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Description  |
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FIELD OF THE INVENTION
This invention relates to methods for increasing the number of copies of a
specific nucleic acid sequence or "target sequence" which may be present
either alone or as a component, large or small, of a homogeneous or
heterogeneous mixture of nucleic acids. The mixture of nucleic acids may
be that found in a sample taken for diagnostic testing, environmental
testing, for research studies, for the preparation of reagents or
materials for other processes such as cloning, or for other purposes.
The selective amplification of specific nucleic acid sequences is of value
in increasing the sensitivity of diagnostic and environmental assays while
maintaining specificity; increasing the sensitivity, convenience, accuracy
and reliability of a variety of research procedures; and providing ample
supplies of specific oligonucleotides for various purposes.
The present invention is particularly suitable for use in environmental and
diagnostic testing due to the convenience with which it may be practiced.
BACKGROUND OF THE INVENTION
The detection and/or quantitation of specific nucleic acid sequences is an
increasingly important technique for identifying and classifying
microorganisms, diagnosing infectious diseases, detecting and
characterizing genetic abnormalities, identifying genetic changes
associated with cancer, studying genetic susceptibility to disease, and
measuring response to various types of treatment. Such procedures have
also found expanding uses in detecting and quantitating microorganisms in
foodstuffs, environmental samples, seed stocks, and other types of
material where the presence of specific microorganisms may need to be
monitored. Other applications are found in the forensic sciences,
anthropology, archaeology, and biology where measurement of the
relatedness of nucleic acid sequences has been used to identify criminal
suspects, resolve paternity disputes, construct genealogical and
phylogenetic trees, and aid in classifying a variety of life forms.
A common method for detecting and quantitating specific nucleic acid
sequences is nucleic acid hybridization. This method is based on the
ability of two nucleic acid strands which contain complementary or
essentially complementary sequences to specifically associate, under
appropriate conditions, to form a double-stranded structure. To detect
and/or quantitate a specific nucleic acid sequence (known as the "target
sequence"), a labelled oligonucleotide (known as a "probe") is prepared
which contains sequences complementary to those of the target sequence.
The probe is mixed with a sample suspected of containing the target
sequence, and conditions suitable for hybrid formation are created. The
probe hybridizes to the target sequence if it is present in the sample.
The probe-target hybrids are then separated from the single-stranded probe
in one of a variety of ways. The amount of label associated with the
hybrids is measured.
The sensitivity of nucleic acid hybridization assays is limited primarily
by the specific activity of the probe, the rate and extent of the
hybridization reaction, the performance of the method for separating
hybridized and unhybridized probe, and the sensitivity with which the
label can be detected. Under the best conditions, direct hybridization
methods such as that described above can detect about 1.times.10.sup.5 to
1.times.10.sup.6 target molecules. The most sensitive procedures may lack
many of the features required for routine clinical and environmental
testing such as speed, convenience, and economy. Furthermore, their
sensitivities may not be sufficient for many desired applications.
Infectious diseases may be associated with as few as one pathogenic
microorganism per 10 ml of blood or other specimen. Forensic investigators
may have available only trace amounts of tissue available from a crime
scene. Researchers may need to detect and/or quantitate a specific gene
sequence that is present as only a tiny fraction of all the sequences
present in an organism's genetic material or in the messenger RNA
population of a group of cells.
As a result of the interactions among the various components and component
steps of this type of assay, there is almost always an inverse
relationship between sensitivity and specificity. Thus, steps taken to
increase the sensitivity of the assay (such as increasing the specific
activity of the probe) may result in a higher percentage of false positive
test results. The linkage between sensitivity and specificity has been a
significant barrier to improving the sensitivity of hybridization assays.
One solution to this problem would be to specifically increase the amount
of target sequence present using an amplification procedure. Amplification
of a unique portion of the target sequence without requiring amplification
of a significant portion of the information encoded in the remaining
sequences of the sample could give an increase in sensitivity while at the
same time not compromising specificity. For example, a nucleic acid
sequence of 25 bases in length has a probability of occurring by chance of
1 in 4.sup.25 or 1 in 10.sup.15 since each of the 25 positions in the
sequence may be occupied by one of four different nucleotides.
A method for specifically amplifying nucleic acid sequences termed the
"polymerase chain reaction" or "PCR" has been described by Mullis et al.
(See U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159 and European patent
applications 86302298.4, 86302299.2, and 87300203.4 and Methods in
Enzymology, Volume 155, 1987, pp. 335-350). The procedure uses repeated
cycles of primer-dependent nucleic acid synthesis occurring simultaneously
using each strand of a complementary sequence as a template. The sequence
which is amplified is defined by the locations of the primer molecules
that initiate synthesis. The primers are complementary to the 3'-terminal
portion of the target sequence or its complement and must complex with
those sites in order for nucleic acid synthesis to begin. After extension
product synthesis, the strands are separated, generally by thermal
denaturation, before the next synthesis step. In the PCR procedure, copies
of both strands of a complementary sequence are synthesized.
The strand separation step used in PCR to separate the newly synthesized
strands at the conclusion of each cycle of the PCR reaction is often
thermal denatured. As a result, either a thermostable enzyme is required
or new enzyme must be added between thermal denaturation steps and the
initiation of the next cycle of DNA synthesis. The requirement of repeated
cycling of reaction temperature between several different and extreme
temperatures is a disadvantage of the PCR procedure. In order to make the
PCR convenient, expensive programmable thermal cycling instruments are
required.
The PCR procedure has been coupled to RNA transcription by incorporating a
promoter sequence into one of the primers used in the PCR reaction and
then, after amplification by the PCR procedure for several cycles, using
the double-stranded DNA as template for the transcription of
single-stranded RNA. (See, e.g. Murakawa et al., DNA 7:287-295 (1988).
Other methods for amplification of a specific nucleic acid sequence
comprise a series of primer hybridization, extending and denaturing steps
to provide an intermediate double stranded DNA molecule containing a
promoter sequence through the use of a primer. The double stranded DNA is
used to produce multiple RNA copies of the target sequence. The resulting
RNA copies can be used as target sequences to produce further copies and
multiple cycles can be performed. (See, e.g., Burg, et al., WO 89/1050 and
Gingeras, et al., WO 88/10315.)
Methods for chemically synthesizing relatively large amounts of DNA of a
specified sequence in vitro are well known to those skilled in the art;
production of DNA in this way is now commonplace. However, these
procedures are time-consuming and cannot be easily used to synthesize
oligonucleotides much greater in length than about 100 bases. Also, the
entire base sequence of the DNA to be synthesized must be known. These
methods require an expensive instrument capable of synthesizing only a
single sequence at one time. Operation of this instrument requires
considerable training and expertise. Methods for the chemical synthesis of
RNA have been more difficult to develop.
Nucleic acids may be synthesized by techniques which involve cloning or
insertion of specific nucleic acid sequences into the genetic material of
microorganisms so that the inserted sequences are replicated when the
organism replicates. If the sequences are inserted next to and downstream
from a suitable promoter sequence, RNA copies of the sequence or protein
products encoded by the sequence may be produced. Although cloning allows
the production of virtually unlimited amounts of specific nucleic acid
sequences, due to the number of manipulations involved it may not be
suitable for use in diagnostic, environmental, or forensic testing. Use of
cloning techniques requires considerable training and expertise. The
cloning of a single sequence may consume several man-months of effort or
more.
Relatively large amounts of certain RNAs may be made using a recombinant
single-stranded-RNA molecule having a recognition sequence for the binding
of an RNA-directed polymerase, preferably Q.beta. replicase. (See, e.g.,
U.S. Pat. No. 4,786,600 to Kramer, et al.) A number of steps are required
to insert the specific sequence into a DNA copy of the variant molecule,
clone it into an expression vector, transcribe it into RNA and then
replicate it with Q.beta. replicase.
SUMMARY OF THE INVENTION
The present invention is directed to novel methods of synthesizing multiple
copies of a target nucleic acid sequence which are autocatalytic (i.e.,
able to cycle automatically without the need to modify reaction conditions
such as temperature, pH, or ionic strength and using the product of one
cycle in the next one).
The present method includes (a) treating an RNA target sequence with a
first oligonucleotide which comprises a first primer which has a
complexing sequence sufficiently complementary to the 3'-terminal portion
of the target to complex therewith and which optionally has a sequence 5'
to the priming sequence which includes a promoter for an RNA polymerase
under conditions whereby an oligonucleotide/target sequence complex is
formed and DNA synthesis may be initiated, (b) extending the first primer
in an extension reaction using the target as a template to give a first
DNA primer extension product complementary to the RNA target, (c)
separating the DNA extension product from the RNA target using an enzyme
which selectively degrades the RNA target; (d) treating the DNA primer
extension product with a second oligonucleotide which comprises a primer
or a splice template and which has a complexing sequence sufficiently
complementary to the 3'-terminal portion of the DNA primer extension
product to complex therewith under conditions whereby an
oligonucleotide/target sequence complex is formed and DNA synthesis may be
initiated, provided that if the first oligonucleotide does not have a
promoter, then the second oligonucleotide is a splice template which has a
sequence 5' to the complexing sequence which includes a promoter for an
RNA polymerase; (e) extending the 3'-terminus of either the second
oligonucleotide or the first primer extension product, or both, in a DNA
extension reaction to produce a template for the RNA polymerase; and (f)
using the template to produce multiple RNA copies of the target sequence
using an RNA polymerase which recognizes the promoter sequence. The
oligonucleotide and RNA copies may be used to autocatalytically synthesize
multiple copies of the target sequence.
In one aspect of the present invention, the general method includes (a)
treating an RNA target sequence with a first oligonucleotide which
comprises a first primer which has a complexing sequence sufficiently
complementary to the 3'-terminal portion of the target to complex
therewith and which has a sequence 5' to the complexing sequence which
includes a promoter for an RNA polymerase under conditions whereby an
oligonucleotide/target complex is formed and DNA synthesis may be
initiated, (b) extending the first primer in an extension reaction using
the target as a template to give a first DNA primer extension product
complementary to the RNA target, (c) separating the first DNA primer
extension product from the RNA target using an enzyme which selectively
degrades the RNA target; (d) treating the DNA primer extension product
with a second oligonucleotide which comprises a second primer which has a
complexing sequence sufficiently complementary to the 3'-terminal portion
of the DNA primer extension product to complex therewith under conditions
whereby an oligonucleotide/target complex is formed and DNA synthesis may
be initiated; (e) extending the 3'-terminus of the second primer in a DNA
extension reaction to give a second DNA primer extension product, thereby
producing a template for the RNA polymerase; and (f) using the template to
produce multiple RNA copies of the target sequence using an RNA polymerase
which recognizes the promoter sequence. The oligonucleotide and RNA copies
may be used to autocatalytically synthesize multiple copies of the target
sequence. This aspect further includes: (g) treating an RNA copy from step
(f) with the second primer under conditions whereby an oligonucleotide/
target sequence complex is formed and DNA synthesis may be initiated; (h)
extending the 3' terminus of the second primer in a DNA extension reaction
to give a second DNA primer extension product using the RNA copy as a
template; (i) separating the second DNA primer extension product from the
RNA copy using an enzyme which selectively degrades the RNA copy; (j)
treating the second DNA primer extension product with the first primer
under conditions whereby an oligonucleotide/target sequence complex is
formed and DNA synthesis may be initiated; (k) extending the 3' terminus
of the second primer extension product in a DNA extension reaction to
produce a template for an RNA polymerase; and (1) using the template of
step (k) to produce multiple copies of the target sequence using an RNA
polymerase which recognizes the promoter. Using the RNA copies of step
(1), steps (g) to (k) may be autocatalytically repeated to synthesize
multiple copies of the target sequence. The first primer which in step (k)
acts as a splice template may also be extended in the DNA extension
reaction of step (k).
Another aspect of the general method of the present invention provides a
method which comprises (a) treating an RNA target sequence with a first
primer which has a complexing sequence sufficiently complementary to the
3' terminal portion of the target sequence to complex therewith under
conditions whereby an oligonucleotide/target sequence complex is formed
and DNA synthesis may be initiated; (b) extending the 3' terminus of the
primer in an extension reaction using the target as a template to give a
DNA primer extension product complementary to the RNA target; (c)
separating the DNA extension product from the RNA target using an enzyme
which selectively degrades the RNA target; (d) treating the DNA primer
extension product with a second oligonucleotide which comprises a splice
template which has a complexing sequence sufficiently complementary to the
3'-terminus of the primer extension product to complex therewith and a
sequence 5' to the complexing sequence which includes a promoter for an
RNA polymerase under conditions whereby an oligonucleotide/target sequence
complex is formed and DNA synthesis may be initiated; (e) extending the 3'
terminus of the DNA primer extension product to add thereto a sequence
complementary to the promoter, thereby producing a template for an RNA
polymerase; (f) using the template to produce multiple RNA copies of the
target sequence using an RNA polymerase which recognizes the promoter
sequence; and (g) using the RNA copies of step (f), autocatalytically
repeating steps (a) to (f) to amplify the target sequence. Optionally, the
splice template of step (d) may also function as a primer and in step (e)
be extended to give a second primer extension product using the first
primer extension product as a template.
In addition, in another aspect of the present invention, where the sequence
sought to be amplified is present as DNA, use of an appropriate
Preliminary Procedure generates RNA copies which may then be amplified
according to the General Method of the present invention.
Accordingly, in another aspect, the present invention is directed to
Preliminary Procedures for use in conjunction with the amplification
method of the present invention which not only can increase the number of
copies present to be amplified, but also can provide RNA copies of a DNA
sequence for amplification.
The present invention is directed to methods for increasing the number of
copies of a specific target nucleic acid sequence in a sample. In one
aspect, the present invention involves cooperative action of a DNA
polymerase (such as a reverse transcriptase) and a DNA-dependent RNA
polymerase (transcriptase) with an enzymatic hybrid-separation step to
produce products that may themselves be used to produce additional
product, thus resulting in an autocatalytic reaction without requiring
manipulation of reaction conditions such as thermal cycling. In some
embodiments of the methods of the present invention which include a
Preliminary Procedure, all but the initial step(s) of the preliminary
procedure are carried out at one temperature.
The methods of the present invention may be used as a component of assays
to detect and/or quantitate specific nucleic acid target sequences in
clinical, environmental, forensic, and similar samples or to produce large
numbers of copies of DNA and/or RNA of specific target sequence for a
variety of uses. These methods may also be used to produce multiple DNA
copies of a DNA target sequence for cloning or to generate probes or to
produce NA and DNA copies for sequencing.
In one example of a typical assay, a sample to be amplified is mixed with a
buffer concentrate containing the buffer, salts, magnesium, nucleotide
triphosphates, primers and/or splice templates, dithiothreitol, and
spermidine. The reaction is then optionally incubated near 100.degree. C.
for two minutes to denature any secondary structure. After cooling to room
temperature, if the target is a DNA target without a defined 3' terminus,
reverse transcriptase is added and the reaction mixture is incubated for
12 minutes at 42.degree. C. The reaction is again denatured near
100.degree. C., this time to separate the primer extension product from
the DNA template. After cooling, reverse transcriptase, RNA polymerase,
and RNAse H are added and the reaction is incubated for two to four hours
at 37.degree. C. The reaction can then be assayed by denaturing the
product, adding a probe solution, incubating 20 minutes at 60.degree. C.,
adding a solution to selectively hydrolyze the unhybridized probe,
incubating the reaction six minutes at 60.degree. C., and measuring the
remaining chemiluminescence in a luminometer. (See, e.g., Arnold, et al.,
PCT US88/02746 (filed Sep. 21, 1988, published Mar. 29, 1989) the
disclosure of which is incorporated herein by reference and is referred to
as "HPA"). The products of the methods of the present invention may be
used in many other assay systems known to those skilled in the art.
If the target has a defined 3' terminus or the target is RNA, a typical
assay includes mixing the target with the buffer concentrate mentioned
above and denaturing any secondary structure. After cooling, reverse
transcriptase, RNA polymerase, and RNAse H are added and the mixture is
incubated for two to four hours at 37.degree. C. The reaction can then be
assayed as described above.
The methods of the present invention and the materials used therein may be
incorporated as part of diagnostic kits for use in diagnostic procedures.
Definitions
As used herein, the following terms have the following meanings unless
expressly stated to the contrary.
1. Template
A "template" is a nucleic acid molecule that is being copied by a nucleic
acid polymerase. A template may be either single-stranded, double-stranded
or partially double-stranded, depending on the polymerase. The synthesized
copy is complementary to the template or to at least one strand of a
double-stranded or partially double-stranded template. Both RNA and DNA
are always synthesized in the 5' to 3' direction and the two strands of a
nucleic acid duplex always are aligned so that the 5' ends of the two
strands are at opposite ends of the duplex (and, by necessity, so then are
the 3' ends).
2. Primer, Splice Template
A "primer" is an oligonucleotide that is complementary to a template which
complexes (by hydrogen bonding or hybridization) with the template to give
a primer/template complex for initiation of synthesis by a DNA polymerase,
and which is extended by the addition of covalently bonded bases linked at
its 3' end which are complementary to the template in the process of DNA
synthesis. The result is a primer extension product. Virtually all DNA
polymerases (including reverse transcriptases) that are known require
complexing of an oligonucleotide to a single-stranded template ("priming")
to initiate DNA synthesis, whereas RNA replication and transcription
(copying of RNA from DNA) generally do not require a primer. Under
appropriate circumstances, a primer may act as a splice template as well
(see definition of "splice template" that follows).
A "splice template" is an oligonucleotide that complexes with a
single-stranded nucleic acid and is used as a template to extend the 3'
terminus of a target nucleic acid to add a specific sequence. The splice
template is sufficiently complementary to the 3' terminus of the target
nucleic acid molecule, which is to be extended, to complex therewith. A
DNA- or RNA-dependent DNA polymerase is then used to extend the target
nucleic acid molecule using the sequence 5' to the complementary region of
the splice template as a template. The extension product of the extended
molecule has the specific sequence at its 3'-terminus which is
complementary to the sequence at the 5'-terminus of the splice template.
If the 3' terminus of the splice template is not blocked and is
complementary to the target nucleic acid, it may also act as a primer and
be extended by the DNA polymerase using the target nucleic acid molecule
as a template. The 3' terminus of the splice template can be blocked in a
variety of ways, including having a 3'-terminal dideoxynucleotide or a
3'-terminal sequence non-complementary to the target, or in other ways
well known to those skilled in the art.
Either a primer or a splice template may complex with a single-stranded
nucleic acid and serve a priming function for a DNA polymerase.
3. Target Nucleic Acid, Target Sequence
A "target nucleic acid" has a "target sequence" to be amplified, and may be
either single-stranded or double-stranded and may include other sequences
besides the target sequence which may not be amplified.
The term "target sequence" refers to the particular nucleotide sequence of
the target nucleic acid which is to be amplified. The "target sequence"
includes the complexing sequences to which the oligonucleotides (primers
and/or splice template) complex during the processes of the present
invention. Where the target nucleic acid is originally single-stranded,
the term "target sequence" will also refer to the sequence complementary
to the "target sequence" as present in the target nucleic acid. Where the
"target nucleic acid" is originally double-stranded, the term "target
sequence" refers to both the (+) and (-) strands.
4. Promoter/Promoter Sequence
A "promoter sequence" is a specific nucleic acid sequence that is
recognized by a DNA-dependent RNA polymerase ("transcriptase") as a signal
to bind to the nucleic acid and begin the transcription of RNA at a
specific site. For binding, such transcriptases generally require DNA
which is double-stranded in the portion comprising the promoter sequence
and its complement; the template portion (sequence to be transcribed) need
not be double-stranded. Individual DNA-dependent RNA polymerases recognize
a variety of different promoter sequences which can vary markedly in their
efficiency in promoting transcription. When an RNA polymerase binds to a
promoter sequence to initiate transcription, that promoter sequence is not
part of the sequence transcribed. Thus, the RNA transcripts produced
thereby will not include that sequence.
5. DNA-dependent DNA Polymerase
A "DNA-dependent DNA polymerase" is an enzyme that synthesizes a
complementary DNA copy from a DNA template. Examples are DNA polymerase I
from E. coli and bacteriophage T7 DNA polymerase. All known DNA-dependent
DNA polymerases require a complementary primer to initiate synthesis. It
is known that under suitable conditions a DNA-dependent DNA polymerase may
synthesize a complementary DNA copy from an RNA template.
6. DNA-dependent RNA Polymerase (Transcriptase)
A "DNA-dependent RNA polymerase" or "transcriptase" is an enzyme that
synthesizes multiple RNA copies from a double-stranded or partially-double
stranded DNA molecule having a (usually double-stranded) promoter
sequence. The RNA molecules ("transcripts") are synthesized in the
5'.fwdarw.3' direction beginning at a specific position just downstream of
the promoter. Examples of transcriptases are the DNA-dependent RNA
polymerase from E. coli and bacteriophages T7, T3. and SP6.
7. RNA-dependent DNA polymerase (Reverse Transcriptase)
An "RNA-dependent DNA polymerase" or "reverse transcriptase" is an enzyme
that synthesizes a complementary DNA copy from an RNA template. All known
reverse transcriptases also have the ability to make a complementary DNA
copy from a DNA template; thus, they are both RNA- and DNA-dependent DNA
polymerases. A primer is required to initiate synthesis with both RNA and
DNA templates.
8. RNAse H
An "RNAse H" is an enzyme that degrades the RNA portion of an RNA:DNA
duplex. RNAse H's may be endonucleases or exonucleases. Most reverse
transcriptase enzymes normally contain an RNAse H activity in addition to
their polymerase activity. However, other sources of the RNAse H are
available without an associated polymerase activity. The degradation may
result in separation of RNA from a RNA:DNA complex. Alternatively, the
RNAse H may simply cut the RNA at various locations such that portions of
the RNA melt off or permit enzymes to unwind portions of the RNA.
9. Plus/Minus Strand(s)
Discussions of nucleic acid synthesis are greatly simplified and clarified
by adopting terms to name the two complementary strands of a nucleic acid
duplex. Traditionally, the strand encoding the sequences used to produce
proteins or structural RNAs was designated as the "plus" strand and its
complement the "minus" strand. It is now known that in many cases, both
strands are functional, and the assignment of the designation "plus" to
one and "minus" to the other must then be arbitrary. Nevertheless, the
terms are very useful for designating the sequence orientation of nucleic
acids and will be employed herein for that purpose.
10. Hybridize, Hybridization
The terms "hybridize" and "hybridization" refer to the formation of
complexes between nucleotide sequences which are sufficiently
complementary to form complexes via Watson-Crick base pairing. Where a
primer (or splice template) "hybridizes" with target (template), such
complexes (or hybrids) are sufficiently stable to serve the priming
function required by the DNA polymerase to initiate DNA synthesis.
11. Primer sequences
The sequences of the primers referred to herein are set forth below.
##STR1##
12. Specificity
Characteristic of a nucleic acid sequence which describes its ability to
distinguish between target and non-target sequences dependent on sequence
and assay conditions.
BRIEF DESCRIPTION OF THE DRAWING
FIGS. 1A to 10 depict the General Methods of the present invention.
FIGS. 2A to 2E depict the embodiment of the present invention referred to
as Preliminary Procedure I.
FIG. 3 depicts the embodiment of the present invention referred to as
Preliminary Procedure II.
FIG. 4A to 4D depicts the improved amplification method.
FIG. 5 shows the results of experiments testing the hypothesis that RNAse H
from AMVandMMLV and E. coli have specific RNA cleavage sites.
FIG. 6 shows the results of incorporation of .sup.32 p-labeled primers
during amplification.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, novel methods and compositions
are provided for the amplification of specific nucleic acid target
sequences for use in assays for the detection and/or quantitation of
specific nucleic acid target sequences or for the production of large
numbers of copies of DNA and/or RNA of specific target sequences for a
variety of uses.
I. General Method
In a preferred aspect, the present invention provides an autocatalytic
amplification method which synthesizes large numbers of DNA and RNA copies
of an RNA target sequence. The target nucleic acid contains the target
sequence to be amplified. The target sequence is that region of the target
nucleic acid which is defined on either end by the primers, splice
templates, and/or the natural target nucleic acid termini and includes
both the (+) and (-) strands.
In one aspect, this method comprises treating a target nucleic acid
comprising an RNA target sequence with a first oligonucleotide which
comprises a first primer which has a complexing sequence sufficiently
complementary to the 3'-terminal portion of the target sequence to complex
therewith and which optionally has a sequence 5' to the complexing
sequence which includes a promoter sequence for an RNA polymerase under
conditions whereby an oligonucleotide/target sequence complex is formed
and DNA synthesis may be initiated. The first oligonucleotide primer may
also have other sequences 5' to the priming sequence. The 3'-end of the
first primer is extended by an appropriate DNA polymerase in an extension
reaction using the RNA as a template to give a first DNA primer extension
product which is complementary to the RNA template. The first primer
extension product is separated (at least partially) from the RNA template
using an enzyme which selectively degrades the RNA template. Suitable
enzymes are those which selectively act on the RNA strand of an RNA-DNA
complex and include enzymes which comprise an RNAse H. Although some
reverse transcriptases include an RNAse H activity, it may be preferable
to add exogenous RNAse H, such as an E. coli RNAse H.
The single-stranded first primer extension product is treated with a second
oligonucleotide which comprises a second primer or a splice template which
has a complexing sequence sufficiently complementary to the 3'-terminal
portion of target sequence contained in the first primer extension product
to complex therewith, under conditions whereby an oligonucleotide/ target
sequence complex is formed and DNA synthesis may be initiated. If the
first primer does not have a promoter then the second oligonucleotide is a
splice template which has a sequence 5' to the complexing region which
includes a promoter for an RNA polymerase. Optionally, the splice template
may be blocked at its 3' terminus. The 3' terminus of the second
oligonucleotide and/or the primer extension product is extended in a DNA
extension reaction to produce a template for a RNA polymerase. The RNA
copies or transcripts produced may autocatalytically multiply without
further manipulation.
Where an oligonucleotide functions as a splice template, its primer
function is not required. Thus, the 3' terminus of the splice template may
be either blocked or unblocked. The components of the resulting reaction
mixture (i.e., an RNA target which allows production of a first primer
extension product with a defined 3' terminus, a first primer, and a splice
template either blocked or unblocked) function to autocatalytically
synthesize large quantities of RNA and DNA.
In one aspect of the present invention, the first and second oligomers both
are primers. The first primer has a sequence 5' to the complexing sequence
which includes a promoter for a RNA polymerase and may include other
sequences. The second primer may also include a sequence 5' to the
complexing sequence which may include a promoter for an RNA polymerase and
optionally other sequences. Where both primers have a promoter sequence,
it is preferred that both sequences are recognized by the same RNA
polymerase unless it is intended to introduce the second promoter for
other purposes, such as cloning. The 3'-end of the second primer is
extended by an appropriate DNA polymerase in an extension reaction to
produce a second DNA primer extension product complementary to the first
primer extension product. Note that as the first primer defined one end of
the target sequence, the second primer now defines the other end. The
double-stranded product of the second extension reaction is a suitable
template for the production of RNA by an RNA polymerase. If the second
primer also has a promoter sequence, transcripts complementary to both
strands of the double-stranded template will be produced during the
autocatalytic reaction. The RNA transcripts may now have different termini
than the target nucleic acid, but the sequence between the first primer
and the second primer remains intact. The RNA transcripts so produced may
automatically recycle in the above system without further manipulation.
Thus, this reaction is autocatalytic.
If the complexing sequence of the second primer complexes with the 3'
terminus of the first primer extension product, the second primer may act
as a splice template and the first primer extension product may be
extended to add any sequence of the second primer 5' to the priming
sequence to the first primer extension product. (See, e.g., FIGS. 1E and
1G) If the second primer acts as a splice template and includes a promoter
sequence 5' to the complexing sequence, extension of the first primer
extension product to add the promoter sequence produces an additional
template for an RNA polymerase which may be transcribed to produce RNA
copies of either strand. (See FIGS. 1E and 1G) Inclusion of promoters in
both primers may enhance the number of copies of the target sequence
synthesized.
Another aspect of the general method of the present invention includes
using a first oligonucleotide which comprises a primer and a second
oligonucleotide which comprises a splice template and which may or may not
be capable of acting as a primer per se (in that it is not itself extended
in a primer extension reaction). This aspect of the general method
comprises treating a target nucleic acid comprising an RNA target sequence
with a first oligonucleotide primer which has a complexing sequence
sufficiently complementary to the 3' terminal portion of the target
sequence to complex therewith under conditions whereby an
oligonucleotide/target sequence complex is formed and DNA synthesis may be
initiated. The first primer may have other sequences 5' to the complexing
sequence, including a promoter. The 3' end of the first primer is extended
by an appropriate DNA polymerase in an extension reaction using the RNA as
a template to give a first primer extension product which is complementary
to the RNA template. The first primer extension product is separated from
the RNA template using an enzyme which selectively degrades the RNA
template. Suitable enzymes are those which selectively act on the RNA
strand of an RNA-DNA complex and include enzymes which comprise an RNAse H
activity. Although some reverse transcriptases include an RNase H
activity, it may be preferable to add exogenous RNAse H, such as an E.
coli RNAse H. The single stranded first primer extension product is
treated with a splice template which has a complexing sequence
sufficiently complementary to the 3'-terminus of the primer extension
product to complex therewith and a sequence 5' to the complexing sequence
which includes a promoter for an RNA polymerase under conditions whereby
an oligonucleotide/target sequence complex is formed and DNA synthesis may
be initiated. The 3' terminus of the splice template may be either blocked
(such as by addition of a dideoxynucleotide) or uncomplementary to the
target nucleic acid (so that it does not function as a primer) or
alternatively unblocked. The 3' terminus of the first primer extension
product is extended using an appropriate DNA polymerase in a DNA extension
reaction to add to the 3' terminus of the first primer extension product a
sequence complementary to the sequence of the splice template 5' to the
complexing sequence which includes the promoter. If the 3' terminus is
unblocked, the splice template may be extended to give a second primer
extension product complementary to the first primer extension product. The
product of the extension reaction with the splice template (whether
blocked or unblocked) can function as a template for RNA synthesis using
an RNA polymerase which recognizes the promoter. As noted above, RNA
transcripts so produced may automatically recycle in the above system
without further manipulation. Thus, the reaction is autocatalytic.
In some embodiments, the target sequence to be amplified is defined at both
ends by the location of specific sequences complementary to the primers
(or splice templates) employed. In other embodiments, the target sequence
is defined at one location of a specific sequence, complementary to a
primer molecule employed and, at the opposite end, by the location of a
specific sequence that is cut by a specific restriction endonuclease, or
by other suitable means, which may include a natural 3' terminus. In other
embodiments, the target sequence is defined at both ends by the location
of specific sequences that are cut by one or more specific restriction
endonuclease(s).
In a preferred embodiment of the present invention, the RNA target sequence
is determined and then analyzed to determine where RNAse H degradation
will cause cuts or removal of sections of RNA from the duplex. Analyses
can be conducted to determine the effect of the RNAse degradation of the
target sequence by RNAse H present in AMV reverse transcriptase and MMLV
reverse transcriptase, by E. coli RNAse H or other sources and by
combinations thereof.
In selecting a primer set, it is preferable that one of the primers be
selected so that it will hybridize to a section of RNA which is
substantially nondegraded by the RNAse H present in the reaction mixture.
If there is substantial degradation, the cuts in the RNA strand in the
region of the primer may inhibit initiation of DNA synthesis and prevent
extension of the primer. Thus, it is preferred to select a primer which
will hybridize with a sequence of the RNA target, located so that when the
RNA is subjected to RNAse H, there is no substantial degradation which
would prevent formation of the primer extension product.
The site for hybridization of the promoter-primer is chosen so that
sufficient degradation of the RNA strand occurs to permit removal of the
portion of the RNA strand hybridized to the portion of the DNA strand to
which the promoter-primer will hybridize. Typically, only portions of RNA
are removed from the RA:DNA duplex through RNAse H degradation and a
substantial part of the RNA strand remains in the duplex.
Formation of the promoter-containing double stranded product for RNA
synthesis is illustrated in FIG. 4. As illustrated in FIG. 4, the target
RNA strand hybridizes to a primer which is selected to hybridize with a
region of the RNA strand which is not substantially degraded by RNAse H
present in the reaction mixture. The primer is then extended to form a DNA
strand complementary to the RNA strand. Thereafter, the RNA strand is cut
or degraded at various locations by the RNAse H present in the reaction
mixture. It is to be understood that this cutting or degradation can occur
at this point or at other times during the course of the reaction. Then
the RNA fragments dissociate from the DNA strand in regions where
significant cuts or degradation occur. The promoter-primer then hybridizes
to the DNA strand at its 3' end, where the RA strand has been
substantially degraded and separated from the DNA strand. Next, the DNA
strand is extended to form a double strand DNA promoter sequence, thus
forming a template for RNA synthesis. It can be seen that this template
contains a double-stranded DNA promoter sequence. When this template is
treated with RNA polymerase, multiple strands of RNA are formed.
Although the exact nature of the RNA degradation resulting from the RNAse H
is not known, it has been shown that the result of RNAse H degradation on
the RNA strand of an RNA:DNA hybrid resulted in dissociation of small
pieces of RNA from the hybrid. It has also been shown that
promoter-primers can be selected which will bind to the DNA after RNAse H
degradation at the area where the small fragments are removed.
FIGS. 1 and 2, as drawn, do not show the RNA which may remain after RNAse H
degradation. It is to be understood that although these figures generally
show complete removal of RNA from the DNA:RNA duplex, under the preferred
conditions only partial removal occurs as illustrated in FIG. 3. By
reference to FIG. 1A, it can be seen that the proposed mechanism may not
occur if a substantial portion of the RNA strand of FIG. 1 remains
undegraded thus preventing hybridization of the second primer or extension
of the hybridized second primer to produce a DNA strand complementary to
the promoter sequence. However, based upon the principles of synthesis
discovered and disclosed in this application, routine modifications can be
made by those skilled in the art according to the teachings of this
invention to provide an effective and efficient procedure for
ampli | | |
