Method for generating directly covalent bonding between interstrand nucleotides

The present invention provides a fast, simple and direct covalent bond formation between two strands of nucleotide sequences. Non-modified first strand nucleotide sequences are hybridized with second strand nucleotide sequences, of which certain specific base structure(s) is modified by chemical reagents in order to generate covalent bonding with the first strand. While the hybridization of these two strand nucleotide sequences generates double-stranded hybrid duplexes between their homologues, covalent bond formation occurs in the region of modified base-pairs. Since neither a polymerase chain restriction nor a restriction enzyme digestion can be performed with the covalently bonded hybrid duplexes, the present invention can be used to subtract common sequences during subtractive hybridization, to inhibit nonspecific contamination during subcloning and to increase binding stability of antisense probes during in situ hybridization as well as gene therapy.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention generally relates to the field of methods for 
generating covalently interstrand bonding between nucleotides. More 
particularly, the present invention relates to the field of improved 
methods of directly covalent bond formation between homologous nucleotide 
sequences. 
2. Description of the Prior Art 
The following references are pertinent to this invention: 
1. Falletta et.al., "Phase 1 Evaluation of Diaziquone in Childhood Cancer", 
Investigational New Drugs 8: 167-170 (1990). 
2. Hartley et.al., "DNA Cross-linking and Sequence Selectivity of 
Aziridinylbenzoquinones", Biochemistry 30: 11719-11724 (1991). 
3. Hampson et.al., "Chemical Crosslinking Subtraction; A New Method for the 
Generation of subtractive hybridization probes", Nucleic Acids Res. 20: 
2899 (1992). 
4. Kimler et.al., "Combination of Aziridinylbenzoquinone and Cis-platinum 
with Radiation Therapy in the 9L Rat Brain Tumor Model", International 
Journal of Radiation Oncology, Biology, Physics 26: 445-450 (1993). 
5. Lehninger et.al., "Principles of Biochemistry, 2nd Edition", Worth 
Press, pp342-343 (1993). 
6. Lisitsyn et.al., "Cloning the Differences Between Two Complex Genomes", 
Science 259: 946-951 (1993). 
7. Sambrook et.al., "Molecular Cloning, 2nd Edition", Cold Spring Harbor 
Laboratory Press, p10.45 (1989). 
8. Solomons et.al., "Organic Chemistry, 6th Edition", John Wiley & Sons 
Press, pp 693, 803-804 (1996). 
9. Tan et.al., "Phase 1 Study of Azinridinylbenzoquinone in Children with 
Cancer", Cancer Research 44: 831-835 (1984). 
10. Wicland et.al., "A Method for Difference Cloning; Gene Amplification 
Following Subtractive Hybridization", Proc. Natl. Acad. Sci. USA 87: 
2720-2724 (1990). 
11. Ueli et.al., "A Simple and Very Efficient Method for Generating cDNA 
Libraries", Gene, 25: pp263-269 (1983). 
12. U.S. Pat. No. 5,589,339 issued to Hampson. 
13. U.S. Pat. No. 5,591,575 issued to Hampson. 
The ability to form covalent bonding between two nucleotide sequences has 
permitted a complete subtraction of common sequences during subtractive 
hybridization as well as a fully stable probing activity during 
in-situ-hybridization and antisense therapy. Because the covalent bonding 
is one of the strongest and most heat-stable interactions between 
molecules, the covalent bonding between two nucleotide sequences can 
sustain some harsh procedures, such as denature, salting and enzyme 
digestion. Based on such property, some methods have been developed either 
to perform chemotherapy or to isolate specific nucleotide sequences with 
external cross-linking chemicals by which two nucleotide strands were 
indirectly bonded. One of the most commonly used cross-linking chemicals 
to accomplish such sequence selectivity is aziridinylbenzoquinone 
(AZQ)-class agent (Hartley et.al., Biochemistry 30: 11719-11724 (1991)), 
involving the cross-linking of guanine and cytosine. 
AZQ-class agents have been used in the chemotherapy of some cancers, such 
as brain tumor in rats (Kimler et.al., International Journal of Radiation 
Oncology, Biology, Physics 26: 445-450 (1993)) and phase 1 childhood 
cancer in human (Falletta et.al., Investigational New Drugs 8: 167-170 
(1990); Tan et.al., Cancer Research 44: 831-835 (1984)). However, although 
AZQ successfully raises the bonding stability of double-stranded genome 
and reduces the replication of cancer cells, the non-specific 
cross-linking feature of AZQ also causes significant toxicity to the 
normal cells. Since the AZQ lacks sequence-specific targeting capability 
in vivo, some in vitro methods have been designed to detect and isolate 
specific nucleotide sequences with AZQ which cross-links common sequences 
of two compared nucleotide libraries. 
Prior art attempts at simplifying subtraction with covalent affinity, such 
as U.S. Pat. No. 5,589,339 and U.S. Pat. No. 5,591,575 to Hampson, also 
uses an AZQ interstrand cross-linking agent to covalently subtract common 
sequences from a tester library. In brief, this method relies upon the 
generation of single-stranded tester and driver libraries which contain 
all sense or all antisense sequences. After the tester is hybridized with 
the driver, resulting in hybrid duplex formation if a sequence is common 
to both libraries, the AZQ is added to generate externally covalent bonds 
between the hybrid duplexes. Because the AZQ cross-links all 
double-stranded sequences, this kind of covalent-bonding nature greatly 
increases the completion of homologue subtraction after hybridization. 
However, in this method, both of the initial tester and driver must be all 
single strands due to the interstrand cross-linking nature of the 
AZQ-class agents, resulting in no use of genomic DNA samples, no detection 
of limited initial materials and no specific-primer amplification of final 
results. These disadvantages cause more restrictions of sample selection, 
less stability of sample storage and less sensitivity of final result 
detection in comparison with traditional methods. Also, the determination 
of final desired sequences is completed by a non-specific random-primer 
extension reaction which lowers the specificity of final results. 
In summary, it is desirable to have a fast, specific and direct covalent 
bonding method for subtracting common sequences in a subtractive 
hybridization procedure as well as for increasing probing specificity in a 
gene targeting system, of which the results may contribute to developing a 
screening method for new genes, a diagnosis for inherent problems, or a 
therapy for diseases. 
SUMMARY OF THE INVENTION 
The present invention is a novel bonding generation method which provides a 
fast, simple, specific and direct covalent bond formation between 
nucleotide homologues. 
Described in detail, a preferred embodiment of the present invention method 
includes the following steps: 
a. providing a first strand of nucleotide sequences, wherein said first 
strand of nucleotide sequences is not modified by amino-blocking agent in 
order to preserve activating amino-groups on its nucleotide base 
structure(s); 
b. contacting said first strand of nucleotide sequences in denatured form 
with a second strand of denatured nucleotide sequences, wherein said 
second strand of denatured nucleotide sequences is single-stranded by 
amino-blocking agent and then modified by carboxylating agent in its 
nucleotide base structure(s), to form a denatured mixture; and 
c. permitting said first strand and said second strand of nucleotide 
sequences in said denatured mixture to form double-stranded hybrid 
duplexes comprising covalent bonding between the activating amino-groups 
of said first strand and the modified carboxyl-groups of said second 
strand of nucleotide sequences. 
The preferred embodiment of the present invention method additionally may 
include the pre-steps of forming double-stranded amplicon DNA of the 
sample, and prior to commencing the aforementioned step (a): 
(1) restricting the initial nucleotide sequences with a restriction 
endonuclease to generate cohesive termini on both ends; 
(2) ligating an adaptor to the ends of the restricted sequences for 
generating a complementary region of a specific primer; and 
(3) incubating the ligated DNA in denatured form with the specific primer 
under conditions sufficient to permit the template-dependent extension of 
the primer to thereby enrich the amount of the initial nucleotide 
sequences. 
In one aspect of this embodiment, step (b) and (c) are repeated on said 
hybrid duplexes at least one once. According to another aspect of this 
preferred embodiment, the initial sequences are amplified, preferably, by 
PCR in the pre-step (3). 
To increase the binding force of heterohybrid duplexes, the second strand 
of nucleotide sequences is preferably carboxylated on the C-4 of 
uracil/thymine or C-5/C-6 of pyrimidines in order to covalently bonding 
with the amino-groups of the first strand sequences on the C-6 of adenine 
or C-6/C-2 of purines respectively. Most preferably, the carboxylated 
group is on the C-5 of uracil which covalently bond to the C-6 amino-group 
of adenine. Advantageously, the covalent bonds of the heterohybrid 
duplexes can not be broken during amplification or cloning by which the 
common sequences of both compared strands can be selected out and the 
unique sequences of the first strand can be isolated. 
To prevent the reassociation of undesired second strand sequence duplexes 
during hybridization, the amino-groups of the second strand sequences are 
blocked or removed by chemically blocking agent(s). Preferably, the 
blocking agent is alkaline acetic chloride reagent which converts the 
activating amino-groups of purines into inactive acetamido-groups for 
preventing hydrogen bond formation between double-stranded nucleotide 
sequences. Advantageously, the denatured and modified second strand 
sequences only covalently hybridize with the homologues of the first 
strand sequences under specific condition, resulting in an increase of 
bonding efficiency. Preferably, the specific condition is under alkaline 
heat-stable buffer in which the blocked amino-groups of the second strand 
sequences are released. 
The present invention also includes a kit for performing improved directly 
covalent bond formation between interstrand nucleotides, comprising some 
or all of the following components: 
a. an amino-blocking agent which makes said second strand of nucleotide 
sequences single-stranded; 
b. a carboxylating agent which generates carboxyl-groups on the nucleotide 
base of said second strand of nucleotide sequences; and 
c. a hybridization buffer which permits said first strand and said second 
strand of nucleotide sequences in a denatured mixture to form directly 
covalent-bonded hybrid duplexes. 
Preferably, the amino-blocking agent is acetic anhydride reagent or 
alkaline acetic chloride reagent, and the carboxylating agent is hot 
alkaline potassium permanganate reagent. Preferably, the hybridization 
buffer is alkaline N-[2-hydroxyethyl]piperazine-N'-[3-propanesulfonic 
acid] (EPPS) and [ethylenediamine]tetraacetic acid (EDTA) mixture buffer. 
Further novel features and other objects of the present invention will 
become apparent from the following detailed description, discussion and 
the appended claims, taken in conjunction with the drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
Although specific embodiments of the present invention will now be 
described with reference to the drawings, it should be understood that 
such embodiments are by way of example only and merely illustrative of but 
a small number of the many possible specific embodiments which can 
represent applications of the principles of the present invention. Various 
changes and modifications obvious to one skilled in the art to which the 
present invention pertains are deemed to be within the spirit, scope and 
contemplation of the present invention as further defined in the appended 
claims. 
The present invention is directed to an improved methods of directly 
covalent bond formation between nucleotide sequences, particularly between 
certain specific base-pairing of two strand homologous nucleotide 
sequences. This method is primarily designed for quickly subtracting 
common sequences during subtractive hybridization, inhibiting nonspecific 
contamination during cloning, and increasing binding stability of 
antisense probes dunng in situ hybridization as well as gene therapy. The 
purpose of the present invention relies on the subtraction force of the 
covalent-bonding between homologous sequences (homologues) during a 
polymerase chain reaction (PCR) or cloning, resulting in no contamination 
of the homologues. The preferred version of the present invention is based 
on: single-stranding of one strand nucleotide sequences, covalent 
modification of the single-stranded nucleotide sequences, and 
hybridization of the modified nucleotide sequences with another 
non-modified nucleotide sequences to form directly covalent bonding in 
specific base-pairs. In conjunction with an adaptor-ligation and a 
specific amplification, a very small amount of nucleotide sequences can be 
used as an initial sample for this method. 
As used herein, the covalent modification refers to a series of redox 
reactions which the capability of directly covalent bonding with 
non-modified nucleotide sequences is render to the modified nucleotide 
sequences and is generated herein by using amino-blocking reagent and 
carboxylating reagent. The amino-blocking reagent refers to a chemical 
which can block or remove the amino-group of a purine base, such as acetic 
anhydride and alkaline acetic chloride. And, the carboxylating reagent 
refers to a chemical that can generate a carboxyl-group on the base 
structure of modified nucleotide sequences in order to form covalent 
bonding with non-modified nucleotide sequences, such as hot alkaline 
potassium permanganate. The homologues refer to the homologous (common) 
sequences which are common to both populations of the modified and 
non-modified nucleotide sequences. 
The advantages of using covalently modified nucleotide sequences are as 
follows: First, during hybridization, the affinity between homologues can 
be greatly enhanced by the covalent modification, such as the 
carboxyl-group on the C-5/C-6 of modified pyrimidines, resulting in 
peptide-bonding with the activating amino-group on the C-6/C-2 of 
non-modified purines respectively (FIG. 1). Such covalent peptide-bonding 
between homologues fully inhibit any further reaction of the homologues 
and therefore reduces the contamination of common nonspecific sequences. 
Second, the covalently modified sequences are single-stranded, resulting 
in high bonding efficiency of heterohybrid strand association between the 
modified and non-modified strand rather than between two modified strands. 
Third, because the covalent bonding is an internal affinity either between 
adenine and modified uracil or between guanine and modified cytosine, such 
covalent pairing feature significantly increases the specificity of 
covalent bonding which is generated only between sequences with highly 
matched base pairing. 
Because the covalent modification can be greatly facilitated by using 
nucleotide analog-incorporated sequences, the nucleotide sequences is 
preferably digested by a restriction-endonuclease on both ends and ligated 
to a specific adaptor for incorporating nucleotide analog(s) with a 
template-dependent primer-extension reaction in the presence of a specific 
primer, preferably using a deoxyuridine triphosphate analog and the 
primer: SEQ ID NO. 2 5'-pCGGTAGTGACTCGGTTAAGATCGC-3'. For example, when 
2'-deoxy-deoxyuridine triphosphate instead of deoxythymidine triphosphate 
is used to generate the modified sequences, the carboxylation reaction 
will occur only on the C-4 of uracil rather than the C-2 which is 
sometimes carboxylated if using deoxythymidine triphosphate. Preferably, 
here listed below are some example compounds of the incorporated analog 
formula: 
##STR1## 
in which A, B, D, E and F are selected from either a N or a CH group, G is 
a 2'-deoxy-D-ribose triphosphates, and X is a methyl group while Y is a H 
group and vice versa. Although specially designed adaptors/primers were 
used to generate analog-incorporated sequences, any oligonucleotide 
capable of being extended into analog-incorporated sequences for the 
purpose of directly covalent bond formation between nucleotide homologues 
is within the scope of the present invention. On the other hand, when 
initial sample is very limited, compared non-modified nucleotide sequences 
will also need to be amplified by the same procedure as aforementioned but 
without incorporated nucleotide analog and using another primer: SEQ ID 
NO. 1 5'-pGCCACCAGAAGAGCGTGTACGTCC-3'. 
In order to prevent the formation of covalent bonding between two modified 
strands, blocking the activating amino-groups of modified nucleotide 
sequences must be completed before covalent modification. Such blocking 
reaction (reduction reaction) is preferably carried out by acetylating the 
amino-group of purines to form an inactive acetamido-group (Solomons et. 
al., 1996) which is incapable of bonding to a carboxyl-group of the 
modified sequences, resulting in single-stranding modified sequences. 
Acetic anhydride and alkaline acetic chloride are major ingredients in the 
preferred amino-blocking reagent of the present invention. Because the 
single-stranded nucleotide sequences do not protect the base structure of 
its nucleotides from oxidative modification (oxidation reaction) any more, 
a carboxylating agent can easily oxidizes the alkene, carbonyl or 
sometimes methyl group (Solomons et.al., 1996) on the bases of the 
single-stranded sequences into a carboxyl-group which then forms a 
covalent peptide-bonding with the activating amino-group of a non-modified 
nucleotide sequences (redox condensing reaction). Hot alkaline potassium 
permanganate is a major ingredient in the preferred carboxylating reagent 
of the present invention based on the reaction of nucleophilic addition. 
Although the adenine (A), guanine (G), cytosine (C), thymine (T) and 
uracil (U) were used in the generation of covalently modified nucleotide 
sequences, any nucleotide or its analog capable of being incorporated and 
modified into nucleotide sequences for the purpose of directly covalent 
bond formation between nucleotide homologues is within the scope of the 
present invention. For example, such possible substitutes could be 
2'-deoxy-uracil derivatives, para-toluene derivatives or else that has the 
same capability of being covalently modified. 
After above covalent modification, the modified nucleotide sequences are 
then mixed with non-modified nucleotide sequences, denatured and 
reassociated at temperature sufficient to inhibit nonspecific 
hybridization, preferably between about 60-80.degree. C., most preferably 
about 68-74.degree. C. It is preferred that the amount of the modified 
sequences is higher than that of the non-modified sequences. In the 
preferred embodiment, the ratio of the modified sequences to the 
non-modified sequences is between 2:1 to about 30:1, most preferably about 
5:1 to about 10:1. If the ratio is too high, successful enrichment of 
unique sequences that only exist in the non-modified sequences will not be 
obtained. If the ratio is too low, common nonspecific sequences will not 
be completely subtracted, and thus cause false-positive contamination. The 
optimal ratio will vary depending on the stringency of wanted covalent 
bonding between compared two strands of nucleotide sequences. 
During the hybridization, two kinds of hybrid duplexes are formed as 
follows: First, homohybrid duplexes formed between two non-modified 
nucleotide sequences; And, heterohybrid duplexes formed between one 
modified and one non-modified nucleotide sequence. Because the heat-stable 
covalent bonding is generated only between the heterohybrid duplexes, the 
heterohybrid duplexes can not be amplified by a PCR or a vector-cloning in 
that those reactions require the separation or restriction of the 
nucleotide duplexes. Contrarily, the homohybrid duplexes are still formed 
by hydrogen-bonding which can be further treated for further reactions, 
such as PCR and cloning. Therefore, the present invention can be used to 
isolate desired unique sequences of one nucleotide library from another 
one by inhibiting the amplification and cloning of unwanted common 
sequences between them. Such isolated sequences are then used to fish out 
the full-length mRNA or cDNA from the nucleotide library, or to locate the 
isolated gene within certain chromosome by in-situ-hybridization. The 
information so obtained will provide further understanding of a variety of 
diseases, physiological phenomena, and genetic functions. 
Alternatively, the present invention may be very useful in an 
in-situ-hybridization as well as antisense gene therapy. Because of 
single-stranding and resistance to restriction-enzyme digestion, if the 
covalently modified nucleotide sequences are labeled with terminal 
transferase and then used as probes in an in-situ-hybridization, the 
targeted nucleotides will be clearly identified in vitro or in vivo due to 
a stable bonding generated between the probes and the targets. In the same 
token, if the covalently modified nucleotide sequences are used as drugs 
in an antisense gene therapy, the targeted gene which we want to 
inactivate will be turned off in that transcription can not be 
accomplished though the covalent bonding region of the targeted gene. 
Examples as mentioned here will be developed into continuity in part of 
the present invention and is not intended in any way to limit the broad 
features or principles of the present invention. 
According to the high reaction rate of covalent modifications in the 
preferred embodiment of the present invention, the labor- and 
time-consuming factors in this directly covalent bonding method can be 
reduced to the minimum. Also, the preparation of the covalently modified 
nucleotide sequences is cheaper and more efficient than that of other 
modified sequences in previous methods. Most importantly, such covalent 
modification can be carried out continuously in microtubes with only few 
changes of buffers. Taken together, these special features make the 
present invention as fast, simple, and inexpensive as a kit for 
specifically generate directly covalent bonding between nucleotide 
homologues. 
Although certain preferred embodiments of the present invention have been 
described, the spirit and scope of the invention is by no means restricted 
to what is described above. For example, within the general framework of 
(a) one or more specific adaptors/primers for nucleotide 
analog-incorporation; (b) one or more nucleotide analogs incorporated into 
modified nucleotide sequences; (c) one or more chemical reagents which can 
be used to accomplish directly covalent modification; (d) one or more 
rounds of hybridization to complete directly covalent bond formation, 
there is a very large number of permutations and combinations possible, 
all of which are within the scope of the present invention. 
EXAMPLE 1 
Preparation of First and Second Strand Nucleotide Sequences 
LNCaP cells, a prostate cancer cell line, were grown in DMEM medium 
supplemented with 2% fetal calf serum. For three-day activin treatment, 6 
dishes of control cells were treated with 1.5 ml 200 ng/ml activin per 
day, while 4 dishes of experimental cells were not treated. On the fifth 
day after the first treatment, 55% reduction in growth was observed in the 
control cells compared to the experimental cells by both microscopy and 
cell counting. All cells were respectively trypsinized and total RNAs were 
isolated with TRIzol reagent (GIBCO/BRL). mRNAs were purified from total 
RNAs with a poly (oligo-dT) dextran column (Oligotex Direct Mini kit, 
Qiagen). After 1 .mu.g mRNAs were mixed with an oligo-dT primer and heated 
to 65.degree. C. (10 min), reverse transcription (RT) was performed with 
cDNA Cycle kit (Invitrogen), and all RT products (2 .mu.g) were 
double-stranded with a DNA polymerase-ligase-RNase cocktail mixture (Ueli 
et.al., Gene, 25: pp263-269 (1983)). 1 .mu.g of double-stranded control 
cDNAs were then digested by a four-cutting enzyme, such as 20U Hpa2 (5h, 
37.degree. C.), and ligated with B-specific primer: SEQ ID NO: 1 
5'-pGCCACCAGAAGAGCGTGTACGTCC-3'in the 5'-end, while experimental cDNAs 
were ligated with A-specific primer: SEQ ID NO. 2 
5'-pCGGTAGTGACTCGGTTAAGATCGC-3' in the same manner. This formed the first 
strand of control cDNA sequences and the second strand of experimental 
cDNA sequences. 
EXAMPLE 2 
Covalent Modification of Second Strand Nucleotide Sequences 
The second strand of experimental cDNA sequences was diluted and amplified 
by polymerase chain reaction (PCR) with A-specific primer. During PCR, the 
recessed 3'-ends were filled in by Taq DNA polymerase (7 min, 72.degree. 
C.) with dATP (2 mM), dCTP (2 mM), dGTP (2 mM), dTTP (0.5 mM) and dUTP 
(3.5 mM). Thirty cycle amplification were performed (1 min, 95.degree. C.; 
1 min, 72.degree. C.; 3 min, 68.degree. C.), and the amplified products, 
named U-DNA, were recovered by Micropure.TM.-EZ columns (Microcon). 50 
.mu.l alkaline acetic chloride reagent was added (6 min, 98.degree. C.) 
into the U-DNA of the second strand sequences to block its activating 
amino-groups by acetylation, by which the second strand sequences also 
become single-stranded. After the acetylated U-DNA was recovered by 
Micropure.TM.-EZ columns and resuspended in total 10 .mu.l 10 mM 
Tris-buffer (pH7.4), 40 .mu.l alkaline potassium permanganate reagent was 
added (5 min, 98.degree. C.; 30 min, 72.degree. C.) to generate 
carboxyl-groups on the C-5/C-6 of uracil/cytosine which can covalently 
bond to the amino-groups on the C-6/C-2 of aderine/guanine of the first 
strand respectively. The carboxylated second strand sequences were finally 
recovered by Micropure.TM.-EZ column and resuspended in total 10 .mu.l 10 
mM N,N'-diisopropylcarbodiimide 
N-[2-hydroxyethyl]piperazine-N'-[3-propanesulfonic acid] and 
[ethylenedinitrilo]tetraacetic acid (EPPS/EDTA) mixture buffer. 
EXAMPLE 3 
Hybridization and Specific Amplification 
For hybridization, the first strand sequences (500 ng) from experimental 
cells was mixed with the covalently modified second strand sequences (3 
.mu.g) in EPPS/EDTA buffer and denatured at 98.degree. C. (6 min) under 
alkaline condition. The mixture was then vortexed, added with 2 .mu.l 5M 
NaCl to adjust salt concentration and incubated at 70.degree. C. (20 h). 
The hybridized DNAs were finally diluted with 20 .mu.l MgCl.sub.2 solution 
(2.5 mM) and amplified by PCR with the B-specific primer. Twenty cycle 
amplification were performed (1 min, 95.degree. C.; 3 min, 73.degree. C.) 
after nick translation with E. coli DNA polymerase 1 plus T.sub.4 DNA 
polymerase 3:1 mixture (5 min, 37.degree. C. without dNTPs; 35 min, 
37.degree. C. with dNTPs), and the resulting products were 
phenol-extracted, isopropanol-precipitated and resuspended in 15 .mu.l 10 
mM Tris buffer for displaying on a 3% agarose gel electrophoresis (FIG. 
2). 
As shown in the FIG. 2, the first strand sequences was amplified with 
B-specific primer (lane 2), the second strand sequences was amplified with 
A-specific primer (lane 3), the first strand sequences was amplified with 
A-specific primer (lane 4), the second strand sequences was amplified with 
A-specific primer (lane 5), the first strand sequences was subtracted by 
modified itself and amplified with B-specific primer (lane 6), the second 
strand sequences was subtracted by modified itself and amplified with 
A-specific primer (lane 7), the first strand sequences was subtracted by 
modified second strand sequences and amplified with B-specific primer 
(lane 8), and the second strand sequences was subtracted by modified first 
strand sequences and amplified with A-specific primer (lane 9). Herein 
said first strand sequences are those nucleotide sequences with B-specific 
primer and said second strand sequences are those nucleotide sequences 
with A-specific primer. Therefore, based on the result of FIG. 2, the 
present invention is so sensitive and specific that all homologues between 
two strands of nucleotide sequences can be covalently subtracted even 
after PCR amplification. 
The present invention has been described with reference to particular 
preferred embodiments; however, the scope of this invention is defined by 
the attached claims and should be constructed to include reasonable 
equivalents. 
Defined in detail, the present invention is a method of performing improved 
directly covalent bond formation between interstrand nucleotides, 
comprising the steps of: 
a. providing a first strand of nucleotide sequences, wherein said first 
strand of nucleotide sequences is not modified by amino-blocking agent in 
order to preserve activating amino-groups on its nucleotide base 
structure(s); 
b. contacting said first strand of nucleotide sequences in denatured form 
with a second strand of denatured nucleotide sequences, wherein said 
second strand of denatured nucleotide sequences is single-stranded by 
amino-blocking agent and then modified by carboxylating conditions in its 
nucleotide base structure(s), to form a denatured mixture; 
c. permitting said first strand and said second strand of nucleotide 
sequences in said denatured mixture to form double-stranded hybrid 
duplexes comprising covalent bonding between the activating amino-groups 
of said first strand and the modified carboxyl-groups of said second 
strand of nucleotide sequences; and 
d. whereby said method provides a fast, simple, specific and direct 
covalent bond formation between said two strands of nucleotide sequences. 
Alternatively defined in detail, the present invention is a kit for 
performing improved directly covalent bond formation between interstrand 
nucleotides, comprising: 
a. an amino-blocking agent which makes said second strand of nucleotide 
sequences single-stranded; 
b. a carboxylating agent which generates carboxyl-groups on the nucleotide 
base of said second strand of nucleotide sequences; 
c. a hybridization buffer which permits said first strand and said second 
strand of nucleotide sequences in a denatured mixture to form directly 
covalent-bonded hybrid duplexes; and 
d. whereby said kit can be used to provide a fast, simple, specific and 
direct covalent bond formation between said two strands of nucleotide 
sequences. 
Defined broadly, the present invention is a method of performing improved 
directly covalent bond formation between interstrand nucleotides, 
comprising the steps of: 
a. providing a first strand of nucleotide sequences, wherein said first 
strand of nucleotide sequences is not modified by redox modification 
reagents in order to preserve activating groups on its nucleotide base 
structure; 
b. contacting said first strand of nucleotide sequences in denatured form 
with a second strand of denatured nucleotide sequences, wherein said 
second strand of nucleotide sequences is single-stranded and covalently 
modified by said redox modification reagents in its nucleotide base 
structure, to form a denatured mixture; 
c. permitting said first strand and said second strand of nucleotide 
sequences in said denatured mixture to form covalently bonded hybrid 
duplexes between the activating groups of said first strand and the 
modified groups of said second strand of nucleotide sequences; and 
d. whereby said method provides a fast, simple, specific and direct 
covalent bond formation between said two strands of nucleotide sequences. 
Attentively defined broadly, the present invention is a kit for performing 
improved directly covalent bond formation between interstrand nucleotides, 
comprising: 
a. a set of redox modification reagents which make said second strand of 
nucleotide sequences single-stranded and modified for covalently bonding 
with said first strand of nucleotide sequences; 
b. a hybridization buffer which permits said first strand and said second 
strand of nucleotide sequences in a denatured mixture to form directly 
covalent-bonded hybrid duplexes; and 
c. whereby said kit can be used to provide a fast, simple, specific and 
direct covalent bond formation between said two strands of nucleotide 
sequences. 
Of course the present invention is not intended to be restricted to any 
particular form or arrangement, or any specific embodiment disclosed 
herein, or any specific use, since the same may be modified in various 
particulars or relations without departing from the spirit or scope of the 
claimed invention hereinabove shown and described of which the apparatus 
shown is intended only for illustration and for disclosure of an operative 
embodiment and not to shown all of the various forms or modifications in 
which the present invention might be embodied or operated. 
The present invention has been described in considerable detail in order to 
comply with the patent laws by providing full public disclosure of at 
least one of its forms. However, such detailed description is not intended 
in any way to limit the broad features or principles of the present 
invention, or the scope of patent monopoly to be granted. 
__________________________________________________________________________ 
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(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
#= "Synthetic DNA"RIPTION: /desc 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
# 24AAGA TCGC 
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