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TABLE OF CONTENTS
1. Field of the Invention
2. Background of the Invention
2.1. DNA Sequencing
2.1.1. Plus/Minus DNA Sequencing Method
2.1.2. Dideoxy Chain Termination Method
2.1.3. Maxam-Gilbert Method
2.1.4. RNA Sequence Determination
2.1.5. Automation of Sequencing
2.2. Site-Specific Mutagenesis
3. Summary of the Invention
4. Brief Description of the Figures
5. Detailed Description of the Invention
5.1. Sequencing
5.2. Site-Specific Mutagenesis
5.3. The Reaction Chamber and Components
5.4. Selection of Nucleotide Precursor to be Added to the Reaction Mixture
5.4.1. Sequencing
5.4.2. Site-Specific Mutagenesis
5.5. Detection of Unicorportated Nucleotide Precuresors in the Effluent
5.5.1. Absorbance Spectroscopy
5.5.2. Detection of Labeled Precursors
5.5.3. Electrochemical Detection
5.5.4. Conductivity Detection
5.6. Automation and Use of Computer
6. Example: Determination of the Sequence of an Olifonucleotide
7. Example: The Computer Programs Used
7.1. The Sequencing Program
7.2. Site-Specific Mutagenesis Program
1. FIELD OF THE INVENTION
This invention relates to a new method for determining the base sequence of
either DNA or RNA. The method of the invention is automatable and does not
require the use of radioactive labels. The method may also be used to
alter the sequence of a DNA or RNA molecule at a specific site, thus
providing for site-specific mutagenesis of the molecule.
2. BACKGROUND OF THE INVENTION
DNA is a long threadlike macromolecule comprising a chain of
deoxyribonucleotides. Similarly, RNA is composed of a chain of
ribonucleotides. A nucleotide consists of a nucleoside, i.e., a
nitrogenous base linked to a pentose sugar, and one or more phosphate
groups which is usually esterified at the hydroxyl group attached to C-5
of the pentose sugar (indicated as 5') of the nucleoside. Such compounds
are called nucleoside 5'-phosphates or 5'-nucleotides. In a molecule of
DNA the pentose sugar is deoxyribose, whereas in a molecule of RNA the
pentose sugar is ribose. The nitrogenous base can be a purine derivative
such as adenine or guanine, or a pyrimidine derivative such as cytosine,
thymine (in deoxyribonucleotides) or uracil (in ribonucleotides). Thus,
the major nucleotides of DNA are deoxyadenosine 5'-triphosphate (dATP),
deoxyguanosine 5'-triphosphate (dGTP), deoxycytidine 5'-triphosphate
(dCTP), and deoxythymidine 5'-triphosphate (dTTP). The major nucleotides
of RNA are adenosine 5'-triphosphate (ATP), guanosine 5'-triphosphate
(GTP), cytidine 5'-triphosphate (CTP) and uridine 5'-triphosphate (UTP).
The sequence of the purine and pyrimidine bases of the DNA or RNA molecule
encodes the genetic information contained in the molecule. The sugar and
phosphate groups of a DNA or RNA molecule perform a structural role,
forming the backbone of the macromolecule. Specifically, the sugar moiety
of each nucleotide is linked by a phosphodiester bridge to the sugar
moiety of the adjacent nucleotide as follows: the 3'-hydroxyl of the
pentose of one nucleotide is joined to the 5'-hydroxyl of the pentose of
the adjacent nucleotide by a phosphodiester bond. One terminus of the
nucleotide chain has a 5-hydroxyl group and the other terminus of the
nucleotide chain has a 3'-hydroxyl group; thus the nucleotide chain has a
polarity. By convention, the base sequence of nucleotide chains are
written in a 5' to 3' direction.
The formation of the phosphodiester bonds between deoxyribonucleotides is
catalyzed by the enzyme DNA polymerase. DNA polymerase requires the
following components to catalyze the synthesis of a chain of DNA: a
template strand (e.g. a single-stranded DNA molecule), a primer (i.e., a
short DNA or RNA chain with a free 3'-hydroxyl group, that is hybridized
to a specific site on the single-stranded template), and activated
deoxyribonucleotide precursors (i.e., nucleoside 5'-triphosphates or
dNTPs). Elongation of the primer strand, catalyzed by DNA polymerase,
proceeds in the 5' to 3' direction along the template. The occurs by means
of nucleophilic attack of the 3'-hydroxyl terminus of the primer on the
innermost phosphorous atom of the incoming nucleotide; a phosphodiester
bridge is formed and pyrophosphate is released. DNA polymerase catalyzes
the formation of a phosphodiester bond only if the base of the incoming
nucleotide is complementary to the base of the nucleotide on the template
strand; that is, the incoming nucleotide must form the correct
Watson-Crick type of basepair with the template. Thus, DNA polymerase is a
template-directed enzyme. Reverse transcriptase is also a
template-directed DNA polymerase, but requires RNA as its template.
Another enzyme, RNA polymerase, catalyzes the polymerization of activated
ribonucleotide precursors that are complementary to the DNA template. Some
polymerases, such as E.coli DNA polymerase I and T4 DNA polymerase, also
have a 3' to 5' exonuclease activity that acts on unpaired termini. This
3' to 5' exonuclease activity serves a "proof-reading" function by
removing mispaired bases before polymerization continues; i.e., the
mispaired bases are edited out of the elongating strand.
2.1. DNA SEQUENCING
A number of different procedures are currently used to determine the base
sequence of DNA or RNA molecules. While the approaches vary considerably,
every one of the methods currently used has the following common elements:
(a) a method for producing a population of radioactive polynucleotides in
which each molecule has one common terminus (either 5' or 3');
(b) a method for producing from this population of radioactive
polynucleotides an array of polynucleotides with one common terminus but
varying in length at the other terminus in increments of a single base;
and
(c) a method for ordering the population of molecules by size, usually by
electrophoretic separation in a high-resolution denaturing polyacrylamide
gel from which an autoradiograph is prepared. The sequence is deduced from
the resulting "bands" or "ladders" on the autoradiogram. Specific
sequencing methods are discussed in the subsections that follow.
2.1.1. PLUS/MINUS DNA SEQUENCING METHOD
The plus/minus DNA sequencing method (Sanger and Coulson, 1975, J. Mol.
Biol. 94: 441-448) involves the following:
DNA polymerase is first used to elongate a primer oligonucleotide and copy
the template in the presence of the four activated nucleotide precursors,
one of which is labeled with .sup.32 P. Ideally, the synthesis is
non-synchronous and as random as possible, so that the maximum number of
olignucleotides of different length, all starting from the primer, are
formed. Excess unreacted nucleotides are removed and the mixture of DNA
strands is divided in two. One half is treated according to the "Minus"
System and the other according to the "Plus" System, as described below:
(a) The "Minus" System: The mixture of random length radiolabeled
oligonucleotides, which are still hybridized to the template DNA, is
divided into four separate reaction mixtures and reincubated with DNA
polymerase in the presence of three activated nucleotide precursors; that
is, one of the four nucleoside 5'-triphosphosphates is missing from each
reaction mixture. Elongation of each chain will proceed as far as it can
along the template; in other words, each chain will terminate at its 3'
end at a position before the site of incorporation of the missing residue.
For example, in the -A system, dATP is the nucleotide missing from the
reaction mixture and each chain will terminate at its 3' end at a position
before a dATP residue would be incorporated into the growing chain.
Therefore, at the end of the incubation period each reaction mixture will
contain a population of DNA molecules each having a common 5' terminus but
varying in length at the 3' terminus. The radiolabeled nucleotides of
varying lengths in each reaction mixture are fractionated according to
size by electrophoresis in a denaturing polyacrylamide gel; each reaction
mixture is fractionated in a separate lane. The relative position of each
residue along the DNA may be located and the sequence of DNA may be
deduced from the autoradiograph of the resulting gel. This system alone is
usually not sufficient to establish a sequence, so a second similar
system, the Plus System, described below, is normally used in conjunction
with it.
(b) The "Plus" System: The mixture of random length radiolabeled
oligonucleotides, which are still hybridized to the template DNA is
divided into four separate reaction mixtures each of which is reincubated
with DNA polymerase in the presence of only one of the four activated
nucleotide precursor. For example, in the +A system only dATP is present
in the reaction mixture. While the population of DNA molecules each has a
common 5' terminus, all the chains will have varying lengths that
terminate with deoxyadenosine residues. The positions of the dATP residues
will be indicated by bands on the autoradiograph obtained after
fractionating the DNA chains in each reaction mixture according to size by
electrophoresis in a denaturing polyacrylamide gel; each reaction mixture
is fractionated in a separate lane. Usually these will be oligonucleotide
products that are one residue larger than the corresponding bands in the
-A system, but if there is more than one consecutive dATP residue, the
distance between the bands in the -A and +A systems will indicate the
number of such consecutive residues.
In order for the plus/minus system to yield reliable results various
criteria must be satisfied. For instance, all DNA fragments must have the
same 5' terminus and the Klenow fragment of DNA polymerase must be used in
order to eliminate the 5' exonuclease activity of DNA polymerase.
Furthermore, it is essential that the nucleotides are fractionated
according to size. Ideally, oligonucleotides of all possible lengths
should be present in the initial reaction mixture so that all residues are
represented in the plus and minus systems, however, it is difficult to
achieve this because certain products are formed in relatively high yield
whereas others are absent. It has been suggested that the polymerase acts
at different rates at different sites or that this effect is partly
related to the secondary structure of the template. While Sanger et al.,
supra, report that best results are obtained if synthesis is carried out
for short times with a relatively high concentration of polymerase,
frequently some expected products are missing. This constitutes a
limitation of the method and is one reason why it is necessary to use both
the plus and minus systems. Consecutive runs of a given nucleotide present
the main difficulty when using the plus/minus method of sequencing.
2.1.2. DIDEOXY CHAIN TERMINATION METHOD
The "dideoxy" chain termination DNA sequencing method of Sanger et al.
1977, Proc. Natl. Acad. Sci. U.S.A. 74: 5463, also makes use of the
ability of DNA polymerase to synthesize a complementary radiolabeled copy
of a single stranded DNA template hybridized to a short DNA primer. The
synthesis is carried out in the presence of all four deoxynucleoside
5'-triphosphates (dNTPs), one or more of which is labeled with .sup.32 P,
and a 2',3'-dideoxynucleoside triphosphate analog of one of the four
dNTPs. Four separate incubation mixtures are prepared each of which
contains only one of the four dideoxynucleotide analogs. Once the analog
is incorported, the 3' end of the growing chain is no longer a substrate
for DNA polymerase and thus cannot be elongated any further. At the end of
the incubation period each reaction mixture will contain a population of
DNA molecules having a common 5' terminus but varying in length to a
nucleotide base-specific 3' terminus. Each population of DNA molecules is
then denatured and fractionated according to size by gel electrophoresis;
each reaction mixture is fractionated in a separate lane. Autoradiography
of the gel allows the sequence to be deduced.
The use of the single-stranded bacteriophage M13 to obtain multiple copies
of the DNA sequence of interest and its "universal primer" sequence has
greatly enhanced the usefulness of the dideoxy chain termination DNA
sequencing method. However, the method absolutely requires fractionation
of the DNA products by size and thus involves gel electrophoresis.
2.1.3. MAXAM-GILBERT METHOD
The Maxam-Gilbert method of DNA sequencing is a chemical sequencing
procedure (Maxam and Gilbert, 1977, Proc. Natl. Acad. Sci. USA 74: 560).
After radioactively labeling either the 3' or the 5' terminus of a
discrete aliquots of the DNA are placed in four separate reaction
mixtures, each of which partially cleaves the DNA in a base-specific
manner. The resulting population of DNA in each reaction mixture is then
denatured and fractionated according to size by gel electrophoresis; each
reaction mixture is fractionated in a separate lane. The DNA sequence is
deduced from the ladders which appear on the resulting autoradiogram.
2.1.4. RNA SEQUENCE DETERMINATION
The major RNA sequence analysis strategies employ 5' and 3' terminal
labeling protocols (England and Uhlenbeck, 1978, Nature 275: 561).
Subsequent to radiolabeling, the RNA molecules are fragmented using
base-specific RNases or chemicals. Similarly to the DNA sequencing
methods, each population of oligoribonucleotides is fractionated by size
via high resolution electrophoresis in a denaturing polyacrylamide gel.
The sequence is then deduced from the autoradiogram corresponding to the
gel.
2.1.5. AUTOMATION OF SEQUENCING
Some major drawbacks to the sequencing methods described above are that
they are labor intensive, time consuming and not readily automated.
Current attempts at automation involve the use of densitometers to "read"
the optical density of the bands or ladders on the autoradiographs. These
techniques require that the gel lanes be straight and also require careful
monitoring by the operator.
2.2. SITE SPECIFIC MUTAGENESIS
Methods currently used to mutagenize DNA include in vivo techniques which
involve treatment with mutagens such as alkylating agents, mitomycin C,
ionizing radiation or ultraviolet radiation, or in vitro techniques such
as deletion loop mutagenesis induced by bisulfite. However, these methods
are likely to yield multiple-base substitutions in a non-specific manner.
Several methods have been developed to generate specific base substitutions
at selected sites in DNA. (For a brief review of the methods used, see
Zakour et al., 1984, Nucleic Acids Research 12(6): 6615-6628 and Abarzua
et al., 984, Proc. Natl. Acad. Sci. 81: 2030-2034) One method involves
inserting into a viral DNA template or recombinant DNA a synthetic
oligonucleotide which encodes a pre-selected change in its nucleotide
sequence. This method is efficient and can produce any type of base
substitution mutation but each different mutation that is introduced
requires the synthesis of a unique oligonucleotide which encodes the
mutation and is complementary to the cohesive ends which must be generated
on the viral or recombinant DNA.
A second method (Shortle et al., 1980, Proc. Natl. Acad. Sci. USA 77:
5375-5379) involves introducing a small single-strand gap in the DNA
molecule followed by mis-repair DNA synthesis; i.e., the mis-incorporation
of a non-complementary nucleotide in the gap. The incorporation of
.alpha.-thiol nucleotides into the gap minimizes the excision of the
non-complementary nucleotide. When deoxyribonucleoside
(1-thio)-triphosphate analogs containing a sulfur atom in place of oxygen
on the phosphorous are used as substrates for the synthesis of a DNA
strand that is complementary to a template DNA, the analog is incorporated
as a thiomonophosphate at rates similar to those of corresponding
unmodified nucleoside triphosphates. However, the phosphorothiate is not
hydrolyzed by the 3' to 5' exonuclease activity of either E. coli DNA
polymerase I or T4 DNA polymerase and, therefore, the mispaired base is
not edited out. Abarzua et al. (1984, Proc. Nat.l. Acad. Sci. 81:
2030-2034) report a modification of this technique using a gapped circular
DNA constructed by annealing viral singlestranded stranded circular DNA
with a mixture of linear duplex DNAs that have had their 3'-hydroxyl
termini processively digested with E. coli exonuclease III under
conditions in which the resulting, newly generated 3'-hydroxyl termini
present in the various hybrid molecules span the region of interest. Base
changes are induced by incorporation of mis-matched
2-thiodeoxyribonucleoside triphosphate analogs, followed by DNA repair
synthesis.
A third method used is based on the infidelity of certain DNA polymerases
and involves the extension of a primer by a non-proofreading DNA
polymerase in the presence of a single non-complementary deoxynucleotide
triphosphate, after which synthesis is completed by a highly accurate DNA
polymerase in the presence of all four deoxyribonucleotide substrates. In
a modification of this method, Zakour et al. (1984, Nucleic Acids Research
12(16): 6615-6628) used T4 DNA polymerase to elongate primer termini to a
position immediately adjacent to two different preselected positions on
.phi.X174 templates. Then, the error-prone DNA polymerase from avian
myeloblastoma virus was used to insert single non-complementary
nucleotides at the designated positions with high efficiency.
3. SUMMARY OF THE INVENTION
This invention presents a new automatable method for sequencing DNA or RNA
that does not require radioactivity or gel electrophoresis. The method may
also be used to accomplish the site-specific mutagenesis of any DNA or RNA
molecule.
The sequencing method of the present invention involves adding an activated
nucleotide precursor (a nucleoside 5'-triphosphate) having a known
nitrogenous base to a reaction mixture comprising a primed single-stranded
nucleotide template to be sequenced and a template-directed polymerase.
The reaction conditions are adjusted to allow incorporation of the
nucleotide precursor only if it is complementary to the single-stranded
template at the site located one nucleotide residue beyond the 3' terminus
of the primer. After allowing sufficient time for the reaction to occur,
the reaction mixture is washed so that unincorporated precursors are
removed while the primed template and polymerase are retained in the
reaction mixture. The wash or effluent is assayed for the incorporation of
precursors. The methods which may be used to detect unincorporated
precursors in the effluent include but are not limited to spectroscopic
methods, radioactive labeling and counting, electrochemical, and
conductivity methods. The detection of all of the of nucleotide precursor
in the effluent that was added to the reaction mixture indicates that the
added precursor was not incorporated into the growing chain and,
therefore, is not part of the nucleotide sequence. If less nucleotide
precursor is detected in the effluent than was added, however, this
indicates that the added precursor was incorporated into the growing chain
and, therefore, is the next nucleotide of the sequence.
The sequencing method of the present invention is readily automated. For
example, the reaction chamber may be attached to five reservoirs--one for
each nucleotide precursor and one for a wash buffer--that feed into the
chamber. The reaction chamber should also have an outlet which feeds the
effluent into the detection instrument used for the assay; for example, a
spectrophotometer, a scintillation counter, Geiger-Muller counter,
conductivity or electrochemical cell, etc. Ideally, the assay instrument
and the valves that regulate the inlet and outlet of the reaction chamber
can be controlled by a computer which is programmed to select the
particular nucleotide precursor to be added to the reaction mixture, to
record the instrument reading of the effluent, and to determine and record
which nucleotides were incorporated into the growing chain, thus
ultimately providing a print-out of the nucleotide sequence.
The sequencing method of the present invention has a number of advantages
over existing methods:
(a) radioactive labels are not required (although they may be used);
(b) fractionation of polynucleotides by size is not required;
(c) gel electrophoresis is not required, therefore, the sequence need not
be deduced by reading bands or ladders on a sequencing gel; and
(d) sequence information is acquired as the reaction proceeds, thus
allowing results to be screened during sequencing.
In another embodiment of the present invention a modification of the
sequencing method may be used to alter or mutagenize a DNA or RNA sequence
at a particular nucleotide site within the sequence. According to this
embodiment, site-specific mutagenesis is accomplished as follows: the
single-step synthesis of a nucleotide strand complementary to a template
strand is accomplished as described above for DNA sequencing, but the
template strand has a known nucleotide sequence. Since the nucleotide
sequence of the template is known, the order of the nucleotide precursors
to be added step-by-step is known. The synthesis is stopped at the
nucleotide residue preceding the residue which is to be altered. The next
nucleotide precursor to be added to the reaction mixture is one which
cannot be edited out by the polymerase under the reaction conditions in
the chamber; this nucleotide base is the mutation desired in the sequence.
Although the nucleotide is mis-paired (i.e. the base is not complementary
to the template strand at that residue) the nucleotide will be
incorporated into the growing strand and will not be edited out by the
template directed polymerase. After each desired site-specific mutation is
accomplished, the synthesis of the remaining portion of the DNA or RNA
molecule need not proceed in a stepwise fashion, therefore, all four
activated nucleotide precursors may be added to the reaction mixture to
complete the elongation.
The reaction chamber and reservoirs used in the embodiment of the invention
are similar to those described for DNA sequencing above except that an
extra reservoir may be required. The site-specific mutagenesis method of
the present invention is automatable and can be controlled by a computer.
In this case the computer is programmed to add each nucleotide of the
known sequence in the proper order, to record the instrument reading of
the effluent to ensure each nucleotide is incorporated, to then add the
mis-matched analog, and finally to add all four nucleotides to the
reaction mixture.
4. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic diagram of the different components (not drawn to
scale) which may be used in the practice of one embodiment of the present
invention.
FIG. 2 is a schematic representation of the reaction and sequence of steps
which may be used for sequencing according to the present invention.
FIG. 3 is a schematic representation of the reaction and sequence of steps
which may be used for site-specific mutagenesis according to the present
invention.
FIG. 4 is a flow chart of a computer program that may be used according to
the present invention.
5 DETAILED DESCRIPTION OF THE INVENTION
This invention presents a new method for sequencing DNA or RNA which is
automatable and does not require the use of radioactivity or gel
electrophoresis. In addition, the present invention involves a method for
the site-specific mutagenesis of a DNA or RNA sequence.
5.1. SEQUENCING
The sequencing method of the invention involves the following: The
single-stranded DNA or RNA molecule to be sequenced is primed at a
specific site with a short oligonucleotide primer. The primed template and
a template-directed polymerase are placed in a reaction chamber that
allows for separation of unreacted nucleotide precursors from the primed
template and the polymerase. If the template is a single-stranded DNA
molecule, a DNA-directed DNA or RNA polymerase may be used; if the
template is a single-stranded RNA molecule, then a reverse transcriptase
(i.e., an RNA directed DNA polymerase) may be used.
One at a time, a particular activated nucleotide precursor is added to the
chamber and allowed to react. The nucleotides added may be either
deoxyribonucleotide or ribonucleotides depending upon the nature of the
template and polymerase used in the reaction mixture. For instance, when
the template is a single-stranded DNA molecule, the polymerase used may be
a DNA-directed DNA polymerase, in which case the nucleotides added should
be deoxyribonucleotides. When double stranded DNA is used, the polymerase
may be a DNA-directed RNA polymerase, in which case the nucleotides added
should be ribonucleotides. Similarly, when the template is a
single-stranded RNA molecule, the polymerase used is a
reverse-transcriptase, i.e., an RNA directed DNA polymerase, in which case
the nucleotides added must be deoxyribonucleotides. In any case, however,
only one type of nucleotide is added at a time. For example, either dATP,
dTTP, dGTP or dCTP is added to the reaction but not a mixture of these
deoxyribonucleotides; similarly, either ATP, UTP, GTP or CTP is added to
the reaction but not a mixture of these ribonucleotides.
If the base of the added nucleotide precursor is complementary to the
template, i.e., if the nucleotide precursor can form a Watson-Crick type
of base pair with the template at the site located one nucleotide residue
beyond the 3' terminus of the primer strand, the nucleotide precursor will
be incorporated into the growing chain. If the base of the nucleotide
precursor is not complementary to the template strand at the site located
one nucleotide residue beyond the 3' terminus of the primer strand, the
nucleotide precursor will not be incorporated into the growing chain.
After an adequate time is allowed for the polymerase reaction to occur the
reaction chamber is "washed" in order to separate any unreacted nucleotide
precursor in the the primed template. Then the wash or effluent is assayed
in order to determine the amount of nucleotide precursor in the effluent.
The methods which may be used to detect unincorporated precursors in the
effluent include but are not limited to spectroscopic methods, such as
absorption or fluorescence spectroscopy; radioactive labeling and counting
(provided the nucleotide precursors are radiolabeled); and electrochemical
or conductivity methods.
The process described above can be automated as follows: The reaction
chamber can be connected to five reservoirs which feed into the reaction
chamber, one of which contains the wash buffer whereas each of the other
four contain a specific nucleotide precursor. The reaction chamber should
also have an outlet valve so that after the addition of a particular
nucleotide precursor and allowing for an appropriate reaction time the
chamber can be washed so that the effluent moves through the outlet valve
into a flow-cell of the detecting instrument used in the assay. The
various components of this embodiment of the invention are illustrated
schematically in FIG. 1. The reservoirs and detector can be attached to a
computer that records which of the nucleotide bases was added to the
reaction chamber and the detector reading of the resultant effluent.
Ideally, the computer can be programmed to choose which nucleotide
precursor to feed into the reaction chamber next and to record the
nucleotides which were incorporated, thus ultimately providing a printout
of the sequence.
5.2. SITE-SPECIFIC MUTAGENESIS
In a second embodiment of the present invention, a modification of the
sequencing method may be used as a basis for site-specific mutagenesis of
any nucleotide sequence. According to this mode of the invention, the
single-stranded DNA or RNA molecule having a known sequence which is to be
mutagenized at a specific site is primed with a short oligonucleotide
primer. The primed template and a template-directed polymerase are placed
in a reaction chamber that allows for separation of unreacted nucleotide
precursors from the primed template and the polymerase. If the template is
a single-stranded DNA molecule, a DNA-directed DNA or RNA polymerase may
be used; if the template is a single-stranded RNA molecule, then a reverse
transcriptase (i.e., and RNA directed DNA polymerase) may be used.
Each activated nucleotide precursor of the known sequence is added to the
chamber and allowed to react. The nucleotides added may be either
deoxyribonucleotides or ribonucleotides depending upon the nature of the
template and polymerase used in the reaction mixture. For instance, when
the template is a single-strand DNA molecule, the polymerase used would be
a DNA-directed DNA polymerase, in which case the nucleotides added should
be deoxyribonucleotides. Similarly, when the template is a single-stranded
RNA molecule, the polymerase used is a reverse-transcriptase, i.e., an
RNA-directed DNA polymerase in which case the nucleotides added must be
deoxyribonucleotides. Alternatively, if a double-stranded DNA template is
used that contains a promoter for which a specifically functional RNA
polymerase is available, i.e, the T7 promoter and polymerase, the
nucleotides added should be ribonucleotides. In one embodiment of the
present invention, only one type of nucleotide is added at a time. For
example, either dATP, dTTP, dGTP or dCTP is added to the reaction but not
a mixture of these deoxyribonucleotides. Similarly, either ATP, UTP, GTP
or CTP is added to the reaction but not a mixture of these
ribonucleotides. In a second embodiment of the method, the nucleotides may
be added up to three at a time in order to speed up the synthesis. This
approach is feasible because the sequence of the template is known.
After an adequate time is allowed for the polymerase reaction to occur the
reaction chamber is "washed" in order to separate any unreacted nucleotide
precursors from the primed template. Then the wash or effluent may be
assayed in order to determine the presence or absence of the nucleotide
precursor in the effluent to ensure that the nucleotide was, in fact,
incorporated into the growing chain. The methods which may be used to
detect unincorporated precursors in the effluent include but are not
limited to spectroscopic methods, such as absorption or fluorescence
spectroscopy; radioactive labeling and counting (provided the nucleotide
precursors are radiolabeled); and electrochemical or conductivity methods.
The stepwise addition and reaction of each nucleotide or groups of up to
three nucleotides is continued and ultimately stopped at the nucleotide
position which precedes the residue which is to be mutagenized. The next
nucleotide to be added to the reaction mixture is a nucleotide that cannot
be edited out of the elongating chain by the polymerase under the
conditions in the reaction chamber. For example, the nucleotide may be an
analog that cannot be edited out by the polymerase; this analog base is
the mutation desired in the sequence. The analog is incorporated into the
growing chain even though the analog base does not form a Watson-Crick
type of base pair with the nucleotide residue in the template to be
mutagenized; and the mis-paired analog will not be edited out of the
growing strand. After an adequate time is allowed for the polymerase
reaction to occur, the reaction chamber is washed and the effluent may be
assayed in order to ensure the incorporation of the analog. After the
desired site-specific mutation is accomplished, the synthesis of the
remaining portion of the DNA or RNA molecule need not proceed in a
step-wise fashion, therefore all four unmodified nucleotide precursors may
be added to the reaction mixture to complete the elongation. An example of
embodiment of the invention is depicted in FIG. 3.
Nucleotide analogs which may be used in the present invention include
deoxyribonucleoside (1-thio)-triphosphates containing a sulfur atom in
place of an oxygen atom on the phosphorus. These analogs are incorporated
as thiomonophosphates at rates similar to those of corresponding
unmodified nucleoside triphosphates. However, the phosphorothioate bond is
not hydroylzed by the 3' to 5' exonuclease of either E. coli DNA
polymerase I or T4 DNA polymerase, therefore, the incorporation of the
analog as a mispaired base cannot be edited out. Other analogs may be used
in the practice of this embodiment of the present invention.
The process described above can be automated as follows: The reaction
chamber can be connected to five reservoirs which feed into the reaction
chamber, one of which contains the wash buffer whereas each of the other
four contains a specific nucleotide precursor. In addition, the reaction
chamber should be connected to one reservoir for each analog. The reaction
chamber should also have an outlet valve so that after the addition of a
particular nucleotide precursor and allowance for an appropriate reaction
time, the chamber can be washed so that the effluent moves through the
outlet valve into a flow-cell of the detecting instrument used in the
assay. The reservoirs and assay instrument can be attached to a computer
that controls the selection of the nucleotides to be added in a step-wise
manner, records the successful incorporation of each nucleotide base added
to the reaction chamber, and records the assay reading of the resultant
effluent. Ideally, the known nucleotide sequence with the desired
site-specific mutation or mutations can simply be fed into the computer,
thus facilitating the process.
The subsections below describe the invention in more detail.
5.3. THE REACTION CHAMBER AND COMPONENTS
The DNA or RNA to be sequenced serves as the template in the polymerase
reaction utilized in the present invention, therefore the molecule to be
sequenced should be single-stranded; however, the template may be linear
or circular. Thus, any single-stranded DNA or RNA molecule may be
sequenced according to the method of the present invention.
Selection of the primer, polymerase and activated nucleotide precursors
used in the practice of the present invention depends upon the nature of
the template to be sequenced. For example, if the template to be sequenced
is DNA, the primer used may be DNA, RNA or a mixture of both. The
polymerase used should be a DNA-directed polymerase. If a DNA-directed DNA
polymerase is used, then deoxyribonucleotide precursors will be used in
the reaction; alternatively a DNA-directed RNA polymerase requires
ribonucleotide precursors to be used in the reaction. However, if the
strand to be sequence is RNA, the polymerase used should be an
RNA-directed DNA polymerase, in which case deoxyribonucleotide precursors
will be used in the reaction.
In an other embodiment of the invention the template can be a
double-stranded DNA molecule such as a chromosome that encodes a promoter.
According to this mode of the invention the polymerase should be one that
recognizes the promoter and initiates transcription of mRNA; therefore,
the nucleotides used are ribonucleotides.
In any case, the polymerase used should have a higher level of
accuracy--that is the polymerase should require correct base pairing
before polymerization to ensure a high level of fidelity of the reaction.
Some polymerases, such as E. coli DNA polymerase I, have a 5' to 3'
exonuclease activity: according to one mode of the present invention, a
polymerase that is low in the 5' to 3' exonuclease is preferred. An
example of a polymerase that is low in the 5' to 3' exonuclease activity
is the Klenow fragment of E. coli DNA polymerase I. Other polymerases
which may be used in the practice of the present invention include but are
not limited to AMV reverse transcriptase, E. coli RNA polymerase, and
wheat germ RNA polymerase II.
Although the volume of the reaction can vary, in the preferred embodiment
of the present invention the volume should be less than one milliliter.
The reaction vessel should be constructed so that unreacted nucleotide
precursors can be separated from the polymerase and reaction products.
Ideally, the reaction vessel is constructed so that each of the four
nucleotide precursors and one or more wash buffers feed into the reaction
chamber which is provided with an outlet that feeds into the flow cell of
an instrument that can be used to assay the nucleotides in the wash or
effluent. The outlet is closed during the reaction time but opened at the
end of the reaction to allow displacement of the effluent when wash buffer
is fed into the reaction chamber.
Alternatively, the reaction vessel can be part of a continuous flow system.
In this embodiment, the flow rate is adjusted such that the nucleotide
precursor injected upstream of the reaction vessel has sufficient time in
the reaction vessel to react (if it is the next base in the sequence) with
the polymerase and nucleotide template, before passing on to the detector.
If the reaction is carried out in a liquid buffer, then a membrane having
an appropriate pore size interposed at the outlet valve could be used to
retain the reaction products and polymerase while allowing unreacted
nucleotide precursors to pass through with the effluent. In this
embodiment, the pore size selected should be large enough to allow the
passage of the unreacted nucleotide precursors but not the primed template
or the polymerase.
In another embodiment, the polymerase or the primed template could be
immobilized on an insoluble inert support, thus, the polymerization
reaction will occur on the surface of the inert support. When the primed
template is immobilized, the pore size of the frit interposed at the
outlet valve need only be small enough to prevent passage of the support
material with the DNA and primer attached. Below the outlet valve would be
a molecular trap of appropriate size to retain the polymerase. By
reversing the flow through this trap, the polymerase could then be passed
back through the reactor with the next nucleotide precursor to be tried.
In yet another embodiment, the reaction vessel may contain a column packing
material that differentially retards the movement of each component in the
reaction mixture.
The porous membrane may be composed of any inert solid material, such as
dialysis membrane material, nitrocellulose, or cellulose acetate, to name
but a few.
5.4. SELECTION OF NUCLEOTIDE PRECURSOR TO BE ADDED TO THE REACTION MIXTURE
The polymerization reaction of the present invention should proceed
synchronously so that substantially all of the primer ends have been
elongated to the same position. Various precautions and approaches may be
taken to ensure that each step of the reaction goes to completion and to
minimize the background or noise reading obtained.
5.4.1. SEQUENCING
In the preferred embodiment of the method for sequencing DNA, a known
excess of the nucleotide precursor is added to the reaction chamber,
allowed to react, and then flushed past a detector. The number of
sequential bases incorporated is determined according to the relative
amount of nucleotide detected in the effluent. For example, if four
equivalents (relative to the molar amount of DNA in the reaction chamber)
of dATP was added and the integration of the effluent signal is 50% of
what would be expected for that amount of dATP, it can be concluded that
two sequential "A" nucleotides were incorporated. This technique serves to
both indicate repeated nucleotides in the sequence and to shorten the
reaction time.
In the preferred embodiment of the method for sequencing DNA, "mistakes" or
primer strands that are not synchronously elongated are carried along with
each addition of a nucleotide precursor for rectification when deemed
necessary because of a deteriorating signal-to-noise ratio, so as to
minimize the background absorbance of the effluent. This may be
accomplished by the proper selection of the appropriate nucleotide to be
added to the reaction mixture for any one step. In one embodiment, in
order to carry along mistakes the penultimate nucleotide that was
successfully incorporated into the primer strand should be the next
nucleotide precursor added to the reaction mixture. If after washing and
assaying the effluent it is determined that the nucleotide precursor was
not incorporated into the elongated primer strand, then each of the other
three nucleotides may be tried, one at a time, in any order. When the
signal-to-noise ratio gets too high the accumulated noise can be corrected
by adding the last nucleotide successfully incorporated into the
elongating strand. This process is repeated whenever required, until the
entire sequence is obtained. For example, if the sequence obtained using
the method of the present invention is determined to be "ATGCTA", the
nucleotide precursor that should be added in the subsequent trial is dTTP.
Thus, if some of the primer strands had not previously been elongated up
to the fifth nucleotide "T", and they are therefore two nucleotide
residues shorter than the rest of the primer strands in the reaction
mixture, the addition of dTTP will carry the shorter primers along so that
they will always be one nucleotide shorter than the majority of the primer
molecules in the population; as a result, the "mistakes" will be carried
along for eliminating when necessary, i.e., when the background or noise
levels are too high. The mistake can be corrected in the example above by
then repeating the addition of dATP.
In an alternate embodiment of the method for sequencing DNA of the present
invention, after each incorporation of a nucleotide precursor an
additional quantity of the same precursor is added to ensure that all the
primer ends have been elongated to the same position. This procedure is
repeated until the entire sequence is obtained.
5.4.2 SITE-SPE MUTAGENESIS
Since the sequence of the DNA or RNA template is known, the nucleotide
precursors are added in the proper sequence in sufficient excess and for
sufficient time to ensure complete reaction. The nucleotide precursors can
be added one at a time, as in the program reproduced in the example, or up
to three nucleotide precursors can be added at a time.
For example, for the synthesis illustrated in FIG. 3, the base that would
normally fit in the site to be modified is a "C". To a solution of the
template, primer and polymerase are added excess amounts of dTTP and dATP.
After the unreacted precursors are flushed out, an excess of dCTP is added
to complete the synthesis through the third base. After the dCTP has been
flushed, excess amounts of dGTP, dATP and dTTP are added to take the
synthesis up to the site to be modified.
Alternatively, a system with the DNA template and primer immobilized on an
insoluble inert support is employed in which the polymerase is passed
through the reactor with the nucleotide precursor, and the polymerase is
retained in a molecular trap below the outlet valve of the reactor. During
the synthesis of the portion of the strand before the nucleotide to be
modified, the polymerase is recycled by back-flushing the trap. At that
point, however, an error-prone polymerase (that is, one with no
proof-reading function, such as avian myeloblastoma virus polymerase) is
used with one equivalent of the nucleotide precursor to be incorporated.
After the error-prone polymerase is flushed from the reactor, the
synthesis sequence continues with the appropriate nucleotide precursors
and the recycled high fidelity polymerase.
5.5. DETECTION OF UNINCORPORATED NUCLEOTIDE PRECURSORS IN THE EFFLUENT
Any quantitative assay for nucleotides may be used to detect the
unincorporated nucleotides in the effluent. Some methods which may be used
to detect unincorporated precursors in the effluent include but are not
limited to spectroscopic methods, such as | | |
