl alcohol (24:1, vol/vol), and then precipitated by 
addition of a half volume of 4 M ammonium acetate (about 35 .mu.l) and 3.7 
volumes of 95% ethanol (about 260 .mu.l). After vortexing, the tube was 
immediately centrifuged at 14,000 r.p.m. in a microcentrifuge for 20 
minutes. The pellet was washed with 80% ethanol without vortexing, 
centrifuged as above for 10 minutes, air dried, and dissolved in 10 .mu.l 
of deionized water. 
The full-length cDNA library was then packaged using standard protocol 
described in the laboratory manual by Sambrook et al. (1989). In order to 
confirm the quality of the library generated by the methods and materials 
of the present invention, we carried out the same quality control 
experiments as in Example 2 for the PCR-based technology. The size 
distribution and high efficiency cloning of full-length cDNAs library were 
similar for both libraries. 
These data show that the methods and compositions of the present invention 
can be used for cDNA library construction based on PRE adaptor-primer 
strategy to generate high quality cDNA libraries with a very high level of 
full-length cDNA clones. 
EXAMPLE 4 
Preferred Method for Amplification ds cDNA using CAPswitch technology and 
Using this ds cDNA in cDNA Subtraction 
The methods and materials of the subject invention were also used for 
producing high-quality cDNA from nanograms of total or poly A+ RNA. The 
flow chart in FIG. 1 (step 1 and 2) shows the main steps of this 
procedure. It will be apparent to those skilled in the art that some 
modification in the preferred protocol, such as structure of CDS primer, 
CAPswitch oligonucleotide, time of addition different reaction component 
can be varied without changing the efficiency of the whole procedure. 
In one embodiment of the subject of invention the ds cDNA amplified by 
methods and materials of the present invention has been used in 
combination with cDNA subtraction technology. When total RNA is used for 
cDNA synthesis by conventional methods, ribosomal RNA is transcribed along 
with poly A+ RNA fraction, even if synthesis is oligo(dT)-primed. If this 
cDNA is used for cDNA subtraction procedure, the excess of ribosomal RNA, 
impurities of genomic DNA and low concentration of cDNA corresponding to 
the poly A+RNA fraction results in inefficient subtractive hybridization. 
However, cDNA generated using methods and materials of the present 
invention can be directly used for subtractive hybridization 
procedure--even if total RNA was used as starting material. In a preferred 
sub-embodiment, tester and driver ds cDNA amplified by methods and 
materials of the present invention was used in combination with 
Suppression Subtractive Hybridization technology (Chenchik et al., U.S. 
Pat. No. 5,565,340). Other methods of subtractive hybridization, described 
for example, by Wigler et al. (U.S. Pat. No. 5,436,142); Hampson et al. 
(Nucl. Acids Res. 20:2899 (1992)); Yang et al. (Anal. Biochem. 
237:109-114(1996)); Balzer et al. (Nucl. Acids Res. 22:2853-2854(1994)), 
and others, can be employed using ss or ds cDNA corresponding tester 
and/or driver and amplified by according to the methods of the present 
invention. 
Step 1. First-strand synthesis--template switching. 
10 pmol of cDNA synthesis primer (oligo d(T) primer) CDS3: 
5'-d(AAGCAGTGGTAACAACGCAGAGTAC(T)30-3' (SEQ ID NO.67), 
and 10 pmol of CAPswitch oligonucleotide (Na1smG3): 
5'-d(AAGCAGTGGTAACAACGCAGAGTACGC)r(GGG)-3' (SEQ ID NO.68), 
were annealed in two separate test tubes to 1 mg of each tester and driver 
Total RNA (CLONTECH Laboratories, Inc.), in a volume of 5 ml of deionized 
water, by heating the mixture for 2 min at 70.degree. C., followed by 
cooling on ice for 2 min. First-strand cDNA synthesis was then initiated 
by mixing the annealed primer-RNA with 200 units of M-MLV RNAse H- reverse 
transcriptase (SuperScript II reverse transcriptase, Life Technologies) in 
a final volume of 10 ml, containing 50 mM Tris-HCl (pH 8.3 at 22.degree. 
C.), 75 mM KCl, 6 mM MgCl2, 1 mM DTT, 1 mM of each dATP, dGTP, dCTP and 
dTTP. The first-strand cDNA synthesis-template switching reaction was 
incubated at 42.degree. C. for 1.5 hr in an air incubator, then diluted by 
adding the 40 ml of TE buffer (10 mM Tris pH 7.6!, 1 mM EDTA) and heated 
at 72.degree. C. for 7 min. 
Step 2. cDNA Amplification 
PCR amplification of full-length cDNA was performed using the Advantage 
KlenTaq Polymerase Mix (CLONTECH Laboratories, Inc.). Amplification was 
conducted in a 100-ml volume containing 5 ml of diluted first-strand cDNA, 
40 mM Tricine-KOH (pH 9.2 at 22.degree. C.), 3.5 mM Mg(OAc)2, 10 mM KOAc, 
75 mg/ml BSA, 200 mM of each dATP, dGTP, dCTP and dTTP, 0.2 mM of PCR 
primer (Na1sm): 
5'-AAGCAGTGGTAACAACGCAGAGT-3' (SEQ ID NO.69) 
and 2 ml of KlenTaq Polymerase mix. Temperature parameters of the PCR 
reactions were as follows: 1 min at 95.degree. C. followed by 17-19 cycles 
of 95.degree. C. for 15 sec and 68.degree. C. for 5 min; followed by a 
10-min final extension at 68.degree. C. PCR products were examined on 1.2% 
agarose/EtBr gels in 1.times. TAE buffer. As a DNA size marker we used a 1 
Kb DNA Ladder (Life Technologies). Terminate reaction by adding 2 ml of 
0.5 M EDTA. 
Step 3. cDNA purification. 
To tester and driver PCR product combined each from two reaction tubes, add 
an equal volume of phenol:choloroform:isoamyl alcohol (25:24:1). Vortex 
thoroughly. Centrifuge the tubes at 14,000 rpm for 10 min to separate the 
phases. Remove the top (aqueous) layer and place it in a clean 1.5-ml 
tube. Add 700 ml of n-butanol and vortex the mix thoroughly. Butanol 
extraction allows you to concentrate PCR product to a volume of 40-70 ml. 
Centrifuge the solution at room temperature at 14,000 rpm for 1 min. 
Remove and discard the upper (n-butanol organic) phase. If you do not end 
up with a volume of 40-70 ml, repeat butanol extraction step with the same 
volume of n-butanol. Invert a 0.75 ml CHROMA SPIN-1000 (CLONTECH 
Laboratories, Inc.) column several times to completely resuspend the gel 
matrix. Remove the top cap from the column, and then remove the bottom 
cap. Place the column into a 1.5-ml centrifuge tube or a 17.times.100 mm 
tube. Discard any column buffer that immediately collects in the tube and 
add 1.5 ml of 1.times. TNE buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 
0.1 mM EDTA). Let the buffer drain through the column by gravity flow 
until you can see the surface of the gel beads in the column matrix. 
Discard the collected buffer and proceed with purification. Carefully and 
slowly apply the sample to the center of the gel beds flat surface. Do not 
allow any sample to flow along the inner wall of the column. Apply 25 ml 
of 1.times. TNE buffer and allow the buffer to completely drain out of the 
column. Apply 150 ml of 1.times. TNE buffer and allow the buffer to 
completely drain out of the column. Transfer column to a clean 1.5-ml 
microcentrifuge tube. Apply 320 ml of 1.times. TNE buffer and collect this 
as your purified cDNA fraction. To confirm that your PCR product is 
present in the purified cDNA fraction, perform the agarose/EtBr gel 
analysis as described in Step 2. 
Step 4. cDNA Subtraction. 
Perform an Rsa I digestion and cDNA subtraction as described in details by 
Chenchik et al. (U.S. Pat. No. 5,565,340). 
It should be understood that the examples and embodiments described herein 
are for illustrative purposes only and that various modifications or 
changes in light thereof will be suggested to persons skilled in the art 
and are to be included within the spirit and purview of this application 
and the scope of the appended claims. 
TABLE 1 
__________________________________________________________________________ 
Designation and 
SEQ ID NO. Sequence Information 
__________________________________________________________________________ 
3'1 5'- d(TGTAGCGTGAAGACGACAGAA)r(N).sub.12 
(SEQ ID NO. 1) 
3'2 5'- d(TGTAGCGTGAAGACGACAGAA(N).sub.11)r(N).sub.1 
(SEQ ID NO. 2) 
3'3 5'- d(TGTAGCGTGAAGACGACAGAA(N).sub.11) 
(SEQ ID NO. 3) 
3'4 5'- d(TGTAGCGTGAAGACGACAGAAGGATG(N).sub.9)r(N).sub.1 
(SEQ ID NO. 4) 
3'1-N4 5'- d(TGTAGCGTGAAGACGACAGAA)r(N).sub.4 
(SEQ ID NO. 5) 
3'1-N8 5'- d(TGTAGCGTGAAGACGACAGAA)r(N).sub.8 
(SEQ ID NO. 6) 
3'1-N12 5'- d(TGTAGCGTGAAGACGACAGAA)r(N).sub.16 
(SEQ ID NO. 7) 
3'1 5'-d(TCTAGAATTCAGCGGCCGC(T).sub.30 VN) 
(SEQ ID NO. 8) 
CSO1 5'-d(CTAATACGACTCACTATAGGGC)r(GGGp)-3' 
(SEQ ID NO. 9) 
3'CGTCATACTCCTGCTTGCTGATCCACATCTGC 
(SEQ ID NO. 10) 
3'CAATGTCCCAAACGTCACCAGAGA 
(SEQ ID NO. 11) 
CSP1 5'- d(CTAATACGACTCACTATAGGGC)- 3' 
(SEQ ID NO. 12) 
CSO2 5'-d(CTAATACGACTCACTATAGGGC)r(GGG)-3' 
(SEQ ID NO. 13) 
3'AATTCGCGGCCGCGTCGAC) 
(SEQ ID NO. 14) 
Complementary strand 
5'GCGCCGGCGCAGCTGp) 
to Ad1 
(SEQ ID NO. 15) 
3'2 5'- d(TCTAGAATTCTCGAGGCGGCCGC(T).sub.30 VN) 
(SEQ ID NO. 16) 
Complementary strand 
3' 3'- d(AGATCTTAAGAGCTCCGCCGGCG) 
to CDS2 
(SEQ ID NO. 17) 
CSO3 5'-d(TGCTGCGAGAAGACGACAGAATTCGG)r(GGG)-3' 
(SEQ ID NO. 18) 
Additional CAPswitch oligonucleotides 
3'1-G 5'- d(TGTAGCGTGAAGACGACAGAA)r(G) 
(SEQ ID NO. 19) 
3'1-G3 5'- d(TGTAGCGTGAAGACGACAGAA)r(GGG) 
(SEQ ID NO. 20) 
3'1-N4G3 5'- d(TGTAGCGTGAAGACGACAGAA)r(N.sub.4 G.sub.3) 
(SEQ ID NO. 21) 
Na21-GCGGCN4G3 
3' 5'- d(TGTAGCGTGAAGACGACAGAA)r(GCGGCN.sub.4 G.sub.3) 
(SEQ ID NO. 22) 
3'1-GTAAG3 5'- d(TGTAGCGTGAAGACGACAGAA)r(GTAAG.sub.3) 
(SEQ ID NO. 23) 
3'1-GATTG3 5'- d(TGTAGCGTGAAGACGACAGAA)r(GATTG.sub.3) 
(SEQ ID NO. 24) 
3'1-TGTTG3 5'- d(TGTAGCGTGAAGACGACAGAA)r(TGTTG.sub.3) 
(SEQ ID NO. 25) 
3'1-CTAAG3 5'- d(TGTAGCGTGAAGACGACAGAA)r(CTAAG.sub.3) 
(SEQ ID NO. 26) 
3'1-GGTAG3 5'- d(TGTAGCGTGAAGACGACAGAA)r(GGTAG.sub.3) 
(SEQ ID NO. 27) 
3'1-G2p 5'- d(TGTAGCGTGAAGACGACAGAA)r(GGp) 
(SEQ ID NO. 28) 
3'1-G3p 5'- d(TGTAGCGTGAAGACGACAGAA)r(GGGp) 
(SEQ ID NO. 29) 
3'1-G5p 5'- d(TGTAGCGTGAAGACGACAGAA)r(GGGGGp) 
(SEQ ID NO. 30) 
3'1N-g3 5'- d(TGATGCGAGTAGACGACAGAA)r(GGG) 
(SEQ ID NO. 31) 
3'1N-G3p 5'- d(TGATGCGAGTAGACGACAGAA)r(GGGp) 
(SEQ ID NO. 32) 
3'1N-G3p 5'- d(TGATGCGAGTAGACGACAGA)r(GGGp) 
(SEQ ID NO. 33) 
3'1B-G3p 5'- d(TACGATGCGAGTAGACGACAGAA)r(GGGp) 
(SEQ ID NO. 34) 
3'2-G3 5'- d(TGCTGCGAGAAGACGACAGAA)r(GGG) 
(SEQ ID NO. 35) 
3'2-G3p 5'- d(TGCTGCGAGAAGACGACAGAA)r(GGGp) 
(SEQ ID NO. 36) 
3'2M-G3 5'- d(TTGCTGGCAGAAGACGACAGA)r(GGG) 
(SEQ ID NO. 37) 
3'G 5'- d(CTAATACGACTCACTATAGGGC)r(G) 
(SEQ ID NO. 38) 
3'G2 5'- d(CTAATACGACTCACTATAGGGC)r(GG) 
(SEQ ID NO. 39) 
3'G3 5'- d(CTAATACGACTCACTATAGGGC)r(GGG) 
(SEQ ID NO. 40) 
3'G5 5'- d(CTAATACGACTCACTATAGGGC)r(GGGGG) 
(SEQ ID NO. 41) 
3'Gp 5'- d(CTAATACGACTCACTATAGGGC)r(Gp) 
(SEQ ID NO. 42) 
3'G2p 5'- d(CTAATACGACTCACTATAGGGC)r(GGp) 
(SEQ ID NO. 43) 
3'G3p 5'- d(CTAATACGACTCACTATAGGGC)r(GGGp) 
(SEQ ID NO. 44) 
3'G5p 5'- d(CTAATACGACTCACTATAGGGC)r(GGGGGp) 
(SEQ ID NO. 45) 
3'GCG 5'- d(CTAATACGACTCACTATAGGGC)r(GCG) 
(SEQ ID NO. 46) 
3'GCG2 5'- d(CTAATACGACTCACTATAGGGC)r(GCGG) 
(SEQ ID NO. 47) 
3'CG 5'- d(CTAATACGACTCACTATAGGGC)r(CG) 
(SEQ ID NO. 48) 
3'DG 5'- d(CTAATACGACTCACTATA)r(GGGCG) 
(SEQ ID NO. 49) 
3'N9G3 5'- d(CTAATACGACTCACTATAGGGC)r(N.sub.9 GGG) 
(SEQ ID NO. 50) 
3'GCG3 5'- d(CTAATACGACTCACTATAGGGC)r(GCGGG) 
(SEQ ID NO. 51) 
T7-SUP1 5'-d(CTAATACGACTCACTATAGGGCGCGGCCGCCCGGG)r(GCG3)-3' 
(SEQ ID NO. 52) 
d(CTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGG) 
(SEQ ID NO. 53) 
r(GCG3)-3' 
3'G3-NH2 5'-d(CTAATACGACTCACTATAGGGC)r(GGG-NH.sub.2) 
(SEQ ID NO. 54) 
(where NH2 is an amino group at the position 
of the ribose residue) 
3'G3-BIO 5'-d(CTAATACGACTCACTATAGGGC)r(GGG-BIO) 
(SEQ ID NO. 55) 
(where BIO is a biotin group at the position 
of the ribose residue) 
3'G3-GLY 5'-d(CTAATACGACTCACTATAGGGC)r(GGG-GLY) 
(SEQ ID NO. 55) 
(where GLY is a glycerol group at the position 
of the ribose residue) 
3'GAG3p 5'-d(CTAATACGACTCACTATAGGGC)r(GAGGGp) 
(SEQ ID NO. 57) 
3'GTG3p 5'-d(CTAATACGACTCACTATAGGGC)r(GTGGGp) 
(SEQ ID NO. 58) 
3'GGAG2p 5'-d(CTAATACGACTCACTATAGGGC)r(GGAGGp) 
(SEQ ID NO. 59) 
3'GGTG2p 5'-d(CTAATACGACTCACTATAGGGC)r(GGTGGp) 
(SEQ ID NO. 60) 
3'GACG2p 5'-d(CTAATACGACTCACTATAGGGC)r(GACGGp) 
(SEQ ID NO. 61) 
3'GATG2p 5'-d(CTAATACGACTCACTATAGGGC)r(GATGGp) 
(SEQ ID NO. 62) 
3'GTTG2p 5'-d(CTAATACGACTCACTATAGGGC)r(GTTGGp) 
(SEQ ID NO. 63) 
3'GAGTGp1 5'-d(CTAATACGACTCACTATAGGGC)r(GAGTGp) 
(SEQ ID NO. 64) 
3'-GGAG3p 5'-d(TCCTAATACGACTCACTATA)r(GGAGGGp) 
(SEQ ID NO. 65) 
3'GAG3p 5'-d(CTAATACGACTCACTATAGGGC)r(GGAGGp) 
(SEQ ID NO. 66) 
3'3 5'-d(AAGCAGTGGTAACAACGCAGAGTAC(T).sub.30) 
(SEQ ID NO. 67) 
3'smG3 5'-d(AAGCAGTGGTAACAACGCAGAGTACGC)r(GGG) 
(SEQ ID NO. 68) 
3'sm 5'- d(AAGCAGTGGTAACAACGCAGAGT) 
(SEQ ID NO. 69) 
3'1-Bio 5'- d(Bio-TGTAGCGTGAAGACGACAGAA) 
(SEQ ID NO. 70) 
3' 5'- d(CTAATACGACTCACTATAGGGC) 
(SEQ ID NO. 71) 
3'7 5'- d(TGCCATCCTAATACGACTCACTA) 
(SEQ ID NO. 72) 
3'Fok 5'- d(CTAATACGACTCACGGATGGGC) 
(SEQ ID NO. 73) 
3'1N 5'- d(TGATGCGAGTAGACGACAGAA) 
(SEQ ID NO. 74) 
Na22 5'- d(TGCTGCGAGAAGACGACAGAA)- 3' 
(SEQ ID NO. 75) 
3'2M 5'- d(TTGCTGGCAGAAGACGACAGA) 
(SEQ ID NO. 76) 
3'1B 5'- d(TACGATGCGAGTAGCGACAGAA) 
(SEQ ID NO. 77) 
3' 5'- d(TGACCAGTGAGCAGAGTGACGA) 
(SEQ ID NO. 78) 
3' 5'- d(CCATCCAATTAACCCTCACTAAAGGGC) 
(SEQ ID NO. 79) 
3'M 5'- d(AAGCAGAGGCAACAACGCAGA) 
(SEQ ID NO. 80) 
3'2 5'- d(ACAAGACGAAGCACAAGAGGGC) 
(SEQ ID NO. 81) 
3'M 5'- d(AAGCAGAGGCAACAACGCAGA) 
(SEQ ID NO. 82) 
3' 5'- d(TTCCGCTTGTCTGCTGGGC) 
(SEQ ID NO. 83) 
3'AS4 5'- d(CGTGCGGCCGCTTCGAG-NH.sub.2) 
(SEQ ID NO. 84) 
3'AS 5'- d(GAGCGGCCGCACGAG-NH.sub.2) 
(SEQ ID NO. 85) 
3'-G3 5'- r(CTAATACGACTCACTATAGGGCGGG) 
(SEQ ID NO. 86) 
3'Fok-G3 5'- r(CTAATACGACTCACTATAGGGCGGATGGG) 
(SEQ ID NO. 87) 
3'In3 5'- r(CTAATACGACTCACTATAGGGC)d(III) 
(SEQ ID NO. 88) 
3'Un3 5'- r(CTAATACGACTCACTATAGGGC)d (UUU) 
(SEQ ID NO. 89) 
Na1Sup2M 5'-(AAGCAGAGGCAACAACGCAGAGAGGGCAGCAGGCAGC)r(GGG)-3' 
(SEQ ID NO. 90) 
RT7NS2M 5'-(AGACGAAGCACAAGAGGGCACGAGCGGCCGCACGGCG)r(GGG)-3' 
(SEQ ID NO. 91) 
RT7NSM-F 5'-(ATACGACTCACTATAGGGCTCGAGCGGCCGCACGGCG)r(GGG-F)-3' 
(SEQ ID NO. 92) 
Na1SMG3-F 5'-(AAGCAGAGTGCTAACAACGCAGAGTACGC)r(GGG-F)-3' 
(SEQ ID NO. 93) 
T7-G3F 5'-(ATACGACTCACTATAGGGC)r(GGG-F)-3' 
(SEQ ID NO. 94) 
T7-GCG3F 5'-(ATACGACTCACTATAGGGC)r(GCGGG-F)-3' 
(SEQ ID NO. 95) 
T3-G3 5'-(ATTAACCCTCACTAAAGGGC)r(GGG)-3' 
(SEQ ID NO. 96) 
T3-NSM 5'-(ATTAACCCTCACTAAAGGGCTCGAGCGGCCGCACGGCG)r(GGG)-3' 
(SEQ ID NO. 97) 
3'SM-GCG3 5'd(AAGCAGTGGTATCAACGCAGAGTAC)r(GCGGG) 
(SEQ ID NO. 98) 
3'-GCG3 5'-d(AAGCAGTGGTAACAACGCAGAGT)r(GCGGG) 
(SEQ ID NO. 99) 
Na1Sup1 5'-d(AAGCAGTGGTAACAACGCAGAGTGGGCAGCAGGCA)r(GCGGG)-3' 
(SEQ ID NO. 100) 
3'M-G3 5'-d(AAGCAGTGGTATCAACGCAGAGTACGC)r(GGG) 
(SEQ ID NO. 101) 
3'SMG3 5'-d(AAGCAGTGGTAACAACGCAGAGTACGC)r(GGG) 
(SEQ ID NO. 102) 
Na1Sup1M 5'-d(AAGCAGTGGTAACAACGCAGAGTGGGCAGCAGCCAGC)r(GGG)-3' 
(SEQ ID NO. 103) 
RT7NSM 5'-d(ATACGACTCACTATAGGGCTCGAGCGGCCGCACGGCG)r(GGG)-3' 
(SEQ ID NO. 104) 
RT7NS2 5'-d(ATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG)r(G 
(SEQ ID NO. 105) 
3' GG) 
RT7Sup4 5'-d(ATACGACTCACTATAGGGCTCGGTCGGGCAGGCACGGCG)r(G 
(SEQ ID NO. 106) 
3' GG) 
3BP-NSMT 5'-d(TTCCGCTTGTCTGCTGGGCTCGTGCGGCCGCTCGGCG)r(GGG)-3' 
(SEQ ID NO. 107) 
RT7-NS3M 5'-d(AGACGAAGCACAAGAGGGCACGAGCAGCGGCACGGCG)r(GGG)-3' 
(SEQ ID NO. 108) 
Fr-T30NN 5'-d(TGACCAGTGAGCAGAGTGACGAGTAC(T).sub.30 VN)-3' 
(SEQ ID NO. 109) 
3'-T30NN 5'-d(CCATCCTAATACGACTCACTATAGGGC(T).sub.30 VN) 
(SEQ ID NO. 110) 
3'o-T7-T30NN 5'-d(Bio-AATACGACTCACTATAGGGC(T).sub.30 VN) 
(SEQ ID NO. 111) 
(SEQ ID NO. 112) 
5'-d(TGCTGCGGAAGACGACAGAA)r(GGG)-3' 
(SEQ ID NO. 113) 
5'-d(TGCTGCGGAAGACGACAGAA)-3' 
(SEQ ID NO. 114) 
5'-d(AATTCGAGCGGCCGC(T).sub.30 VN)-3' 
__________________________________________________________________________ 
V = G or A or C 
N = G or A or T or C 
p is 3phosphate group 
NH2 is an amino group 
BIO is a biotin group 
GLY is a glycerol group 
I is inosine 
U is deoxyribofuranosyl5-nitroindole phosphate 
F is a fluoro group at the 3' position of the ribose residue 
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Gelfand, D. H. "Reverse transcription with thermostable DNA 
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5,322,770 Issued Jun. 21, 1994. 
Li, W. -B., Gruber, C. E., Jessee, J. A. and Lin, J. -J. "Method of nucleic 
acid sequence selection" U.S. Pat. No. 5,500,356 Issued Mar. 19, 1996. 
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specific nucleic acid sequence and subsequent transformation with 
preselected clones. U.S. Pat. No. 5,484,702 Issued Jan. 16, 1996. 
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Gurskaya, N., Tarabykin, V. and Sverdlov, E. Method for Suppressing DNA 
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Patent: Oct. 15, 1996. 
Wigler, M. and Lisitsyn, N. Methods for Producing Probes Capable of 
Distingushing Variant Genomic Sequences. U.S. Pat. No. 5,436,142. Date of 
Patent: Jul. 25, 1995. 
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__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 114 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
# 33 CAGA ANNNNNNNNN NNN 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
# 33 CAGA ANNNNNNNNN NNN 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
# 32 CAGA ANNNNNNNNN NN 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 36 CAGA AGGATGNNNN NNNNNN 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
# 25 CAGA ANNNN 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
# 29 CAGA ANNNNNNNN 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
# 37 CAGA ANNNNNNNNN NNNNNNN 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
# 51GGCCGCT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTV N 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
# 25 TAGG GCGGG 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
# 35 CTTG CTGATCCACA TCTGC 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
# 26 TCAC CAGAGA 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
# 22AGG GC 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
# 25 TAGG GCGGG 
- (2) INFORMATION FOR SEQ ID NO:14: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
# 19 GAC 
- (2) INFORMATION FOR SEQ ID NO:15: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
# 15 
- (2) INFORMATION FOR SEQ ID NO:16: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- TCTAGAATTC TCGAGGCGGC CGCTTTTTTT TTTTTTTTTT TTTTTTTTTT TT - #TVN 
55 
- (2) INFORMATION FOR SEQ ID NO:17: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
# 23TTCT AGA 
- (2) INFORMATION FOR SEQ ID NO:18: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
# 29 CAGA ATTCGGGGG 
- (2) INFORMATION FOR SEQ ID NO:19: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
# 22AGA AG 
- (2) INFORMATION FOR SEQ ID NO:20: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
# 24CAGA AGGG 
- (2) INFORMATION FOR SEQ ID NO:21: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
# 28 CAGA ANNNNGGG 
- (2) INFORMATION FOR SEQ ID NO:22: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
# 33 CAGA AGCGGCNNNN GGG 
- (2) INFORMATION FOR SEQ ID NO:23: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
# 28 CAGA AGTAAGGG 
- (2) INFORMATION FOR SEQ ID NO:24: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
# 28 CAGA AGATTGGG 
- (2) INFORMATION FOR SEQ ID NO:25: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
# 28 CAGA ATGTTGGG 
- (2) INFORMATION FOR SEQ ID NO:26: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
# 28 CAGA ACTAAGGG 
- (2) INFORMATION FOR SEQ ID NO:27: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
# 28 CAGA AGGTAGGG 
- (2) INFORMATION FOR SEQ ID NO:28: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
# 23CAGA AGG 
- (2) INFORMATION FOR SEQ ID NO:29: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
# 24CAGA AGGG 
- (2) INFORMATION FOR SEQ ID NO:30: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
# 26 CAGA AGGGGG 
- (2) INFORMATION FOR SEQ ID NO:31: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
# 24CAGA AGGG 
- (2) INFORMATION FOR SEQ ID NO:32: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
# 24CAGA AGGG 
- (2) INFORMATION FOR SEQ ID NO:33: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
# 23CAGA GGG 
- (2) INFORMATION FOR SEQ ID NO:34: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
# 26 GACA GAAGGG 
- (2) INFORMATION FOR SEQ ID NO:35: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
# 24CAGA AGGG 
- (2) INFORMATION FOR SEQ ID NO:36: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
# 24CAGA AGGG 
- (2) INFORMATION FOR SEQ ID NO:37: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
# 24ACAG AGGG 
- (2) INFORMATION FOR SEQ ID NO:38: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
# 23TAGG GCG 
- (2) INFORMATION FOR SEQ ID NO:39: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
# 24TAGG GCGG 
- (2) INFORMATION FOR SEQ ID NO:40: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
# 25 TAGG GCGGG 
- (2) INFORMATION FOR SEQ ID NO:41: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
# 27 TAGG GCGGGGG 
- (2) INFORMATION FOR SEQ ID NO:42: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
# 23TAGG GCG 
- (2) INFORMATION FOR SEQ ID NO:43: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
# 24TAGG GCGG 
- (2) INFORMATION FOR SEQ ID NO:44: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
# 25 TAGG GCGGG 
- (2) INFORMATION FOR SEQ ID NO:45: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
# 27 TAGG GCGGGGG 
- (2) INFORMATION FOR SEQ ID NO:46: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
# 25 TAGG GCGCG 
- (2) INFORMATION FOR SEQ ID NO:47: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
# 26 TAGG GCGCGG 
- (2) INFORMATION FOR SEQ ID NO:48: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
# 24TAGG GCCG 
- (2) INFORMATION FOR SEQ ID NO:49: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
# 23TAGG GCG 
- (2) INFORMATION FOR SEQ ID NO:50: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
# 34 TAGG GCNNNNNNNN NGGG 
- (2) INFORMATION FOR SEQ ID NO:51: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
# 27 TAGG GCGCGGG 
- (2) INFORMATION FOR SEQ ID NO:52: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 
# 40 TAGG GCGCGGCCGC CCGGGGCGGG 
- (2) INFORMATION FOR SEQ ID NO:53: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 47 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: 
# 47TAGG GCACGCGTGG TCGACGGCCC GGGCGGG 
- (2) INFORMATION FOR SEQ ID NO:54: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: 
# 25 TAGG GCGGG 
- (2) INFORMATION FOR SEQ ID NO:55: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: 
# 25 TAGG GCGGG 
- (2) INFORMATION FOR SEQ ID NO:56: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: 
# 25 TAGG GCGGG 
- (2) INFORMATION FOR SEQ ID NO:57: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: 
# 27 TAGG GCGAGGG 
- (2) INFORMATION FOR SEQ ID NO:58: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: 
# 27 TAGG GCGTGGG 
- (2) INFORMATION FOR SEQ ID NO:59: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: 
# 27 TAGG GCGGAGG 
- (2) INFORMATION FOR SEQ ID NO:60: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: 
# 27 TAGG GCGGTGG 
- (2) INFORMATION FOR SEQ ID NO:61: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 
# 27 TAGG GCGACGG 
- (2) INFORMATION FOR SEQ ID NO:62: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: 
# 27 TAGG GCGATGG 
- (2) INFORMATION FOR SEQ ID NO:63: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: 
# 27 TAGG GCGTTGG 
- (2) INFORMATION FOR SEQ ID NO:64: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: 
# 27 TAGG GCGAGTG 
- (2) INFORMATION FOR SEQ ID NO:65: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: 
# 26 TATA GGAGGG 
- (2) INFORMATION FOR SEQ ID NO:66: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: 
# 27 TAGG GCGAGGG 
- (2) INFORMATION FOR SEQ ID NO:67: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: 
- AAGCAGTGGT AACAACGCAG AGTACTTTTT TTTTTTTTTT TTTTTTTTTT TT - #TTT 
55 
- (2) INFORMATION FOR SEQ ID NO:68: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 
# 30 GCAG AGTACGCGGG 
- (2) INFORMATION FOR SEQ ID NO:69: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 
# 23GCAG AGT 
- (2) INFORMATION FOR SEQ ID NO:70: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: 
#21 CAGA A 
- (2) INFORMATION FOR SEQ ID NO:71: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 
# 22AGG GC 
- (2) INFORMATION FOR SEQ ID NO:72: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: 
# 23CTCA CTA 
- (2) INFORMATION FOR SEQ ID NO:73: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: 
# 22TGG GC 
- (2) INFORMATION FOR SEQ ID NO:74: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: 
#21 CAGA A 
- (2) INFORMATION FOR SEQ ID NO:75: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: 
#21 CAGA A 
- (2) INFORMATION FOR SEQ ID NO:76: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: 
#21 ACAG A 
- (2) INFORMATION FOR SEQ ID NO:77: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: 
# 23GACA GAA 
- (2) INFORMATION FOR SEQ ID NO:78: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: 
# 22GAC GA 
- (2) INFORMATION FOR SEQ ID NO:79: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: 
# 27 CACT AAAGGGC 
- (2) INFORMATION FOR SEQ ID NO:80: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: 
#21 GCAG A 
- (2) INFORMATION FOR SEQ ID NO:81: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: 
# 22AGG GC 
- (2) INFORMATION FOR SEQ ID NO:82: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: 
#21 GCAG A 
- (2) INFORMATION FOR SEQ ID NO:83: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: 
# 19 GGC 
- (2) INFORMATION FOR SEQ ID NO:84: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: 
# 17 G 
- (2) INFORMATION FOR SEQ ID NO:85: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: 
# 15 
- (2) INFORMATION FOR SEQ ID NO:86: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: RNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: 
# 25 TAGG GCGGG 
- (2) INFORMATION FOR SEQ ID NO:87: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: RNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: 
# 29 TAGG GCGGATGGG 
- (2) INFORMATION FOR SEQ ID NO:88: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: RNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: 
# 25 TAGG GCNNN 
- (2) INFORMATION FOR SEQ ID NO:89: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: RNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: 
# 25 TAGG GCNNN 
- (2) INFORMATION FOR SEQ ID NO:90: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: 
# 40 GCAG AGAGGGCAGC AGGCAGCGGG 
- (2) INFORMATION FOR SEQ ID NO:91: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: 
# 40 GGCA CGAGCGGCCG CACGGCGGGG 
- (2) INFORMATION FOR SEQ ID NO:92: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: 
# 40 GGCT CGAGCGGCCG CACGGCGGGG 
- (2) INFORMATION FOR SEQ ID NO:93: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: 
# 32 ACGC AGAGTACGCG GG 
- (2) INFORMATION FOR SEQ ID NO:94: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: 
# 22GCG GG 
- (2) INFORMATION FOR SEQ ID NO:95: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95: 
# 24GGCG CGGG 
- (2) INFORMATION FOR SEQ ID NO:96: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: 
# 23GGGC GGG 
- (2) INFORMATION FOR SEQ ID NO:97: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: 
# 41 GGGC TCGAGCGGCC GCACGGCGGG G 
- (2) INFORMATION FOR SEQ ID NO:98: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: 
# 30 GCAG AGTACGCGGG 
- (2) INFORMATION FOR SEQ ID NO:99: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: 
# 28 GCAG AGTGCGGG 
- (2) INFORMATION FOR SEQ ID NO:100: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: 
# 40 GCAG AGTGGGCAGC AGGCAGCGGG 
- (2) INFORMATION FOR SEQ ID NO:101: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: 
# 30 GCAG AGTACGCGGG 
- (2) INFORMATION FOR SEQ ID NO:102: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: 
# 30 GCAG AGTACGCGGG 
- (2) INFORMATION FOR SEQ ID NO:103: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: 
# 40 GCAG AGTGGGCAGC AGCCAGCGGG 
- (2) INFORMATION FOR SEQ ID NO:104: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: 
# 40 GGCT CGAGCGGCCG CACGGCGGGG 
- (2) INFORMATION FOR SEQ ID NO:105: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: 
# 42 GGCT CGAGCGGCCG CCCGGGCAGG GG 
- (2) INFORMATION FOR SEQ ID NO:106: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: 
# 42 GGCT CGGTCGGGCA GGCACGGCGG GG 
- (2) INFORMATION FOR SEQ ID NO:107: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: 
# 40 GGCT CGTGCGGCCG CTCGGCGGGG 
- (2) INFORMATION FOR SEQ ID NO:108: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: 
# 40 GGCA CGAGCAGCGG CACGGCGGGG 
- (2) INFORMATION FOR SEQ ID NO:109: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 58 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: 
- TGACCAGTGA GCAGAGTGAC GAGTACTTTT TTTTTTTTTT TTTTTTTTTT TT - #TTTTVN 
58 
- (2) INFORMATION FOR SEQ ID NO:110: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 59 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: 
- CCATCCTAAT ACGACTCACT ATAGGGCTTT TTTTTTTTTT TTTTTTTTTT TT - #TTTTTVN 
59 
- (2) INFORMATION FOR SEQ ID NO:111: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 52 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: 
- AATACGACTC ACTATAGGGC TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT VN - # 
52 
- (2) INFORMATION FOR SEQ ID NO:112: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: 
# 23AGAA GGG 
- (2) INFORMATION FOR SEQ ID NO:113: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: 
# 20 AGAA 
- (2) INFORMATION FOR SEQ ID NO:114: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 47 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (synthetic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: 
# 47TTTT TTTTTTTTTT TTTTTTTTTT TTTTTVN 
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