Exchangeable template reaction

The invention provides a method for the synthesis of DNA based on a cyclic mechanism of combining deoxyoligonucleotides comprising combining: (a) a series of unique single-stranded deoxypolynucleotides, each having a 5' sequence which, when in double-stranded form, can be enzymatically treated to form a unique 3' single-stranded protrusion for selective cyclic hybridization with another unique single-stranded deoxypolynucleotide of the series; (b) a unique deoxypolynucleotide having a 3' sequence which can selectively hybridize with one of the unique single-stranded deoxypolynucleotides of (a); (c) a polymerase which can direct the formation of double-stranded deoxypolynucleotides from the single-stranded deoxypolynucleotides; and (d) an enzyme which can form a unique single-stranded 3' protrusion from the double-stranded deoxypolynucleotides; under conditions which hybridize the unique single-stranded deoxypolynucleotides in a cyclic manner to form the DNA. Also provided is a kit comprising a series of unique synthesized single-stranded deoxypolynucleotides, each having a 5' sequence which, when in double-stranded form, can be enzymatically treated to form a unique 3' single-stranded protrusion for selective cyclic hybridization with another unique single-stranded deoxypolynucleotide of the series.

Various references are cited herein. These references are hereby 
incorporated by reference into the application to more fully describe the 
state of the art to which the invention pertains. 
BACKGROUND OF THE INVENTION 
The technology for the functional expression of DNA fragments in 
heterologic genetic systems depends to a great extent on an accessible 
source of DNA. There are two ways to obtain genetic material for genetic 
engineering manipulations: (1) isolation and purification of DNA in an 
appropriate form from natural sources (this technique is well-elaborated 
and constitutes the backbone of genetic engineering and molecular 
biology), or (2) the synthesis of DNA using various chemical-enzymatic 
approaches, a discipline that has been intensively researched over the 
last 15 years. The former approach is limited to naturally-occurring 
sequences which do not easily lend themselves to specific modification. 
The latter approach is much more complicated and labor-intensive. However, 
the chemical-enzymatic approach has many attractive features including the 
possibility of preparing, without any significant limitations, any 
desirable DNA sequence. 
Two general methods currently exist for the synthetic assembly of 
oligonucleotides into long DNA fragments. First, oligonucleotides covering 
the entire sequence to be synthesized are first allowed to anneal, and 
then the nicks are repaired with DNA ligase. The fragment is then cloned 
directly, or cloned after amplification by the polymerase chain reaction 
(PCR). The DNA is subsequently used for in vitro assembly into longer 
sequences. This approach is very sensitive to the secondary structure of 
oligonucleotides, which interferes with the synthesis. Therefore, the 
approach has low efficiency and is not reliable for assembly of long DNA 
fragments. 
The second general method for gene synthesis utilizes polymerase to fill in 
single-stranded gaps in the annealed pairs of oligonucleotides. After the 
polymerase reaction, single-stranded regions of oligonucleotides become 
double-stranded, and after digestion with restriction endonuclease, can be 
cloned directly or used for further assembly of longer sequences by 
ligating different double-stranded fragments. This approach is relatively 
independent of the secondary structure of oligonucleotides; however, after 
the polymerase reaction, each segment must be cloned. The cloning step 
significantly delays the synthesis of long DNA fragments and greatly 
decreases the efficiency of the approach. Additionally, this approach can 
be used for only relatively small DNA fragments and requires restriction 
endonuclease recognition sites to be introduced into the sequence. 
Thus, the major essential disadvantages of existing approaches for the 
synthesis of DNA is low efficacy and the requirement that synthesized DNA 
must be amplified by cloning procedures, or by the PCR, before use. The 
main problem with existing approaches is that the long polynucleotide must 
be assembled from relatively short oligonucleotides utilizing either 
inefficient chemical or enzymatic synthesis. The use of short 
oligonucleotides for the synthesis of long polynucleotides can cause many 
problems due to multiple interactions of complementary bases, as well as 
problems related to adverse secondary structure of oligonucleotides. These 
problems lower the efficiency and widespread use of existing synthetic 
approaches. 
Therefore, there exists a great need for an efficient means to make 
synthetic DNA of any desired sequence. Such a method could be universally 
applied. For example, the method could be used to efficiently make an 
array of DNA having specific substitutions in a known sequence which are 
expressed and screened for improved function. The present invention 
satisfies these needs by providing an efficient and powerful method for 
the synthesis of DNA. The method is generally referred to as the 
Exchangeable Template Reaction (ETR). 
SUMMARY OF THE INVENTION 
The invention provides a method for the synthesis of DNA based on a cyclic 
mechanism of combining deoxyoligonucleotides comprising combining: (a) a 
series of unique single-stranded deoxypolynucleotides, each having a 5' 
sequence which, when in double-stranded form, can be enzymatically treated 
to form a unique 3' single-stranded protrusion for selective cyclic 
hybridization with another unique single-stranded deoxypolynucleotide of 
the series; (b) a unique deoxypolynucleotide having a 3' sequence which 
can selectively hybridize with one of the unique single-stranded 
deoxypolynucleotides of (a); (c) a polymerase which can direct the 
formation of double-stranded deoxypolynucleotides from the single-stranded 
deoxypolynucleotides; and (d) an enzyme which can form a unique 
single-stranded 3' protrusion from the double-stranded 
deoxypolynucleotides; under conditions which hybridize the unique 
single-stranded deoxypolynucleotides in a cyclic manner to form the DNA. 
Also provided is a kit comprising a series of unique synthesized 
single-stranded deoxypolynucleotides, each having a 5' sequence which, 
when in double-stranded form, can be enzymatically treated to form a 
unique 3' single-stranded protrusion for selective cyclic hybridization 
with another unique single-stranded deoxypolynucleotide of the series.

DETAILED DESCRIPTION OF THE INVENTION 
Description of the Exchangeable Template Reaction (ETR) mechanism. The ETR 
is a method for the synthesis of long polynucleotide DNA fragments using 
short synthetic oligonucleotides as templates for DNA polymerase. The 
method is based on a cyclic mechanism involving three main components: (1) 
polymerase activity to synthesize double-stranded DNA, (2) enzymatic 
activity to create 3' terminal single-stranded regions, and (3) 
specifically designed synthetic deoxyoligonucleotides used as templates 
for the polymerase reaction. The critical step is the enzymatic creation 
of a 3' terminal single-stranded region at the "growing point" of the 
synthesizing polynucleotide chain, which is used for the complementary 
binding of the next oligonucleotide as a template to continue the 
polymerase reaction. 
The order of oligonucleotide additions for each cycle is encoded in each 3' 
terminal sequence. At the 3' terminus of the growing DNA molecule a 
specific sequence of nucleotides can anneal with a complementary sequence 
of nucleotides from the synthetic oligonucleotide. Thus, it is possible to 
synthesize a long DNA fragment in one step by simply combining the entire 
set of deoxyoligonucleotides in one reaction tube containing all the 
required enzymatic activities and incubating the mixture at the optimal 
temperature and optimal buffer. 
Each cycle begins with the complementary binding of the 3' terminal region 
of a synthetic oligonucleotide with the 3' protruding region of 
double-stranded DNA (step 1 in FIG. 1). After annealing, a DNA polymerase 
reaction occurs to create a second strand of DNA using the short synthetic 
oligonucleotide as a template for DNA polymerase (step 2 in FIG. 1). After 
polymerization is complete, the double-stranded DNA has been extended by 
the length of the synthetic oligonucleotide. To initiate the second round 
in the cycle of DNA synthesis, another enzymatic reaction occurs that 
creates a 3' protruding single-stranded region by removing several 
nucleotides from the 5' terminus leaving a 3' protrusion. This protrusion 
is used to anneal another short synthetic oligonucleotide (step 3 in FIG. 
1). 
Thus, this invention provides a method for the synthesis of DNA based on a 
cyclic mechanism of combining deoxyoligonucleotides comprising combining 
in any order: 
(a) a series of unique single-stranded deoxypolynucleotides, each having a 
5' sequence which, when polymerized to double-stranded form, can be 
enzymatically treated to form a unique 3' single-stranded protrusion for 
selective cyclic hybridization with another unique single-stranded 
deoxypolynucleotide of the series; 
(b) a unique deoxypolynucleotide having a 3' sequence which can selectively 
hybridize with one of the unique single-stranded deoxypolynucleotides of 
(a); 
(c) a polymerase which can direct the formation of double-stranded 
deoxypolynucleotides from the single-stranded deoxypolynucleotides; and 
(d) an enzyme which can form a unique single-stranded 3' protrusion from 
the double-stranded deoxypolynucleotides; under conditions which hybridize 
the unique deoxypolynucleotides in a cyclic manner and polymerize the 
hybridized deoxypolynucleotides to form the DNA. 
"Cyclic" as used herein means a sequential hybridization in a regularly 
repeated order. Thus, as noted above, hybridization of 
deoxypolynucleotides (hereinafter "DPNTs") occurs only in a specified 
controlled order. For example, a series of DPNTs (two or more), each of 
which encodes a unique segment of a desired long DPNT, are synthesized. 
During the synthesis, the sequence of each DPNT is selected to produce, 
when later cleaved by an enzyme, a unique 3' protrusion which will 
hybridize with only one other member of the DPNT series. When the DPNTs 
are combined, only two of the DPNTs initially hybridize. Once this 
hybridization occurs, the sequence of the remaining synthesis is set. A 
polymerase utilizes the two hybridized DPNTs to form double strands. The 
appropriate enzyme then acts on the double-stranded DPNTs to form the 
unique 3' single-stranded protrusion. The next DPNT which hybridizes only 
with this unique 3' protrusion then hybridizes. Once this hybridization 
occurs, the polymerase again directs the synthesis of double strands. 
After the double strands are completed, the enzyme again produces a unique 
3' single protrusion which was previously synthesized to hybridize only 
with the next unique DPNT. The sequence is then repeated the desired 
number of times. 
This invention also provides hybridization and cleavage which proceeds in 
both directions, e.g., first hybridize DPNTs in the middle of the desired 
sequence with cleavage sites on both subsequently-formed ends. The 
selection of DPNTs and enzymes follows the procedure of unidirectional 
synthesis but enzyme sites on both ends of the double-stranded DNA are 
created. 
Once a long DPNT is made by the above method, a new series of DPNTs can be 
added, each having a 5' sequence which, when in double-stranded form, can 
be enzymatically treated to form a unique 3' single-stranded protrusion 
for selective cyclic hybridization with another unique single-stranded 
DPNT of the series. This procedure can be repeated many times. The number 
of DPNTs in the reaction is only limited by undesired interference of 
hybridization. This can be avoided by creating unique 3' protrusions and 
hybridizing DPNTs which have minimal sequence similarity. Very long DPNTs 
including genes and entire genomes can thereby be synthesized by this 
method. 
As can be appreciated from the above, the method works so long as a unique 
3' single-stranded protrusion is formed by an enzymatically-treated 
hybridized unique DPNT. By "unique" is meant a nucleotide sequence on one 
DPNT which is absent on another DPNT so that selective hybridization can 
occur. The number of unique nucleotides necessary for selective 
hybridization depends on hybridization conditions. For example, for a 3' 
protrusion of four nucleotides, the optimal temperature of the reaction is 
about 37.degree. C. This optimal temperature may be different if a 
different polymerase is utilized in the synthesis. This is true because 
different polymerases have different affinities to complementary 
complexes. Thermostable enzymes also have a rather high affinity to such 
complexes. A longer 3' protrusion should be more reactive and more 
specific in hybridization and utilize a higher annealing temperature. 
However, the single-stranded region must be of a size to avoid being 
involved in secondary structure formation. This region, to be effective in 
hybridization, should be represented in a single-stranded form at the 
reaction temperature. From this point of view, thermostable enzymes can be 
more effective in ETR because a higher reaction temperature can be 
utilized. Thus, very effective single-stranded terminal regions can be 
about 7-9 nucleotides long. For such lengths it is routine to find 
conditions to maintain single-stranded form. Specific complementary 
complexes between DPNTs can be effectively organized at higher 
temperatures, which decreases the possibility of improper complex 
formation. The optimal temperature for the 7-9 nucleotide 3' protrusion 
may be around 55.degree.-65.degree. C., the optimal temperature for the 
activity of thermostable polymerases. Thus, a preferred range of 3' 
protrusion length is about 3-12 nucleotides. Longer protrusions can be 
made and routinely tested by the methods described in the Experimental 
section to optimize length and conditions for a particular system. 
The precise 5' sequence of a member of the series will depend on the 
desired sequence for the ultimate DNA and the type of enzyme utilized to 
form the protrusion. Thus, once an ultimate desired sequence is selected, 
a 5' sequence is synthesized which corresponds to the desired sequence and 
which will either be cleaved or exposed such that the desired sequences 
remain and the undesired sequences, if any, are removed prior to 
hybridization of the next member of the series. For example, if a 
restriction endonuclease is utilized, it must cleave in such a way that 
unique sequences for each member of the series to be hybridized are 
produced. BstXI, as described in detail in the Experimental section, 
provides one example of such a restriction endonuclease because the 
endonuclease allows for four unique nucleotides to be synthesized in each 
member of the series which remains after cleavage. 
Because of the unique nature of the 5' sequence which is treated to produce 
the unique 3' protrusion, the members of the series of DPNTs must be 
synthesized if a restriction endonuclease is utilized, for example with a 
DNA synthesizer. Since the DPNT which starts the hybridization can 
hybridize directly with the second DPNT, it is not affected by the 
enzymatic treatment. Therefore, the first unique DPNT can be obtained, if 
desired, by means other than synthesis and can be single- or 
double-stranded. For example, the DPNT can be a fragment excised from 
natural DNA, e.g. plasmid, phage genome, or viral genome by restriction 
endonucleases. Likewise, the fragment can be obtained by specific 
amplification using PCR. PCR fragments are more suitable because terminal 
sequences of the amplified fragment can be easily modified with primers 
used for amplification with the introduction of desirable nucleotide 
modifications, including artificially synthesized non-natural derivatives 
of nucleotides. Any suitable number of nucleotides sufficient for 
efficient hybridization under the selected conditions can be utilized for 
this initial hybridization. 
This unique synthesis-initiating DPNT, which begins synthesis by providing 
a template for hybridization of the second DPNT from the series, can be 
bound to a solid support for improved efficiency. The solid phase allows 
for the efficient separation of the synthesized DNA from other components 
of the reaction. Different supports can be applied in the method. For 
example, supports can be magnetic latex beads or magnetic control pore 
glass beads. Being attached to the first DPNT, these beads allow the 
desirable product from the reaction mixture to be magnetically separated. 
Binding the DPNT to the beads can be accomplished by a variety of known 
methods, for example carbodiimide treatment (Gilham, Biochemistry 
7:2809-2813 (1968); Mizutani and Tachbana, J. Chromatography 356:202-205 
(1986); Wolf et al., Nucleic Acids Res. 15:2911-2926 (1987); Musso, 
Nucleic Acids Res. 15:5353-5372 (1987); Lund et al., Nucleic Acids Res. 
16:10861-10880 (1988)). The DPNT attached to the solid phase is the primer 
for synthesis of the whole DNA molecule. Synthesis can be accomplished by 
addition of sets of compatible oligonucleotides together with enzymes. 
After the appropriate incubation time, unbound components of the method 
can be washed out and the reaction can be repeated again to improve the 
efficiency of each oligonucleotide to be utilized as a template. 
Alternatively, another set of oligonucleotides can be added to continue 
the synthesis This "set principle," barely applicable to solution 
synthesis, turns the method into a very powerful method for the synthesis 
of a long DNA molecule that is not possible with any other methods. 
Solid phase, to be efficiently used for the synthesis, can contain pores 
with sufficient room for synthesis of the long DNA molecules. The solid 
phase can be composed of material that cannot non-specifically bind any 
undesired components of the reaction. One way to solve the problem is to 
use control pore glass beads appropriate for long DNA molecules. The 
initial primer can be attached to the beads through a long connector. The 
role of the connector is to position the primer from the surface of the 
solid support at a desirable distance. 
Any polymerase which can direct the synthesis of double strands from 
partially hybridized single strands is appropriate. Suitable polymerases, 
for example, may include Taq polymerase, large fragments of E. coli DNA 
polymerase I, DNA polymerase of T7 phase. The optimal conditions of the 
polymerization vary with the type of polymerase used. Likewise, the 
optimal polymerase can vary with the conditions necessary for the 
synthesis (Bej et al., Crit. Rev. Biochem. Mol. Biol. 26(3-4): 301-334 
(1991); Tabor and Richardson, Proc. Natl. Acad. Sci. USA. 86:4076-4080 
(1989); Petruska et al., Proc. Natl. Acad. Sci. USA 85:6252-6256 (1988)). 
One example of an enzyme capable of removing several nucleotides from the 
5' terminus is the restriction endonuclease BstXI. This restriction 
endonuclease is compatible with ETR for the following reasons: (1) a 3' 
protrusion is produced, (2) the single-stranded 3' protrusion does not 
have any sequence restrictions, and (3) after cleavage the restriction 
site cannot be restored by the interaction of the next synthetic 
oligonucleotides. 
While the Experimental section is directed towards the use of BstXI, the 
discovery is the production of a unique 3' protrusion however it is 
obtained. Therefore, in the subject method, any enzyme can be utilized 
which can form a unique 3' protrusion from double-stranded DNA. Other 
presently known enzymes useful in the method include 5' exonucleases 
specific for double-stranded DNA, such as the exonuclease of T7 and lambda 
phage, and an enzyme of DNA recombination, such as recA. 
The method utilizing a 5' exonuclease specific for double-stranded DNA can 
be performed as follows: oligonucleotides to be used in the reaction as 
templates for polymerase reaction are chemically modified at a defined 
point to prevent T7 exonucleases from jumping over the modified 
nucleotides. For example, oligonucleotide phosphorodithioates can be 
utilized using methods described in Caruthers, Nucl. Acids Symp. Ser. 
21:119-120 (1989). As described above, polymerase first fills gaps in 
hybridized DPNTs. When the reaction is finished, the exonuclease of the T7 
starts cutting double-stranded DNA beginning from the 5' end (the opposite 
5' end should be modified or attached to solid phase to prevent cleavage 
from the end). This reaction goes until the modified position where it 
stops. The 3' protrusion created by the exonuclease activity can then be 
used for hybridization with the next oligonucleotide in the cycle 
reaction. T7 is well known to have a relatively strong preference for 
double-stranded DNA (Kerr and Sadowski, J. Biol. Chem. 247:311-318 (1972); 
Thomas and Olivera, J. Biol. Chem. 253:424-429 (1978); Shon et al., J. 
Biol. Chem. 25:13823-13827 (1982)). 
Another double-stranded specific exonuclease is encoded by lambda phage 
(Sayers et al., Nucleic Acids Res. 16:791-802 (1988)). This enzyme can 
also be utilized in the method. 
The main advantage of these exonucleases is the possibility of creating a 
single-stranded 3' protrusion of any necessary size to allow the use of 
higher temperatures in the reaction. Additionally, because the exonuclease 
recognizes any blunt end, its use eliminates the need to synthesize DPNT 
having a restriction site when polymerized to double-stranded form. 
The method can also be performed utilizing an enzyme of DNA recombination. 
It is known that recA can replace one strand of double-stranded DNA, in a 
strong sequence-specific manner, with a single-stranded DNA from solution 
creating D-loop structures (Cox and Lehman, Ann. Rev. Biochem. 56:229-262 
(1987); Tadi-Laskowski et al., Nucleic Acids Res. 16:8157-8169 (1988); 
Hahn et al., J. Biol. Chem. 263:7431-7436 (1988)). In this modification of 
the method, DPNTs are combined in one reaction with polymerase and recA. 
Polymerase fills single-stranded gaps and recA replaces the terminal 
region of one of the strands of double-stranded DNA with a single-stranded 
DPNT from solution which provides the polymerase with a new template. An 
advantage of the reaction is strong specificity of the hybridization which 
is due to enzymatic support. In any other variations of the method, for 
example with restriction endonucleases and exonucleases, the hybridization 
is the only step without enzymatic support. While restriction 
endonucleases and exonucleases can only create a 3' protrusion, recA can 
create a single-stranded region at the ends of double-stranded DNA and 
anneals oligonucleotides to the 3' protrusion. 
The invention also provides various novel compositions used in the 
invention. Provided is a kit comprising a series of unique synthesized 
single-stranded DPNTs, each having a 5' sequence which, when polymerized 
to double-stranded form, can be enzymatically treated to form a unique 3' 
single-stranded protrusion for selective cyclic hybridization with another 
unique single-stranded DPNT of the series. The DPNTs can exist in 
lyophilized form or in a suitable carrier such as saline. The kit can 
further comprise a unique DPNT having a 3' sequence which can selectively 
hybridize with one of the series of unique single-stranded DPNTs. The kit 
can still further comprise a polymerase which can direct the formation of 
double-stranded polynucleotides from the single-stranded DPNTs. Finally, 
the kit can comprise an enzyme which can form a unique single-stranded 
protrusion from the double-stranded DPNTs. 
The invention also provides an automated synthesizer programmed to perform 
the method of claim 1 and remove undesired components. This synthesizer 
can be programmed to perform repeat cycles of the synthesis. 
EXPERIMENTAL 
MATERIALS AND METHODS 
Deoxyoligonucleotides were synthesized using an automatic synthesizer 
(Applied Biosystem Model 380B, Foster City, Calif.) and purified by 
polyacrylamide gel electrophoresis (PAGE) in 10% polyacrylamide in TBE 
buffer (0.045M Tris-borate, pH 8.3, containing 0.001M EDTA and 7M urea). 
Oligonucleotides were recovered from the gel by electroelution. 
The ETR was carried out at 37.degree. C. for 0.5-5 hrs. in a volume of 50 
.mu.l of 10 mM Tris-HCl buffer, pH 7.9, containing 10 mM MgCl; 50 mM NaCl; 
1 mM DTT; 0.25 mM each of dATP, dGTP, dTTP, and dCTP (Pharmacia-LKB, 
Uppsala, Sweden); 5 units of native Taq DNA polymerase (Cetus Corp., 
Emeryville, Calif.); 30 units of BstXI (New England BioLabs, Beverly, 
Mass.); and 0.5-100 pmol of each deoxyoligonucleotide. Analysis of the 
reaction course was accomplished by utilizing one of the 
deoxyoligonucleotides without a BstXI site radiolabeled with 
gamma-.sup.32 P!ATP in 50 mM Tris-HC1, pH 7.6, containing 10 mM MgCl, 5 
mM DTT, 10 .mu.CI gamma-.sup.32 P! ATP (5,000 Ci/mmole, New England 
Nuclear, Wilmington, Del.), and 10-20 pmol of oligonucleotide. After the 
completion of the ETR, the products were analyzed by PAGE in 8% 
polyacrylamide containing 8M urea, and the specific products were revealed 
by autoradiography. 
RESULTS 
Verification of ETR using BstXI. The BstXI is a commercially available 
endonuclease that satisfies the requirements stated above. The major 
drawback of this enzyme is that it produces only a 4 nucleotide 
single-stranded 3' protrusion for annealing to the next oligonucleotide. 
We anticipated that this short protrusion may lower the overall efficiency 
of the ETR relative to the use of an exonuclease, which would yield a much 
longer protrusion. Nevertheless, we decided to explore this approach since 
it represented the easiest way to verify the cyclic mechanism involved in 
the synthesis of DNA by the ETR. Accordingly, four sets of 
oligonucleotides were designed and synthesized (FIGS. 2-5). 
Set 1. Synthesis of a DNA fragment of the hepatitis B virus (HBV) genome. 
One of the most powerful applications of synthetic DNA fragments is in 
site-specific mutagenesis of DNA, especially if the introduction of 
multiple mutations is desired in a long sequence. Using ETR, a DNA 
fragment corresponding to the sequence encoding the terminal protein of 
the HBV genome was synthesized and modified by changing the nucleotide 
sequence of one of the deoxynucleotides. This fragment was created from 
three deoxynucleotides (FIG. 2-I) (SEQ ID Nos: 2-4) and synthesized by the 
ETR as shown in FIG. 2-II (SEQ ID Nos. 5-9). All three deoxynucleotides 
were combined in one tube with Taq DNA polymerase and BstXI in the 
presence of DPNTs. Different relative concentrations of the 
oligonucleotides were used in the reaction. Deoxynucleotide A (SEQ ID No: 
2) was radiolabeled. The concentrations of deoxynucleotide A and B (SEQ ID 
No: 3) were fixed at 1 pmol, while the concentration of deoxynucleotide 
C(SEQ ID No: 4) was used at 1 pmol, 10 pmol, and 100 pmol. Reactions 
containing 10 pmol and 100 pmol of C were more efficient than reactions 
containing 1 pmol of C with no significant differences in efficiency 
between reactions containing 10 pmol and 100 pmol. When the amount of B 
was increased to 10 pmol, there was no improvement in the efficiency of 
synthesizing a full-size fragment. Although a 10-fold molar excess of B 
and C over labeled A did not improve the efficiency of the ETR, these 
conditions did, however, make the reaction more reproducible. In all 
subsequent experiments, at least a 10-fold molar excess of the unlabeled 
to labeled oligonucleotides were used for monitoring the reactions. In 
control experiments without B or C, no DNA fragment of the expected size 
was found. Reactions were carried out at constant temperatures of 
4.degree. C., 10.degree. C., 20.degree. C., 37.degree. C., 42.degree. C., 
and 65.degree. C. The best yield was obtained at 37.degree. C. No 
full-size fragment was obtained at 4.degree. C., 10.degree. C., or 
65.degree. C. Only a dimer of A and B was found at these temperatures. At 
37.degree. C, a full-size fragment was obtained after a 5 min. incubation. 
After a 5 h. incubation, the full-size fragment gave a strong band by 
autoradiography. This fragment was cleaved with restriction endonucleases, 
and amplified by the PCR, which produced a fragment of the correct size 
measured by electrophoresis. 
In experiments using radiolabeled deoxynucleotide A, a full-size fragment 
was identified after electrophoresis under denaturating conditions. When 
radiolabeled deoxynucleotide C was used, no synthesis occurred. This 
result was reproducible and suggested that only A can initiate the 
polymerase synthesis of full-size DNA fragments using B and C as 
templates. The double-stranded DNA product of the ETR contains a 
non-interrupted strand synthesized by the polymerase reaction and primed 
with A, and a second strand with nicks between the other oligonucleotides 
that participated in the reaction as templates for the polymerase 
reaction. These nicks can be repaired with DNA ligase. Alternatively, the 
DNA fragments can be used directly for cloning, amplified by the PCR, or 
treated with other DNA-modifying enzymes such as restriction 
endonucleases. 
Set 2. Synthesis of the DNA fragments encoding for the nucleocapsid protein 
of the hepatitis C virus (HCV). The DNA sequence encoding the HCV 
nucleocapsid protein was divided into 3 fragments. Each fragment was 
synthesized separately by the ETR (FIGS. 3-5). The first fragment was 
synthesized from 5 deoxynucleotides (FIG. 3) (SEQ ID No: 10-14), the 
second fragment from 3 (FIG. 4) (SEQ ID Nos: 15-17), and the third from 4 
deoxynucleotides (FIG. 5) (SEQ ID Nos: 18-21). All reactions were carried 
out as described above. The longest synthesized fragment contained 228 
base pairs (bp). The yield of full-size fragments was estimated to be 
approximately 5-10%. 
Different buffers were tested (Table 1) for the ETR using oligonucleotides 
to synthesize the first segment of the gene (FIG. 3). Buffer NEB3 is the 
optimal buffer for BstXI, whereas the various Taq buffers are optimal for 
Taq DNA polymerase. The best result for the ETR reaction utilized, 
however, was obtained with buffers NEB2 and NEB4. 
Both BstXI and Taq polymerase have high optimal temperature conditions. 
Because of the short single-stranded protrusion formed by BstXI, however, 
the ETR was found to be optimal at 37.degree. C. rather than the optimal 
temperatures for these enzymes. 
For the ETR synthesis of the first segment, corresponding to the HCV 
nucleocapsid gene (core protein), the relative concentrations of the 
deoxynucleotides was 1:4:20:40:60. When the relative concentrations were 
changed to 1:1:20:40:60, the rate of ETR was changed as well. At 
1:4:20:40:60 relative concentrations of oligonucleotides, the full-size 
fragment could be detected after a 3 hr. incubation period at 37.degree. 
C. in NEB2. At the 1:1 relative concentrations of deoxynucleotides 1 and 
2, the fragment was synthesized in detectable amounts after only a 30 min. 
incubation period. 
Each of the three fragments synthesized by the ETR was purified by PAGE and 
amplified by the PCR. Amplified products were digested with the 
appropriate restriction endonuclease and treated with DNA ligase. The 
whole gene was amplified again and analyzed by restriction endonuclease 
mapping. The amplified product was inserted into an expression vector 
under the control of the T7 promoter. Briefly, this DNA fragment and 
vectorp TS7 (especially constructed for the expression of the HCV core 
protein) were cleaved with NdeI and HindIII. After removal of the enzymes 
these DNA components were mixed and treated with DNA ligase. The ligation 
mixture was used to transform E. coli that produce T7 RNA polymerase. 
Transformed E. coli cells expressed an immunologically active product 
detected by Western Blot analysis using sera previously shown reactive for 
HCV anticore activity (MATRIX, Abbott Laboratories, Abbott Park, Ill.). In 
addition, the expressed product possessed the correct molecular weight 
based on SDS-PAGE analysis. Thus, all three segments corresponding to the 
HCV nucleocapsid gene were correctly synthesized by the ETR. 
The fragment was then sequenced using standard techniques. The sequence 
confirmed the success of the ETR. The sequence was found to be exactly as 
designed. DNA synthesis utilizing ETR is a method of producing DNA of 
precise fidelity. 
It should be understood that the foregoing relates only to preferred 
embodiments of the present invention and that numerous changes and 
modifications may be made therein as described in the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 21 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CCANNNNNNTGG12 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 59 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CCCAGATCTCAATCTCGGGAATCTCAATGTTAGTATTCCTTGGACTCATAAGGTGGGAA59 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 68 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii ) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CCCCCACCACTCTGGATTAAAGATAGGTACTGTAGAGGAAAAAAGCGCCGTAAAGTTTCC60 
CACCTTAT68 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 79 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CCCGGGCCCACAAATTGTTGACACCTATTAATAATGTCCTCTTGTAAATGAATCTTAGGA60 
AAGGAAGGAG TTTGCCACT79 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CCCAGATCTATAAGGTGGGAA21 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(x i) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CCCCCACCACTCTGGTTCCCACCTTAT27 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CCCAGATCTATAAGGTGGGAACCAGAGTGGTGGGGG36 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CCCAGATCTATAAGGTGGGAACCAGAGTG29 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
CCCGGGCCCCACTCTGGTTCCCACCTTATAGATCTGGG38 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 76 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
CCCCATATGAGCACGATTCCTAAACCACAAAGAAAAACCAAACGTAACACCAATCGACGA60 
CCACAAGATGTAAAGT 76 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 69 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CCCCCACCTCCGTGGAAGCAAATAGACTCCACCAACGATCTGACCGCCACCCG GGAACTT60 
TACATCTTG69 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CCCCCATCTTCCTGGTCGCGCGCACACCCAACCTAGGTCCCCTCC45 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D ) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CCCCCAACCTCGTGGTTGCGAGCGCTCGGAAGTCTTC37 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
CCCCCTCAGGCCGACGCACTTTAGGGATAGGCTGTCGTCTACCTC45 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 75 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
CCCCCTGAGGGCAGGACCTGGGCTCAACCCGGTTACCCCTGGCCCCTCTATGGCAATGAG60 
GGCTGCGGGTGGGCG 75 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 71 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
CCCCCAGATCAGTGGGTCCCCAACTCGGTCGAGAGCCGCGGGGAGAC AGGAGCCATCCCG60 
CCCACCCGCAG71 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 46 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
CCCATCGATGACCTTACCCAAATTTCGCGACCTACGTCGCGGATCA46 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
CCCATCGATACCCTCACGTGCGGCTTCGCCGACCTCATGGGGTACATACCGCTCGTC57 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 62 base pairs 
(B) TYPE: nucleic acid 
(C ) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
CCCCCAACTCCATGGGCAAGGGCTCTGGCGGCACCTCCAAGAGGGGCGCCGACGAGCGGT60 
AT 62 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 79 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
CCCCCAGGAAGATGGAGAAAGAGCAACCAGGAAGGTTTCCTGTTGCATAA TTGACGCCGT60 
CTTCTAGAACCCGTACTCC79 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 73 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
CCCAAGCTTTTAGTTTCGAACTTGGTAGGCTGAAGCGGGCACAGTCAGGCAAGAGAGCAG60 
GGCCAGAAGGAAG73