U.S. patent number 5,503,995 [Application Number 08/261,670] was granted by the patent office on 1996-04-02 for exchangeable template reaction.
This patent grant is currently assigned to The United States of America as represented by the Department of Health. Invention is credited to Howard A. Fields, Yury Khudyakov.
United States Patent |
5,503,995 |
Khudyakov , et al. |
April 2, 1996 |
Exchangeable template reaction
Abstract
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.
Inventors: |
Khudyakov; Yury (Atlanta,
GA), Fields; Howard A. (Marietta, GA) |
Assignee: |
The United States of America as
represented by the Department of Health (Washington,
DC)
|
Family
ID: |
25305501 |
Appl.
No.: |
08/261,670 |
Filed: |
June 17, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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849294 |
Mar 10, 1992 |
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Current U.S.
Class: |
435/91.1;
435/91.5; 435/91.52 |
Current CPC
Class: |
C07K
14/005 (20130101); C12N 15/10 (20130101); C12Q
1/6853 (20130101); C12Q 1/6853 (20130101); C12N
2730/10122 (20130101); C12N 2770/24222 (20130101); C12Q
2525/131 (20130101); C12Q 2521/301 (20130101); C12Q
2521/101 (20130101) |
Current International
Class: |
C07K
14/005 (20060101); C07K 14/02 (20060101); C07K
14/18 (20060101); C12Q 1/68 (20060101); C12N
15/10 (20060101); C12P 019/34 (); C12Q
001/68 () |
Field of
Search: |
;435/91.1,91.5,6,172.1,172.3,91.52 ;935/16,17 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0359545 |
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Mar 1990 |
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EP |
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0438292 |
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Jul 1991 |
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EP |
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9000626 |
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Jan 1990 |
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WO |
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WO91/17267 |
|
Nov 1991 |
|
WO |
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WO92/05287 |
|
Apr 1992 |
|
WO |
|
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|
Primary Examiner: Jones; W. Gary
Assistant Examiner: Myers; Carla
Attorney, Agent or Firm: Needle & Rosenberg
Parent Case Text
This application is a continuation of application Ser. No.
07/849,294, filed Mar. 10, 1992 and now abandoned.
Claims
What is claimed is:
1. A method for the synthesis of DNA based on a cyclic mechanism of
combining deoxypolynucleotides comprising admixing:
(a) a plurality of unique single-stranded deoxypolynucleotides,
wherein each unique deoxypolynucleotide has one or more copies and
a 5' sequence which, when the deoxypolynucleotide is in
double-stranded form, can be enzymatically treated to form a unique
3' single-stranded protrusion for selective cyclic hybridization of
the enzymatically-treated double-stranded deoxypolynucleotide with
another unique single-stranded deoxypolynucleotide;
(b) a unique single-stranded deoxypolynucleotide having a 3'
sequence which can selectively hybridize with only one of the
unique single-stranded deoxypolynucleotides of (a);
(c) a polymerase which can direct the formation of double-stranded
deoxypolynucleotides from the hybridization product of the unique
single-stranded deoxypolynucleotides of (a) and (b); and
(d) a single enzyme which forms a unique single-stranded 3'
protrusion from each unique member of the double-stranded
deoxypolynucleotides formed by the polymerization of the
single-stranded deoxypolynucleotides of (a) and (b) with the
polymerase of (c);
under conditions which (1) hybridize the unique single-stranded
deoxypolynucleotide of (b) with the first member of the plurality
of the unique single-stranded deoxypolynucleotides of (a), (2)
polymerize the hybridized deoxypolynucleotides to form a
double-stranded DNA and (3) Can form the unique 3' single-stranded
protrusion of step (d) for cyclic hybridization, polymerization and
unique 3' single-stranded protrusion formation with the remaining
members of the plurality of unique single-stranded
deoxypolynucleotides of (a) in a single reaction mixture.
2. The method of claim 1, further comprising combining the DNA
resulting from the synthesis based on the cyclic mechanism of
combining deoxypolynucleotides of claim 1 with a second plurality
of unique synthesized single-stranded deoxypolynucleotides, wherein
each unique deoxypolynucleotide has one or more copies and a 5'
sequence which, when the deoxypolynucleotide is in double-stranded
form, can be enzymatically treated to form a unique 3'
single-stranded protrusion for selective cyclic hybridization of
the enzymatically-treated double-stranded deoxypolynucleotide with
another unique single-stranded deoxypolynucleotide of the second
plurality; under conditions which hybridize the unique
single-stranded deoxypolynucleotides in a cyclic manner to the DNA
resulting from the synthesis based on the cyclic mechanism of
combining deoxypolynucleotides of claim 1.
3. The method of claim 2, repeated a plurality of times.
4. The method of claim 1, wherein the unique single-stranded
deoxypolynucleotides are synthesized.
5. The method of claim 1, wherein each of the unique
single-stranded deoxypolynucleotides encodes a unique portion of a
gene.
6. The method of claim 1, wherein the plurality of unique
single-stranded deoxypolynucleotides comprises at least three
deoxypolynucleotides.
7. The method of claim 1, wherein the 5' sequence, when in
double-stranded form, can be enzymatically cleaved with a
restriction endonuclease to form a 3' protrusion.
8. The method of claim 7, wherein the 5' sequence comprises (SEQ ID
No: 1)
wherein N is any nucleotide.
9. The method of claim 1, wherein the 5' sequence, when in
double-stranded form, can be enzymatically cleaved by a 5'
exonuclease specific for double-stranded deoxypolynucleotides to
form a 3' protrusion.
10. The method of claim 1, wherein the deoxypolynucleotide of (b)
is bound to a solid support prior to combining with the
deoxypolynucleotides of (a).
11. The method of claim 10, wherein solid support is comprised of
beads.
12. The method of claim 11, wherein the beads are solid phase
controlled pore glass.
13. The method of claim 12, wherein the beads are coated with
glycerol.
14. The method of claim 11, wherein the beads are coated with
avidin.
15. The method of claim 14, wherein the avidin beads are bound to
biotin.
16. The method of claim 1, wherein the combining is performed
substantially simultaneously.
17. The method of claim 1, wherein the polymerase is Taq
polymerase.
18. The method of claim 1, wherein the enzyme is a restriction
endonuclease.
19. The method of claim 18, wherein the restriction endonuclease is
BstxI.
20. The method of claim 1, wherein the enzyme is a 5' exonuclease
specific for double-stranded deoxypolynucleotides.
21. The method of claim 20, wherein the exonuclease is selected
from the group consisting of the exonuclease of T7 and lambda
phage.
22. The method of claim 1, wherein the enzyme is an enzyme of DNA
recombination.
23. The method of claim 22, wherein the enzyme of DNA recombination
is recA.
24. A kit comprising (1) a plurality of unique synthesized
single-stranded deoxypolynucleotides, wherein each unique
synthesized deoxypolynucleotide has one or more copies and a 5'
sequence which, when the synthesized deoxypolynucleotide is in
double-stranded form, can be enzymatically treated to form a unique
3' single-stranded protrusion for selective cyclic hybridization of
the enzymatically treated double-stranded deoxypolynucleotide with
another unique single-stranded deoxypolynucleotide and (2) an
enzyme selected from the group consisting of exonuclease and
restriction endonuclease, which can form a unique single-stranded
3' protrusion from each of the double-stranded deoxypolynucleotides
formed by the polymerization of the hybridized unique
single-stranded deoxypolynucleotides.
25. The kit of claim 24, further comprising a unique
deoxypolynucleotide having a 3' sequence which can selectively
hybridize with one of the unique single-stranded
deoxypolynucleotides.
26. The kit of claim 24, further comprising a polymerase which can
direct the formation of double-stranded polynucleotides from the
single-stranded deoxypolynucleotides.
Description
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.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic showing the general mechanism for the cyclic
ETR.
FIG. 2-I shows the sequence of deoxyoligonucleotides designed for
the ETR synthesis of a fragment corresponding to the HBV.
Recognition sites for restriction endonucleases used for the ETR
(BstXI) and for cloning (BgIII and ApaI) are indicated.
FIG. 2-II shows the stepwise description of the mechanism of the
ETR for three deoxyoligonucleotides corresponding to the HBV
genome.
FIG. 3-I shows the primary structure of the deoxyoligonucleotides
corresponding to the 5' terminal region of the HCV nucleocapsid
gene. Sites for restriction endonucleases used for the ETR (BstXI),
for assembly of the gene (DdeI), and for cloning (NdeI) are
shown.
FIG. 3-II shows a schematic representation of the ETR.
Deoxyoligonucleotides are shown as solid lines. Points represent
DNA polymerase synthesized regions of the double-stranded fragment.
The upper strand consists of Oligo 1 and newly-synthesized
sequences. The lower strand is composed of oligonucleotide
sequences that remain after BstXI digestion and after synthesis of
new sequences at the very 3' terminus of the strand. The order of
the deoxyoligonucleotides involved in the reaction is
indicated.
FIG. 4-I shows the primary structure of the deoxyoligonucleotides
corresponding to the middle part of the HCV nucleocapsid gene.
FIG. 4-II shows a schematic representation of the ETR corresponding
to the middle part of the HCV nucleocapsid gene.
FIG. 5-I shows the primary structure of the deoxyoligonucleotides
corresponding to the 3' terminal region of the HCV nucleocapsid
gene.
FIG. 5-II shows a schematic representation of the ETR corresponding
to the 3' terminal region of the HCV nucleocapsid gene.
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
* * * * *