U.S. patent number RE39,885 [Application Number 09/082,247] was granted by the patent office on 2007-10-16 for detection of nucleic acid amplification.
This patent grant is currently assigned to Becton, Dickinson and Company. Invention is credited to James G. Nadeau, George T. Walker.
United States Patent |
RE39,885 |
Nadeau , et al. |
October 16, 2007 |
Detection of nucleic acid amplification
Abstract
Methods for detecting, immobilizing or localizing primer
extension products of a Strand Displacement Amplification reaction
which are coupled to, and an indication of, amplification of the
target sequence. The primer extension products are secondary,
target-specific DNA products generated concurrently with SDA of the
target sequence and can therefore be used to detect and/or measure
target sequence amplification in real-time. In general, the
secondary amplification products are not amplifiable and remain
inert in the SDA reaction after they are formed without interfering
with amplification of the target sequence. The secondary
amplification products may be designed or modified to contain
special features to facilitate their detection, immobilization or
localization.
Inventors: |
Nadeau; James G. (Ellicott
City, MD), Walker; George T. (Chapel Hill, NC) |
Assignee: |
Becton, Dickinson and Company
(Franklin Lakes, NJ)
|
Family
ID: |
22860539 |
Appl.
No.: |
09/082,247 |
Filed: |
May 20, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
08229281 |
Apr 18, 1994 |
05547861 |
Aug 20, 1996 |
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Current U.S.
Class: |
435/6.12;
435/91.2 |
Current CPC
Class: |
C12Q
1/6844 (20130101); C12Q 1/6844 (20130101); C12Q
2565/525 (20130101); C12Q 2561/113 (20130101); C12Q
2531/119 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12P 19/34 (20060101) |
Field of
Search: |
;435/6,91.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 420 260 |
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Apr 1991 |
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EP |
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WO 90/06374 |
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Aug 1990 |
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WO |
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WO 92/01812 |
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Feb 1992 |
|
WO |
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WO 92/02638 |
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Feb 1992 |
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WO |
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WO 92/11390 |
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Jul 1992 |
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WO |
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WO 92/18650 |
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Oct 1992 |
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WO |
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WO 95/32306 |
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Nov 1995 |
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WO |
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Other References
Walker et al, "A DNA Probe Assay Using Strand Displacement
Amplification (SDA) and Filtration to Separate Reacted and
Unreacted Detector Probes", Molecular and Cellular Probes,
9:399-402, 1995. cited by other .
"Becton Dickenson Announces Licensing Agreement with Otsuka
Pharmaceutical", http://www.newtech/sda/otsuka.html, 1998. cited by
other .
Letter from David W. Highet, Esq. of Becton Dickinson and Company
dated Aug. 17, 1998. cited by other .
G.T. Walker, et al. "Strand Displacement Amplification--An
Isothermal, in vitro DNA Amplification Technique" Nucl. Acids Res.
20, 1691-1696 (1992). cited by other .
G. T. Walker, et al. "Isothermal in vitro amplification of DNA by a
restriction enzyme/DNA polymerase system" Proc. Natl. Acad. Sci USA
89, 392-396 (1992). cited by other .
C.P.H. Vary "Triple-Helical Capture Assay for Quantification of
Polymerase Chain Reaction Products" Clin. Chem. 38, 687-694 (1992).
cited by other .
J. Wahlberg, et al. "General Colorimetric Method for DNA
Diagnostics Allowing Direct Solid-Phase Genomic Reactions" Proc.
Natl. Acad. Sci USA 87, 6569-6573 (1990). cited by other .
D.J. Kemp, et al. "Colorimetric Detection of Specific DNA Segments
Amplified by Polymerase Chain Reactions" Proc. Natl. Acad. Sci. USA
86, 2423-2427 (1989). cited by other .
F.F. Chehab, et al. "Detection of Specific DNA Sequences by
Fluorescence Amplification: A Color Complementation Assay" Proc.
Natl. Acad. Sci. USA 86, 9178-9182 (1989). cited by other .
A.C. Syvanen, et al. "Quantification of Polymerase Chain Reaction
Products by Affinity-Based Hybrid Collection" Nucl. Acids Res. 16,
11327-11338 (1988). cited by other .
A. Chan, et al. "Quantification of Polymerase Chain Reaction
Products in Agarose Gels with a Fluorescent Europium Chelate as
Label and Time-Resolved Fluorescence Spectroscopy" Anal. Chem. 65,
158-163 (1993). cited by other .
C.R. Newton, et al. "The Production of PCR Products with 5' Single
Stranded Tails Using Primers that Incorporate Novel Phosphoramidite
Intermediates" Nucl. Acids. Res. 21, 1155-1162 (1993). cited by
other .
P.M. Holland, et al. "Detection of Specific Polymerase Chain
Reaction Product by Utilizing the 5'-3' Exonuclease Activity of
Thermus Aquaticus DNA Polymerase" Clin. Chem. 38, 462-463 (1992).
cited by other .
P.M. Holland, et al. "Detection of Specific Polymerase Chain
Reaction Product by Utilizing the 5'-3' Exonnuclease Activity of
Thermus Aquaticus DNA Polymerase" Proc. Natl. Acad. Sci. USA 88,
7276-7280 (1991). cited by other .
WO9201812--Uhlen et al. Competitive PCR for quantitations of DNA,
pp. 1-19, pub. Feb. 6, 1992. cited by examiner.
|
Primary Examiner: Horlick; Kenneth R.
Assistant Examiner: Tung; Joyce
Attorney, Agent or Firm: Kiang; Allan M.
Claims
What is claimed is:
1. A method for concurrently generating a secondary amplification
product and an amplification product in a Strand Displacement
Amplification (SDA) reaction, wherein the SDA reaction comprises
(i) a DNA polymerase having strand displacing activity and lacking
5'-3' exonuclease activity and (ii) a restriction endonuclease
which nicks a hemimodified double stranded restriction endonuclease
recognition site, the method comprising: a) hybridizing a signal
primer to a target sequence and hybridizing a first SDA
amplification primer to the target sequence upstream of the signal
primer; b) extending the hybridized signal primer on the target
sequence to produce a signal primer extension product and extending
the hybridized first SDA amplification primer on the target
sequence such that extension of the first SDA amplification primer
displaces the signal primer extension product from the target
sequence; c) hybridizing a second SDA amplification primer to the
signal primer extension product and extending the hybridized second
SDA amplification primer on the signal primer extension product to
produce a second SDA amplification primer extension product
comprising a newly synthesized strand and double stranded
hemimodified recognition site for the restriction endonuclease; d)
nicking the hemimodified recognition site and displacing the newly
synthesized strand from the signal primer extension product using
the DNA polymerase; e) hybridizing the signal primer to the
displaced newly synthesized strand and extending the signal primer
such that a double stranded secondary amplification product is
generated.
2. The method of claim 1 further comprising detecting the secondary
amplification product by means of a chemical modification or
special nucleotide sequence incorporated into the signal
primer.
3. The method of claim 2 wherein the secondary amplification
product is detected by means of an affinity ligand or reporter
group incorporated into the signal primer.
4. The method of claim 2 wherein the secondary amplification
product is detected by means of a nucleotide sequence incorporated
into the signal primer, the nucleotide sequence comprising a
recognition site for a double-stranded DNA binding protein.
5. The method of claim 2 wherein the secondary amplification
product is detected by means of a nucleotide sequence incorporated
into the signal primer, the nucleotide sequence comprising a
restriction endonuclease recognition site.
6. The method of claim 5 wherein the secondary amplification
product is detected by cleaving the restriction endonuclease
recognition site with a restriction endonuclease to generate a
cleavage product, separating the cleavage product on the basis of
size and detecting the cleavage product.
7. The method of claim 6 wherein the cleavage product is separated
by filtration.
8. A method for concurrently generating a secondary amplification
product and an amplification product in a Strand Displacement
Amplification (SDA) reaction, wherein the SDA reaction comprises
(i) a DNA polymerase having strand displacing activity and lacking
5'-3' exonuclease activity and (ii) a restriction enzyme which
nicks a hemimodified double stranded restriction endonuclease
recognition site, the method comprising: a) hybridizing a first
signal primer to a first strand of a double-stranded target
sequence and hybridizing a first SDA amplification primer to the
first strand of the target sequence upstream of the first signal
primer; b) extending the hybridized first signal primer on the
first strand to produce a first extension product and extending the
hybridized first SDA amplification primer on the first strand such
that extension of the first SDA amplification primer displaces the
first extension product from the target sequence; c) hybridizing a
second signal primer to the first extension product and hybridizing
a second SDA amplification primer to the first extension product
upstream of the second signal primer; d) extending the hybridized
second signal primer on the first extension product to produce a
second SDA extension product and extending the hybridized second
amplification primer on the first extension product such that
extension of the second SDA amplification primer displaces the
second extension product from the first extension product; e)
hybridizing the first signal primer to the displaced second
extension product and extending the hybridized first signal primer
on the second extension product such that a double stranded
secondary amplification product is generated.
9. The method of claim 8 further comprising detecting the secondary
amplification product by means of a reporter group incorporated
into the first signal primer and a modification to facilitate
capture of the secondary amplification product incorporated into
the second signal primer.
10. The method of claim 8 further comprising the steps of: a)
hybridizing the second SDA signal primer to a second strand of the
double stranded target sequence and hybridizing the second
amplification primer to the second strand of the target sequence
upstream of the second signal primer; b) extending the hybridized
second signal primer on the second strand to produce a third
extension product and extending the hybridized second SDA
amplification primer on the second SDA strand such that extension
of the second amplification primer displaces the third extension
product from the second strand of the target sequence; c)
hybridizing the first signal primer to the displaced third
extension product and hybridizing the first SDA amplification
primer to the displaced third extension product upstream of the
first signal primer; d) extending the hybridized first signal
primer on the third extension product to produce a fourth extension
product and extending the hybridized first SDA amplification primer
on the third extension product such that extension of the first SDA
amplification primer displaces the fourth extension product from
the third extension product; e) hybridizing the second signal
primer to the displaced fourth extension product and extending the
second signal primer on the fourth extension product such that a
double stranded secondary amplification product is generated.
11. The method of claim 10 further comprising detecting the
secondary amplification product by means of a chemical modification
or special nucleotide sequence incorporated into the signal
primer.
12. The method of claim 11 wherein the secondary amplification
product is detected by means of an affinity ligand or reporter
group incorporated into the signal primer.
13. The method of claim 11 wherein the secondary amplification
product is detected by means of a nucleotide sequence incorporated
into the signal primer, the nucleotide sequence comprising a
recognition site for a double-stranded DNA binding protein.
14. The method of claim 11 wherein the secondary amplification
product is detected by means of a nucleotide sequence incorporated
into the signal primer, the nucleotide sequence comprising a
restriction endonuclease recognition site.
15. The method of claim 14 wherein the secondary amplification
product is detected by cleaving the restriction endonuclease
recognition site with a restriction endonuclease to generate a
cleavage product, separating the cleavage product on the basis of
size and detecting the cleavage product.
16. The method of claim 15 wherein the cleavage product is
separated by filtration.
17. The method of claim 2 wherein the secondary amplification
products are detected in concurrently with amplification of the
target sequence in real-time.
18. The method of claim 2 wherein the secondary amplification
products are detected post-amplification.
19. The method of claim 9 wherein the secondary amplification
products are detected in concurrently with amplification of the
target sequence in real-time.
20. The method of claim 9 wherein the secondary amplification
products are detected post-amplification.
.Iadd.21. A method for concurrently generating a secondary
amplification product and an amplification product in a primer
based nucleic acid amplification reaction, the method comprising:
a) hybridizing a signal primer to a target sequence and hybridizing
a first amplification primer to the target sequence upstream of the
signal primer, wherein a characteristic of said signal primer is
that it may function as an amplifiable primer in a linear fashion;
b) extending the hybridized signal primer on the target sequence to
produce a signal primer extension product and extending the
hybridized first amplification primer on the target sequence such
that extension of the first amplification primer displaces the
signal primer extension product from the target sequence; c)
hybridizing a second amplification primer to the signal primer
extension product and extending the hybridized second amplification
primer on the signal primer extension product to produce a second
amplification primer extension product comprising a newly
synthesized strand; d) displacing the newly synthesized strand from
the signal primer extension product; and e) hybridizing the signal
primer to the displaced newly synthesized strand and extending the
signal primer such that a double stranded secondary amplification
product is generated..Iaddend.
.Iadd.22. The method of claim 21 further comprising detecting the
secondary amplification product by means of a chemical modification
or special nucleotide sequence incorporated into the signal
primer..Iaddend.
.Iadd.23. The method of claim 22 wherein the secondary
amplification product is detected by means of an affinity ligand or
reporter group incorporated into the signal primer..Iaddend.
.Iadd.24. The method of claim 22 wherein the secondary
amplification product is detected by means of a nucleotide sequence
incorporated into the signal primer, the nucleotide sequence
comprising a recognition site for a double-stranded DNA binding
protein..Iaddend.
.Iadd.25. The method of claim 22 wherein the secondary
amplification product is detected by means of a nucleotide sequence
incorporated into the signal primer, the nucleotide sequence
comprising a restriction endonuclease recognition
site..Iaddend.
.Iadd.26. The method of claim 25 wherein the secondary
amplification product is detected by cleaving the restriction
endonuclease recognition site with a restriction endonuclease to
generate a cleavage product..Iaddend.
.Iadd.27. The method of claim 26 wherein the secondary
amplification product is detected by separating the cleavage
product on the basis of size and detecting the cleavage
product..Iaddend.
.Iadd.28. The method of claim 27 wherein the cleavage product is
separated by filtration..Iaddend.
.Iadd.29. A method for concurrently generating a secondary
amplification product and an amplification product in a primer
based nucleic acid amplification reaction, the method comprising:
a) hybridizing a first signal primer to a first strand of a
double-stranded target sequence and hybridizing a first
amplification primer to the first strand of the target sequence
upstream of the first signal primer, wherein a characteristic of
said signal primer is that it may function as an amplifiable primer
in a linear fashion; b) extending the hybridized first signal
primer on the first strand to produce a first extension product and
extending the hybridized first amplification primer on the first
strand such that extension of the first amplification primer
displaces the first extension product from the target sequence; c)
hybridizing a second signal primer to the first extension product
and hybridizing a second amplification primer to the first
extension product upstream of the second signal primer; d)
extending the hybridized second signal primer on the first
extension product to produce a second extension product and
extending the hybridized second amplification primer on the first
extension product such that extension of the second amplification
primer displaces the second extension product from the first
extension product; and e) hybridizing the first signal primer to
the displaced second extension product and extending the hybridized
first signal primer on the second extension product such that a
double stranded secondary amplification product is
generated..Iaddend.
.Iadd.30. The method of claim 29 further comprising detecting the
secondary amplification product by means of a reporter group
incorporated into the first signal primer and a modification to
facilitate capture of the secondary amplification product
incorporated into the second signal primer..Iaddend.
.Iadd.31. The method of claim 29 further comprising: a) hybridizing
the second signal primer to a second strand of the double stranded
target sequence and hybridizing the second amplification primer to
the second strand of the target sequence upstream of the second
signal primer; b) extending the hybridized second signal primer on
the second strand to produce a third extension product and
extending the hybridized second amplification primer on the second
strand such that extension of the second amplification primer
displaces the third extension product from the second strand of the
target sequence; c) hybridizing the first signal primer to the
displaced third extension product and hybridizing the first
amplification primer to the displaced third extension product
upstream of the first signal primer; d) extending the hybridized
first signal primer on the third extension product to produce a
fourth extension product and extending the hybridized first
amplification primer on the third extension product such that
extension of the first amplification primer displaces the fourth
extension product from the third extension product; and e)
hybridizing the second signal primer to the displaced fourth
extension product and extending the second signal primer on the
fourth extension product such that a double stranded secondary
amplification product is generated..Iaddend.
.Iadd.32. The method of claim 31 further comprising detecting the
secondary amplification product by means of a chemical modification
or special nucleotide sequence incorporated into the signal
primer..Iaddend.
.Iadd.33. The method of claim 32 wherein the secondary
amplification product is detected by means of an affinity ligand or
reporter group incorporated into the signal primer..Iaddend.
.Iadd.34. The method of claim 32 wherein the secondary
amplification product is detected by means of a nucleotide sequence
incorporated into the signal primer, the nucleotide sequence
comprising a recognition site for a double-stranded DNA binding
protein..Iaddend.
.Iadd.35. The method of claim 32 wherein the secondary
amplification product is detected by means of a nucleotide sequence
incorporated into the signal primer, the nucleotide sequence
comprising a restriction endonuclease recognition
site..Iaddend.
.Iadd.36. The method of claim 35 wherein the secondary
amplification product is detected by cleaving the restriction
endonuclease recognition site with a restriction endonuclease to
generate a cleavage product..Iaddend.
.Iadd.37. The method of claim 36 wherein the secondary
amplification product is detected by separating the cleavage
product on the basis of size and detecting the cleavage
product..Iaddend.
.Iadd.38. The method of claim 37 wherein the cleavage product is
separated by filtration..Iaddend.
.Iadd.39. The method of claim 22 wherein the secondary
amplification products are detected in real-time..Iaddend.
.Iadd.40. The method of claim 22 wherein the secondary
amplification products are detected
post-amplification..Iaddend.
.Iadd.41. The method of claim 29 wherein the secondary
amplification products are detected in real-time..Iaddend.
.Iadd.42. The method of claim 29 wherein the secondary
amplification products are detected post-amplification..Iaddend.
Description
FIELD OF THE INVENTION
The present invention relates to methods for detecting and
measuring amplification of a nucleic acid target sequence.
BACKGROUND OF THE INVENTION
In vitro nucleic acid amplification techniques have provided
powerful tools for detection and analysis of small amounts of
nucleic acids. The extreme sensitivity of such methods has lead to
attempts to develop them for diagnosis of infectious and genetic
diseases, isolation of genes for analysis, and detection of
specific nucleic adds as in forensic medicine. Nucleic acid
amplification techniques can be grouped according to the
temperature requirements of the procedure. The polymerase chain
reaction (PCR; R. K. Saiki, et al. 1985, Science 230, 1350-1354),
ligase chain reaction (LCR; D. Y. Wu, et al. 1989, Genomics 4,
560-569; K. Barfinger, et al. 1990. Gene 89, 117-122; F. Barany.
1991. Proc. Natl. Acad. Sci. USA 88, 189-193) and
transcription-based amplification (D. Y. Kwoh, et al. 1989. Proc.
Natl. Acad. Sci. USA 86, 1173-1177) require temperature cycling. In
contrast, methods such as strand displacement amplification (SDA;
G. T. Walker, et al. 1992. Proc. Natl. Acad Sci. USA 89, 392-396
and G. T. Walker, et al. 1992. Nuc. Acids. Res. 20, 1691-1696, both
disclosures being incorporated herein by reference), self-sustained
sequence replication (3SR; J. C. Guatelli, et al. 1990. Proc. Natl.
Acad. Sci. USA 87, 1874-1878) and the Q.beta. replicase system (P.
M. Lizardi, et al. 1988. BioTechnology 6, 1197-1202) are isothermal
reactions. In addition, WO 90/10064 and WO 91/03573 describe use of
the bacteriophage phi29 replication origin for isothermal
replication of nucleic acids.
A variety of methods have also been developed to detect and/or
measure nucleic acid amplification. For the most part, these
methods are primer-based, meaning that they depend on hybridization
of a primer to the target sequence, in some cases followed by
extension of the primer. Primer-based detection of amplified
nucleic acids in PCR often relies on incorporation of an
amplification primer into the amplified product (amplicon) during
the amplification reaction. Features engineered into the PCR
amplification primer therefore appear in the amplification product
and can be used either to detect the amplified target sequence or
to immobilize the amplicon for detection by other means. For
example, Syvanen, et al. (1988. Nucleic Acids Res. 16, 11327-11338)
report the use of biotinylated PCR amplification primers to produce
biotin-containing amplification products. These amplicons can then
be hybridized to a second probe containing a fluorescent dye or
other reporter group. The hybridized complex is then selectively
isolated from other components of the reaction mixture by
affinity-based immobilization of the biotin-containing complex and
is detected by means of the reporter group. Laongiaru, et al.
(1991. European Patent Application No. 0 420 260) describe a
similar use of biotin-containing PCR amplification primers
conjugated to fluorescent dyes for detection of PCR amplification
products. The amplicons containing the primers are separated from
unextended primers on the basis of size, and multiplex
amplification was detected using different fluorescent dyes on two
amplification primer sets. Kemp, et al. (1989. Proc. Natl. Acad.
Sci. USA 86, 2423-2427; 1990. PCT Patent Application No. WO
90/06374) describe a method for capturing amplified DNA by
incorporation of one modified amplification primer and use of a
second modified amplification primer as a means for detection. The
Kemp "capture primer[ contains a 5' tail which is the single
stranded form of the recognition sequence for the double-stranded
DNA binding protein GCN4. The Kemp "detector primer" includes a
biotin moiety on its 5' end. The amplified product is immobilized
by binding to the double-stranded GCN4 recognition sequence
generated by amplification using the capture primer. The biotin
moiety introduced by the detector primer is bound to an
avidin-peroxidase complex to provide colorimetric detection of the
immobilized PCR amplification product. Wahlberg, et al. (1990.
Proc. Natl. Acad. Sci. USA 87, 6569-6573) report a similar method
in which one PCR amplification primer is biotinylated and the other
contains a 5' tail encoding the E. coli lac operator sequence.
Double stranded amplification products are immobilized by binding
to streptavidin and detected colorimetrically by binding of a lac
repressor-.beta.-galactosidase fusion protein to the
double-stranded lac operator generated by amplification. The
Wahlberg, et al. method differs from the Kemp, et al. method in
that the biotin-streptavidin interaction rather than the
double-stranded binding protein provides immobilization of the
amplification products and the double-stranded binding protein
provides colorimetric detection. This suggests that the two methods
could be combined by using two amplification primers, each with a
5' tail encoding the recognition sequence of a different
double-stranded binding protein. Amplified products could then be
immobilized by binding to one double stranded binding protein and
detected by binding to the other. C. A. Vary (1992. Clinical
Chemistry 38, 687-694; 1992. PCT Patent Application No. WO
92/11390) describes the use of amplification primers containing 5'
tails which form hybridization sites for a third oligonucleotide
when incorporated into otherwise double-stranded amplicons.
Hybridization of one tail was used to capture the amplified product
and the other was used to detect it by hybridization to a probe
conjugated to a fluorescent dye.
All of these primer-based methods of detecting PCR amplification
products require two amplification reactions to achieve high
sensitivity, i.e., detection of fewer than 100 copies of the target
sequence. That is, a first amplification of the target sequence is
followed by a second amplification using nested primers
incorporating the desired modifications for capture and/or
detection. Two consecutive amplifications in this manner are needed
to avoid unacceptably high levels of background signal produced by
amplification of non-target DNA spuriously primed with the
modified, signal-generating primers. This feature of the prior art
methods makes them time-consuming and cumbersome, and the
advantages of primer-based detection methods are therefore often
offset by the requirement for a second consecutive amplification
reaction.
Non-specific amplification of DNA would be expected to present
particular problems for primer-based detection of amplification
products in SDA reactions because these amplifications are carried
out at a relatively low temperature (about 37.degree.-40.degree.
C.) which would allow increased mispriming as compared to PCR,
resulting in even higher levels of background signal. Unexpectedly,
the instant methods for primer-based detection of SDA resulting in
low levels of background signal in spite of the use of only a
single amplification reaction which generates products for
detection concurrently with amplification of the target sequence.
Simultaneous or concurrent generation of a secondary amplification
product and the amplified target sequence is referred to herein as
real-time primer extension, real-time detection of amplification,
etc.
SUMMARY OF THE INVENTION
The instant invention provides methods for detecting, immobilizing
(capturing) or localizing primer extension products of an SDA
reaction which are coupled to, and an indication of, amplification
of the target sequence. The primer extension products are
secondary, target-specific DNA products generated during SDA of the
target sequence and can therefore be used to detect and/or measure
target sequence amplification. The secondary products, however, are
not amplifiable and remain inert in the SDA reaction after they are
formed without interfering with the exponential amplification of
the target sequence. The secondary product can be designed or
modified to contain special features to facilitate its detection,
immobilization (capture) or localization. The inventive methods are
useful for real-time monitoring of SDA reactions, especially in
situations where detection of target sequence amplicons would
interfere with further amplification or manipulation. The instant
methods will also be useful for detection of amplification products
in fixed cells after in situ SDA, especially when the secondary
products contain 5' tail sequences to facilitate detection or
localization of amplification products.
DESCRIPTION OF THE DRAWINGS
FIG. 1A and FIG. 1B illustrate the steps of the methods of the
invention. FIG. 1A illustrates the production of the secondary
amplification product from a single stranded target sequence using
two signal primers. FIG. 1B shows the analogous process originating
from the complementary strand when the original target sequence is
double stranded.
FIG. 2 illustrates the production of the secondary product using a
single signal primer.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is a method for detecting, monitoring or
localizing amplification products of SDA reactions by real-time
primer extension. Amplification of a target sequence by SDA is
detected, monitored or localized by simultaneously generating a
secondary amplification product, the production of which is tightly
coupled to amplification of the target sequence. This secondary
amplification product is produced during the SDA reaction without
requiring any additional additional amplification steps or
manipulations. Once generated, the secondary amplification product
is inert in the reaction mixture and does not interfere with or
inhibit normal SDA of the desired target sequence. The methods are
therefore useful for real-time monitoring of SDA and detecting
amplification of the target sequence, especially in situations
where detection of the amplified target sequence itself would
inhibit or prevent further reaction or manipulation of the
amplicons.
The present invention provides a primer-based amplification
detection method in which the need for a second amplification
reaction is eliminated. The method employs signal primers which are
similar to capture and detector probes and do not function as
amplification primers in the SDA reaction. Consequently, any
extension products formed through errant extension of these signal
primers on non-target templates cannot undergo subsequent
amplification. Because misprimings itself is comparatively rare, it
is detectable only after subsequent amplification of the misprimed
sequence. In the absence of such subsequent amplification, as in
the methods of the present invention, the signal primers may be
added to the amplification reaction prior to initiation of
amplification with no apparent increase in background signal
levels. This greatly simplifies the detection procedure and makes
possible homogeneous real-time analysis of SDA reactions.
As used herein, the following terms and phrases are defined as
follows:
An amplification primer is a primer for amplification of a target
sequence by primer extension. For SDA, the 3' end of the
amplification primer (the target binding sequence) hybridizes at
the 3' end of the target sequence. The amplification primer
comprises a recognition site for a restriction endonuclease near
its 5' end. The recognition site is for a restriction endonuclease
which will cleave one strand of a DNA duplex when the recognition
site is hemimodified ("nicking"), as described by Walker, et al.
(1992. PNAS, supra). A hemimodified recognition site is a double
stranded recognition site for a restriction endonuclease in which
one strand contains at least one derivatized nucleotide which
causes the restriction endonuclease to nick the primer strand
rather than cleave both strands of the recognition site. Usually,
the primer strand of the hemimodified recognition site does not
contain derivatized nucleotides and is nicked by the restriction
endonuclease. Alternatively, the primer may contain derivatized
nucleotides which cause the unmodified target strand to be
protected from cleavage while the modified primer strand is nicked.
The preferred hemimodified recognition sites are
hemiphosphorothioated recognition sites for the restriction
endonucleases HincII, HindII, AvaI, NciI and Fnu4HI. The
amplification primer also comprises a 3'-OH group which is
extendable by DNA polymerase when the target binding sequence of
the amplification primer is hybridized to the target sequence. For
the majority of the SDA reaction, the amplification primer is
responsible for exponential amplification of the target
sequence.
Extension products are nucleic acids which comprise a primer and a
newly synthesized strand which is the complement of the target
sequence downstream of the primer binding site. Extension products
result from hybridization of a primer to a target sequence and
extension of the primer by polymerase using the target sequence as
a template.
A bumper primer is a primer which anneals to a target sequence
upstream of the amplification primer, such that extension of the
bumper primer displaces the downstream amplification primer and its
extension product. Extension of bumper primers is one method for
displacing the extension products of amplification primers, but
heating is also suitable.
Identical sequences will hybridize to the same complementary
nucleotide sequence. Substantially identical sequences are
sufficiently similar in their nucleotide sequence that they also
hybridize to the same partially complementary nucleotide
sequence.
The terms target or target sequence refer to nucleic acid sequences
to be amplified. These include the original nucleic acid sequence
to be amplified, its complementary second strand and either strand
of a copy of the original sequence which is produced in the
amplification reaction. The target sequence may also be referred to
as a template for extension of hybridized amplification
primers.
A signal primer is a primer which hybridizes to a target sequence
downstream of an amplification primer such that extension of the
amplification primer displaces the signal primer and its extension
product. The signal primer comprises a 3'-OH group which can be
extended by DNA polymerase when the signal primer is hybridized to
the target sequence. The signal primer may be unmodified, e.g., for
detection of secondary amplification products based on their size.
Alternatively, the signal primer may include a reporter group or
label, or a structural feature to facilitate detection of its
extension product.
Amplification products, amplified products or amplicons are copies
of the target sequence generated by hybridization and extension of
an amplification primer. This term refers to both single stranded
and double stranded amplification primer extension products which
contain a copy of the original target sequence, including
intermediates of the amplification reaction.
Secondary amplification products or secondary products are copies
of the target sequence generated by hybridization and extension of
a signal primer. The secondary amplification product comprises an
internal segment of the amplified target sequence. These terms also
refer to both single stranded and double stranded extension
products of signal primers, including intermediates in the process
which generates the final double stranded form. In contrast to
amplification products, the double stranded secondary amplification
product is generally not available for further amplification,
although some secondary amplification products may be amplifiable
in a linear fashion.
In the methods of the invention, amplification primers for SDA are
hybridized to a target sequence and the target sequence is
amplified generally as described by Walker, et al., 1993 PNAS or
Walker, et al. 1993 Nuc. Acids Res., supra. As described in these
two publications, the target sequence may be prepared for SDA
either by restricting total DNA with an appropriate restriction
endonuclease (e.g., HincII) or by generating target fragments
having the appropriate restriction endonuclease recognition sites
at the ends using bumper primers and amplification primers.
Prepared fragments containing the target sequence are then
amplified by SDA as described. However, the SDA reaction of the
invention further comprises at least one signal primer which
results in simultaneous or concurrent generation of a secondary
amplification product for use in detecting, monitoring or
localizing amplification products produced by the SDA reaction. The
secondary amplification products may also contain features which
facilitate their capture or immobilization, so that they may be
isolated for detection, quantitation or further manipulation. The
secondary amplification products are produced in the SDA reaction
by inclusion of at least one signal primer in the reaction mixture.
For certain applications, it may be preferable to include a pair of
signal primers. The signal primer or signal primers hybridize to
the target sequence downstream of the hybridization site of the
amplification primers. They are extended by polymerase in a manner
similar to extension of the amplification primers. The signal
primer hybridizes at a site in the target sequence such that
extension of the amplification primer displaces the extension
product of the signal primer. At least the 3' end of the signal
primer comprises a sequence which hybridizes to the target
sequence. The entire signal primer may hybridize to the target
sequence, for example when it is unmodified or chemically modified
for detection by addition of a reporter group, label or affinity
ligand. Alternatively, the 5' end of the signal primer may comprise
a sequence which does not hybridize to the target sequence but
which contains special nucleotide sequences (often involving
structural features) which facilitate detection or capture of the
secondary amplification product. These chemical modifications and
special sequences are incorporated into the secondary amplification
products when the signal primers are hybridized and extended on a
template. Examples of chemical modifications include affinity
ligands (e.g., avidin, streptavidin, biotin, haptens, antigens and
antibodies) and reporter groups (labels, e.g., radioisotopes,
fluorescent dyes, enzymes which react to produce detectable
reaction products, and visible dyes). Examples of special
nucleotide sequences include (i) sequences which will form a triple
helix by hybridization of a labeled oligonucleotide probe to the
double stranded secondary amplification product, and (ii)
recognition sites for double-stranded DNA binding proteins which
become capable of binding the double-stranded DNA binding protein
when rendered double stranded during amplification (e.g.,
repressors, regulatory proteins, restriction endonucleases, RNA
polymerase). Nucleotide sequences which result in double stranded
restriction endonuclease recognition sites are a preferred
structural feature for use in signal primers, as subsequent
restriction may be used to generate a secondary amplification
product which is recognizable by a characteristic size.
When the inventive methods employ two signal primers which
hybridize to opposite strands of a double stranded target sequence,
as illustrated in FIGS. 1A and 1B, one of the signal primers may
contain a special nucleotide sequence or chemical modification to
facilitate capture or immobilization of the secondary amplification
product and the other may contain a detectable reporter group or
label for detection of the captured or immobilized secondary
amplification product. The use of labels and reporter groups for
detecting nucleic acids as well as the use of ligands, chemical
modifications and nucleic acid structural features for capture or
immobilization of nucleic acids is well known in the art.
Alternatively, the signal primer may be unmodified, i.e., without
reporter groups, capture groups or structural features to
facilitate detection or capture of the secondary amplification
products. The secondary amplification products may then be detected
based on their size, e.g., by gel electrophoresis and ethidium
bromide staining. All of these methods are useful in the present
invention and one skilled in the art can routinely select
appropriate methods for use in any particular amplification assay
system.
It is an important feature of the invention that the signal primers
do not function as amplification primers in the SDA reaction in
which they are employed. Without wishing to be bound by any
specific mechanism by which the inventive methods work. Applicants
believe it is this feature which allows the signal primers to be
added to the amplification reaction mixture without promoting the
high levels of background signal generated by other primer-based
methods. High levels of background signal are believed to be due to
non-specific priming and subsequent amplification of spuriously
primed non-target DNA when the primers are capable of functioning
as amplification primers. The present invention therefore greatly
simplifies the procedures for primer-based detection methods, which
previously relied on two consecutive amplification reactions to
attain high sensitivity and specificity, the second reaction being
performed with internally nested signal-generating amplification
primers.
As stated above, nucleic acid fragments having appropriate
restriction endonuclease recognition sequences at the ends and
containing the target sequence may be prepared for amplification
either as described by Walker, et al. 1992. PNAS, supra or as
described by Walker, et al. 1992 Nuc. Acids Res., supra. For
simplicity, the illustrations of the inventive methods in FIG. 1A,
FIG. 1B and FIG. 2 begin with a nucleic acid fragment containing
the target sequence. If prepared according to Walker, et al. 1992.
PNAS, supra, it represents restricted double stranded DNA which has
been denatured. If prepared according to Walker, et al. 1992. Nuc.
Acids Res., supra, appropriate restriction endonuclease recognition
sites are added to the fragment according to the disclosed target
generation scheme. It is believed that bumper, amplification and
signal primers may simultaneously hybridize to a target sequence in
the target generation scheme of Walker, et al. (1992. Nuc. Acids
Res., supra), extension of each upstream primer displacing the
extension product of the downstream primer and simultaneously
generating amplifiable target fragments and secondary amplification
products.
FIG. 1A and FIG. 1B illustrate one embodiment of the invention in
which a pair of signal primers are used for detecting amplification
of a double-stranded target sequence (5'-A-B-C-D/5'-D'-C'-B'-A').
FIG. 1A illustrates the method of the invention for the first of
the two complementary strands of the target sequence
(5'-D'-C'-B'-A'). The raised portion of the amplification primers
illustrated in FIGS. 1A, 1B and 2 indicates a nickable restriction
endonuclease recognition site as described above and by Walker, et
al. (1992. PNAS and Nuc. Acids Res.). Long raised portions
illustrate full-length restriction endonuclease recognition sites
and short raised portions illustrate partial restriction
endonuclease recognition sites, generally produced alter nicking
and displacing a strand. The nucleic acid fragments comprising the
target sequence may be generated either by endonuclease restriction
of larger nucleic acids (Walker, et al. 1992. PNAS, supra) or by
target generation as described by Walker, et al. (1992. Nuc. Acids
Res., supra). However, for purposes of illustration and to simplify
the diagrams, FIGS. 1A, 1B and 2 begin with the target sequence
contained on a nucleic acid fragment previously restricted with a
restriction endonuclease which does not cut the target
sequence.
In FIG. 1A, signal primer R-B is included in the SDA reaction
mixture and hybridizes to the target sequence downstream of a first
amplification primer by hybridization of the B portion of the
signal primer to B'. The R portion of the R-B signal primer
sequence includes a reporter group or label, or is a structural
feature to facilitate detection or capture. R may or may not
hybridize, as discussed above, but is shown here as not hybridizing
to clarify the different functional features of the signal primer.
For the purposes of this illustration, R will contain a reporter
group, but may contain other chemical modifications or structural
features as discussed above. Both amplification primer A and signal
primer R-B are extended by DNA polymerase using the target sequence
as a template. The signal primer extension product R-B-C-D
(structure #1) is displaced from the template by extension of
amplification primer A and in turn serves as a template for
hybridization and extension of a second signal primer Q'-C' and a
second amplification primer D'. The C' portion of the Q'-C'
sequence hybridizes to C. The Q' portion of the second signal
primer is analogous to R, and for purposes of this illustration Q'
will contain a modification or sequence to facilitate capture of
the secondary amplification product. The Q'-C' extension product is
displaced by extension of the second amplification primer. The
displaced Q'-C' extension product (structure #2) then serves as a
template for hybridization and extension of R-B, resulting in a
double stranded, target-specific secondary amplification product
(structure #3) which comprises the terminal segment (R and Q') of
the signal primers and the internal segment B'-C' of the target
sequence. As the secondary amplification product does not contain
nickable restriction endonuclease recognition sites, it is not
amplifiable in the SDA reaction and remains effectively inert
throughout the remainder of the amplification reaction, but
additional copies of the secondary amplification product are
generated from the target sequence.
Hybridization and extension of the second amplification primer
(D'), in addition to displacing the R'-B'-C'-Q' extension product,
generates a double stranded fragment with the R/R' sequence at one
end and a hemimodified restriction endonuclease recognition site at
the other end (structure #4). This restriction endonuclease
recognition site is nickable by the restriction endonuclease
present in the SDA reaction. The DNA polymerase present in the SDA
reaction can then initiate polymerization and displacement at the
nick, resulting in the illustrated R'-B'-C'-D' product comprising a
portion of the restriction endonuclease recognition site. This
product can be made double-stranded by hybridization and extension
of R-B (structure #5). Although cyclically repeating the nicking,
polymerizing and displacing cycle amplifies this fragment at a
linear rate, generally neither the single-stranded or
double-stranded product will be detectable by virtue of the absence
of the Q/Q' portion containing the modification or sequence to
facilitate capture. If the functions of Q/Q' and R/R' are reversed,
(i.e., Q/Q' contains the reporter group or label and R/R' contains
the modification or sequence to facilitate capture), these
products, though captured, would not be detectable by virtue of the
absence of the reporter group or label. It should be understood,
however, that structure #5 may be detectable when the reporter
group is detectable independent of capture, e.g., when the reporter
group is a fluorescent label detectable by anisotropy or
fluorescence polarization (WO 92/18650; R. Devlin, et al. 1993.
Clin. Chem. 39, 1939-1943) or a radioisotope which can be detected
by gel electrophoresis and autoradiography.
FIG. 1A also shows how extension of the first amplification primer
on the target sequence, in addition to displacing the extension
product of R-B, generates the double-stranded target sequence with
the hemimodified, nickable restriction endonuclease recognition
site which is required for amplification of the target sequence by
SDA (structure #6). These reaction products enter the conventional
SDA reaction and are amplified. Formation of the secondary
amplification product is therefore tightly coupled to amplification
of the target sequence and is useful to monitor whether or not
amplification has taken place as well as to provide a measure of
target amplification. In spite of the tight linkage of generation
of the secondary amplification product and generation of
amplification products, however, amplification of the target
sequence is not inhibited provided essential reaction components
are present in excess. In addition, amplified target sequences may
also bind signal primers, resulting in generation of additional
copies of the secondary amplification products.
FIG. 1B illustrates generation of secondary amplification products
from the complementary second strand of the double-stranded target
sequence (5'-A-B-C-D). In general, the reaction steps for the
complementary second strand are similar to those for the first
strand. However, the second amplification primer (D') and signal
primer C'-Q' hybridize first to the complementary strand and are
extended. The first amplification primer (A) and signal primer R-B
then hybridize to the displaced extension product of C'-Q'
(A'-B'-C'-Q', structure #7) and are extended to produce R-B-C-Q
(structure #8). Hybridization of Q'-C' to R-B-C-Q and extension
results in the double stranded secondary amplification products
R'-B'-C'-Q'/R-B-C-Q (structure #9). This secondary amplification
product is detectable in systems requiring both capture and
reporter groups due to the presence of both features in structure
#9. The reaction for the complementary strand also produces a
reaction product which can be linearly amplified by nicking,
polymerizing and displacing (structure #10). The displaced single
strand of this linear amplification becomes double stranded by
hybridization and extension of Q'-C' (structure # 11). Generally,
neither the single or double stranded reaction products of this
linear amplification are detectable due to the absence of either
the reporter group or the capture group. They are not further
amplifiable because they lack an intact restriction endonuclease
recognition site. However, if Q comprises a reporter group which is
detectable independent of capture (e.g., a fluorescent label or a
radioisotope as described above), structures # 10 and # 11 will
also be detectable.
Detection specificity will generally be improved when two signal
primers are employed as in FIG. 1A and FIG. 1B, but a single signal
primer may also be used. This method is illustrated in FIG. 2. In
this case, the signal primer may contain either a capture group or
a reporter group, and the target sequence itself or an
amplification primer may optionally provide a second capture or
reporter group. Alternatively, when both a capture and reporter
group are required, the signal primer may contain both a capture
and a reporter, group which act in conjunction only when the signal
oligonucleotide becomes double-stranded. This structure is formed
only when the presence of target sequences induces priming,
extension, displacement and re-priming as shown in FIG. 2. Such
bi-functional signal primers may also form the basis for a variety
of homogeneous detection methods such as fluorescence anisotropy or
fluorescence energy transfer.
To generate secondary amplification products using a single signal
primer according to FIG. 2, a first amplification primer (A) and
the signal primer R-B are hybridized to a single stranded target
sequence A'-B'-C'-D'. Both primers are extended, and extension of
the first amplification primer displaces the extension product of
signal primer R-B (R-B-C-D), producing structure #1. As there is no
second signal primer, only the second amplification primer (D')
hybridizes to P-B-C-D and is extended, generating structure #2 with
a nickable, hemimodified restriction endonuclease recognition site.
Linear amplification of this product by nicking, polymerizing and
displacing, as shown, generates fragments to which the signal
primer can hybridize and be extended. This generates the double
stranded secondary amplification product, structure #3. It is not
amplifiable due to the lack of an intact restriction endonuclease
recognition site, but is detectable by virtue of R/R' when the
reporter or capture group is detectable only in double stranded
form or by virtue of R when the reporter group is detectable alone.
When detection of the reporter group does not require
double-strandedness (e.g., a fluorescent label), structures #1, #2
and #3 are detectable as secondary amplification products.
EXAMPLE 1
The real-time detection of amplification of the instant invention
was compared to conventional post-amplification detection of
amplified target sequences. Fragments of the IS6110 sequence of
Mycobacterium tuberculosis (M.tb) were amplified in SDA reactions
performed essentially as described by Walker, et al. (1992, Nuc.
Acids Res.), except that each 60 .mu.L reaction mixture contained
0.2 .mu.g of human placental DNA and varying amounts of genomic
M.tb DNA. Amplification primer sequences (S.sub.1 and S.sub.2) and
bumper primer sequences (B.sub.1 and B.sub.2) were also as in
Walker, et al. (1992, Nuc. Acids Res.) For the amplification
reactions incorporating signal primers, the .sup.32P-labeled signal
primer .sup.32P-CGTTATCCACCATAC (SEQ ID NO:1) was added to the
reactions prior to amplification at a final concentration of 60 nM.
Predicted secondary amplification products produced in these
reactions were 35 and 56 nucleotides in length. For
post-amplification detection of amplified target sequences,
one-tenth of the reaction mixture was used to detect amplification
products by primer extension of SEQ ID NO:1 as described by Walker,
et a. (1992, Nucl. Acids. Res., supra), producing extension
products either 35 or 56 nucleotides in length.
Amplification was allowed to proceed for 2 hr. at 37.degree. C. in
the presence of 1 to 500,000 genome copies of M.tb. After stopping
the amplification, one-tenth of each reaction was subjected to
electrophoresis on denaturing polyacrylamide gels. As little as one
copy of M.tb genomic DNA was detected using the signal primer
according to the invention. Also, the signal intensity decreased
with decreasing target levels, indicating that the levels of
secondary amplification product reflect the degree of target
sequence amplification. The real-time extension of the signal
primer appeared on the gel to be several fold less sensitive than
the conventional post-amplification primer extension method,
possibly because the .sup.32P-labeled signal primer was present
during SDA at concentrations about 10-fold less than the SDA
primers. If SDA primers are extended on the target sequence before
a signal primer binds and is extended, no signal will result. Thus,
higher concentrations of signal primer should increase the method's
sensitivity by improving hybridization kinetics for the signal
primer. Higher signal primer concentrations are therefore preferred
when reaction products are separated for detection, but the
concentration of amplification primers may be kept similar to the
concentrations used in conventional SDA. However, lower signal
primer concentrations are preferred to keep background low for
homogeneous detection methods such as fluorescence anisotropy. The
lower concentrations of signal primer are preferably used with
lower concentrations of polymerase and the amplification primer
which hybridizes upstream of the signal primer than is customary in
conventional SDA. This experiment also demonstrated that the
presence of the signal primer in the amplification reaction mixture
does not lead to significant levels of background signal. In fact,
background signal levels appeared to be lower in samples detected
by real-time signal-primer extension as compared to
post-amplification primer extension.
EXAMPLE 2
SDA reactions were performed generally as previously described
(Walker, 1993, PCR--Methods and Applications 3. 1) in 50 mM
KiPO.sub.4 (pH 7.5), 0.1 mg/mL bovine serum albumin, 0.5 mM dUTP,
0.2 mM each dGTP, dCTP and dATP.alpha.S, 7 mM MgCl.sub.2, 11% (v/v)
glycerol, the indicated concentrations of amplification primers, 25
nM bumper primers, 50 ng human placental DNA, the indicated amount
of exonuclease deficient Klenow (United States Biochemicals), 150
units HincII (New England Biolabs). Reactions were run for the
indicated time at 41.degree. C. SDA reactions contained varying
amounts of M.tb DNA, which contains the IS6110 target sequence for
amplification. The S.sub.1 amplification primer sequence, the
B.sub.1 bumper primer sequence and the B.sub.2 bumper primer
sequence used were as described by Walker, et al. (1992, Nuc. Acids
Res., supra). The S.sub.2 amplification primer (SEQ ID NO:2) had
the target binding sequence and HincII site disclosed by these
authors, but comprised a different sequence at the 5' end. The
amplification primers hybridize to nucleotide positions 972-984 and
1011-1023 of the IS6110 sequence. The bumper primers hybridize to
nucleotide positions 954-966 and 1032-1044. Secondary amplification
products were visualized by autoradiography after electrophoresis
on denaturing polyacrylamide gels.
SDA reactions were performed for 3 hrs. in the presence of 0.1 nM
of a 5'-.sup.32P-labeled signal primer (SEQ ID NO:3). This signal
primer is 28 nucleotides in length and hybridizes to nucleotide
positions 985-1012 of the IS6110 target sequence, between the
amplification primers. S.sub.1 and S.sub.2 were present at 180 and
30 nM, respectively. Exonuclease deficient Klenow was used at 0.25
units. Samples 1-4 contained 100, 10, 1 and 0 M.tb genome
molecules, respectively. During the SDA reaction, SEQ ID NO:3 is
extended by polymerase to a length of 44 nucleotides using the
target sequence as a template. As discussed above, this template is
most likely primarily the displaced, amplified target strand
generated during SDA, but concurrent extension of the bumper,
amplification and signal primers on the original target sequence
has not been ruled out and would be expected to occur as well. The
44-mer is displaced from the target sequence by extension of the
upstream amplification primer (S.sub.2). The 3'-end of the 44-mer
hybridizes to the 3'-end of the second amplification primer
(S.sub.1) and a double-stranded 65-mer is formed after extension by
polymerase. The 44-mer and 65-mer secondary amplification products
were observed only in the presence of the M.tb target sequence
(samples 1-3), indicating signal primer extension and
transformation to double stranded form.
The preceding SDA reactions were repeated in the presence of 0.1 nM
(samples 1-3) or 1 nM (samples 4-6) of a 5'-.sup.32-P-signal primer
which was 15 nucleotides in length (SEQ ID NO:4). This signal
primer hybridizes at nucleotide positions 999-1013 of the IS6110
target sequence, between the amplification primers. S.sub.1 and
S.sub.2 were used at 500 nM. Two units of exonuclease deficient
Klenow were used and SDA was performed for 2 hrs. The three
nucleotides at the 5'-end of SEQ ID NO:4 and the three nucleotides
at the 3'-end of SEQ ID NO:2 (the S.sub.2 amplification primer) are
identical and therefore compete for the same IS6110 binding site.
Samples 1 and 4 contained 10000 M.tb genome molecules while samples
2 and 5 contained 100 genome molecules. Samples 3 and 6 did not
contain M.tb DNA.
During the SDA reaction, 45-mer and 66-mer secondary products are
produced when the target sequence is amplified. They were observed
only in the presence of M.tb DNA, indicating extension of the
signal primer and transformation into double stranded form (samples
1, 2, 4 and 5). In the absence of M.tb DNA (samples 3 and 6), no
radiolabeled products were seen. More sensitive detection was
obtained when using a concentration of 1 nM of signal primer
(samples 4-6) as compared to 0.1 nM, most likely due to more
favorable hybridization kinetics for the signal primer and improved
thermodynamic stability of the signal primer/target sequence hybrid
during SDA.
SDA was repeated in the presence of 0.1 nM of a 5'-.sup.32P-labeled
signal primer which was 42 nucleotides in length (SEQ ID NO:5). The
26 nucleotides at the 3'-end of the signal primer (the target
binding sequence) hybridize to the IS6110 target sequence at
nucleotide positions 985-1010, between the amplification primers.
5' to the target binding sequence is a recognition site for the
restriction endonuclease HincII. S.sub.1 and S.sub.2 were present
at 180 and 30 nM. Exonuclease deficient Klenow was used at 0.25
units and SDA was performed for 3 hrs. Samples 1-4 contained 100,
10, 1 and 0 M.tb genome molecules.
During the SDA reaction, the signal primer is extended by the
polymerase to a length of 58 nucleotides. This 58-mer is displaced
by extension of the upstream amplification primer (SEQ ID NO:2).
The 3'-end of the 58-mer hybridizes to the 3'-end of the other
amplification primer (S.sub.1), forming a double-stranded 79-mer
after extension by polymerase. The HincII recognition site at the
5'-end of the signal primer becomes cleavable by HincII upon
formation of the double-stranded 79-mer. That is, in the
double-stranded 79-mer, both the strand comprising the original
signal primer and the strand formed through polymerase extension
using dGTP, dCTP, TTP and dATP.alpha.S are cleavable. HincII does
not cleave the signal primer in its original single-stranded form.
Cleavage of the double stranded 79-mer during the SDA reaction
produces a 5'-.sup.32P-labeled 13-mer which is detectable as a
secondary amplification product.
58-mer and 79-mer primer extension secondary amplification products
and the 13-mer cleavage secondary amplification product were
observed only in the presence of M.tb target DNA (samples 1-3),
indicating extension of the signal primer and transformation to
double-stranded form. In the absence of M.tb DNA no secondary
amplification products (extension products or cleavage products)
were observed.
SDA was repeated in the presence of 0.1 nM of a 5'-.sup.32P-labeled
signal primer which was 33 nucleotides in length (SEQ ID NO:6). The
26 nucleotides at the 3'-end of the signal primer (the target
binding sequence) hybridize to the IS6110 target sequence at
nucleotide positions 985-1010, between the amplification primers.
5' to the target binding sequence is a recognition site for the
restriction endonuclease EcoRI. S.sub.1 and S.sub.2 were present at
180 and 30 nM, respectively. Exonuclease deficient Klenow was used
at 0.25 units. SDA was performed for 3 hrs. Samples 1-4 contained
100, 10, 1 and 0 M.tb genome molecules. After SDA, 20 units of
EcoRI were added to each SDA reaction and the samples were
incubated for 30 min. at 37.degree. C.
During the SDA reaction, the signal primer is extended by the
polymerase to a length of 49 nucleotides. This 49-mer is displaced
by extension of the upstream amplification primer (SEQ ID NO:2).
The 3'-end of the 49-mer hybridizes to the 3'-end of the other
amplification primer (S.sub.1), forming a double-stranded 70-mer
after extension by polymerase. The EcoRI recognition site at the
5'-end of the signal primer becomes cleavable by EcoRI upon
formation of the 70-mer and addition of EcoRI. EcoRI cleavage of
the double-stranded 70-mer produces a cleavage product which is a
5'-.sup.32P-labeled dinucleotide. This dinucleotide is detectable
by autoradiography as a secondary amplification product.
49-mer and 70-mer extension products and the dinucleotide cleavage
secondary amplification product were observed only in the presence
of M.tb target DNA (samples 1-3, indicating extension of the signal
primer and transformation to double-stranded form. In the absence
of M.tb DNA no secondary amplification products (extension products
or cleavage products) were observed.
The .sup.32P-dinucleotide cleavage product was alternatively
detected by liquid scintillation counting. SDA was repeated in the
presence of 0.5 nM 5'-.sup.32P-labeled SEQ ID NO:6 signal primer.
Prior to its use in SDA, the .sup.32P-labeled signal primer was
purified away from the gamma-.sup.32P-ATP used in the kinase
labeling reaction by denaturing gel electrophoresis. S.sub.1 and
S.sub.2 were present at 180 and 30 nM. Exonuclease deficient Klenow
was used at 0.25 units. SDA was performed for 3 hrs. Samples 1-7
contained 10.sup.5, 10.sup.4, 10.sup.3, 10.sup.2, 10, 1 and 0 M.tb
genome molecules. After SDA, 40 units of EcoRI were added to each
SDA reaction and the samples were incubated at 37.degree. C. for 30
min.
A 12.5 .mu.l aliquot from each 50 .mu.l SDA reaction was diluted to
75 .mu.l in 20 mM TRIS-HCL (pH 7.4), 50 mM KCl, 5 mM MgCl.sub.2.
Each of these samples was then filtered using a MICROCON-10
microconcentrator (icon, Beverly, Mass.) and .sup.32P activity was
detected in the filtrate and on the filter by liquid scintillation
counting. The results are shown below:
TABLE-US-00001 Initial # of M tb Genome Filtrate Sample Molecules
(cpm) Filter (cpm) 1 10.sup.5 13,449 44,802 2 10.sup.4 12,299
49,366 3 10.sup.3 9,006 50,739 4 10.sup.2 6,689 52,712 5 10.sup.
3,153 57,732 6 1 2,072 55,835 7 0 2,120 57,995
The .sup.32P-dinucleotide released by EcoRI cleavage of the
double-stranded 70-mer extension product is small enough that it
passes through the MICROCON-10 filter, while the larger initial
.sup.32P-labeled 33-mer signal primer and .sup.32P-labeled 49-mer
extension product are retained on the filter. Using this filtration
detection method, the IS6110 target sequence could be detected in a
sample which contained as few as 10 M.tb genomes prior to SDA.
EXAMPLE 3
Two signal primers, one modified to facilitate capture and one
modified to facilitate detection, were used to generate secondary
amplification products in an SDA reaction. In this experiment, one
signal primer had an affinity ligand (Q', three biotin moieties)
attached to its 5' end and the second signal primer was 5'-end
labeled with a reporter group (R, a .sup.32P-containing phosphate
group). Thus, double-stranded secondary amplification products
which comprised both the reporter group and the affinity ligand
(such as structure #3 and structure #9 of FIG. 1A and FIG. 1B)
could be captured and detected. In this example, streptavidin
coated magnetic beads were used to capture and separate the
secondary amplification products, which were then detected by
scintillation counting.
Biotinylated signal primers were prepared as follows.
Oligonucleotide SEQ ID NO:7 was synthesized on an Applied
Biosystems DNA Synthesizer Model 380B, using standard
phosphoramidite chemistry. The instrument was then used to attach
three biotin groups to the oligonucleotide by three successive
couplings with BIOTIN ON phosphoramidite (Clonetech). Following
synthesis, the oligonucleotide was deprotected by treatment with
concentrated ammonia and purified by denaturing gel
electrophoresis. The biotinylated signal primers with attached
affinity ligands for capture of the secondary amplification
products are referred to as capture signal primers and, for the
purposes of this example, are analogous to the Q'-C' signal primer
of FIG. 1A and FIG. 1B wherein Q' comprises biotin.
To prepare .sup.32P-labeled signal primers, oligonucleotides SEQ ID
NO:1 and SEQ ID NO:8 were labeled with radioactive phosphate as
described by Walker, et al. (1992, Nucl. Acids Res., supra). The
radiolabeled signal primers are referred to as detection signal
primers and, for the purposes of this example, are analogous to the
R-B signal primer of FIG. 1A and FIG. 1B wherein R comprises a
radiolabel. In the experiment, one of these two detection signal
primers was used in conjunction with the capture signal primer to
generate secondary amplification products.
SDA was carded out essentially as described by Walker, et al.
(1992, Nucl. Acids Res., supra) with the following modifications.
Amplification primers were SEQ ID NO:9 and SEQ ID NO:10 (analogous
to A and D' in FIG. 1A and FIG. 1B). These primers amplify a 103
nucleotide fragment (nucleotide positions 944-1046) of the IS6110
insertion element of M.tb. Each 50 .mu.l reaction contained the
following components: 45 mM K.sub.iPO.sub.4, pH 7.5; 6 mM
MgCl.sub.2; 0.1 mg/ml acetylated BSA; 12% dimethylsulfoxide; 0.5 mM
dUTP; 0.2 mM each dCTP, dGTP, dATP.alpha.S; 500 nM amplification
primers: 50 nM bumper primers (SEQ ID NO:11 and SEQ ID NO:12); 75
nM detection signal primer and capture signal primer (SEQ ID NO:1
and SEQ ID NO:7 or SEQ ID NO:8 and SEQ ID NO:7); 100 ng human
placental DNA; 150 units HincII (New England Biolabs); 2.5 units
exo.sup.-- Klenow DNA polymerase (US Biochemicals); 3% (v/v)
glycerol added with enzymes; and either 0 or 10.sup.5 copies of the
M.tb genome.
All reaction components, except MgCl.sub.2, HincII and polymerase,
were assembled and the mixtures were heated to 95.degree. C. for
two minutes. The samples were then placed in a water bath at
40.degree. C. for two minutes, 3 .mu.l of 0.1M MgCl.sub.2 were
added to each sample and the samples were mixed. Three .mu.l of an
enzyme mixture containing 50 units/.mu.l HincII, 0.833 units/.mu.l
polymerase and 50% (v/v) glycerol were added and the samples were
incubated at 40.degree. C. for 2 hrs.
After incubation, a 5 .mu.l aliquot of each reaction mixture was
analyzed by denaturing gel electrophoresis. Because detection on
gels requires only the presence of R, various products appeared in
the range of 50-120 nucleotides for samples containing genomic M.tb
DNA. These bands were absent in reactions lacking M.tb DNA,
indicating that the reaction products in this size range were
target-specific. The secondary amplification products predicted for
this example, determined by calculation of the known sizes and
binding positions of the signal primers according to the reaction
scheme outlined in FIG. 1A and FIG. 1B, are shown in the following
Table. As can be seen from FIG. 1A and FIG. 1B, structure #3 (in
single-stranded form on denaturing gels) contains R and is
detectable. In addition, the detectable single strand of structure
#3 is identical to structure #2 and to the detectable single strand
of structure #9. These secondary amplification products are
therefore indistinguishable on the gel. Structure #4 and structure
#5 (also in single-stranded form on denaturing gels) are also
detectable by virtue of the presence of R.
TABLE-US-00002 EXPECTED SECONDARY PRODUCT SIZES (nucleotides, nt)
.sup.32P-Labeled Signal Primer Structure (FIGS. 1A and 1B) SEQ ID
NO 1 SEQ ID NO 8 #3, #9 and #8 52 nt 92 nt #4 58 nt 98 nt #5 79 nt
119 nt
Streptavidin-coated magnetic beads (Streptavidin Paramagnetic
Particles, Nucleic Acid Qualified, 1 mg/ml, Promega Corporation,
Madison, Wis.) were washed three times with 1X PBS as recommended
by the manufacturer. For each analysis, 50 .mu.g of the beads were
suspended in 180 .mu.l of 1X PBS in a 1.5 ml eppendorf tube and
combined with 20 .mu.l of the SDA reaction mixture. These samples
were incubated with occasional mixing for 10 min. at room
temperature. A magnet was then used to gather the beads on one side
of the tube and the supernatant was removed. The beads were then
washed by resuspending them in 1X PBS (200 .mu.l), gathering them
magnetically on the side of the tube and removing the supernatant.
This washing process was repeated three more times, and the
.sup.32P activity remaining on the beads was detected by liquid
scintillation counting. The results are shown in the following
Table:
TABLE-US-00003 10.sup.5 Initial Genome 0 Initial Genome Detector
Signal Primer Molecules Molecules SEQ ID NO 1 80,547 cpm 1,080 cpm
SEQ ID NO 8 54,385 cpm 961 cpm
Small aliquots of the beads (10%), removed prior to scintillation
counting, were heated to 95.degree. C. in the presence of 50% urea
and subjected to electrophoresis on a denaturing polyacrylamide
gel. Only structure #3 and structure #9 contain both the biotin
modification and the .sup.32P label, and it was predicted that only
these structures would bind to the beads and be detectable.
Autoradiography of the gel did show that the structure #3 and
structure #9 secondary amplification products represent the
predominant radioactive species retained by the beads during the
magnetic separation process. However, smaller amounts of a species
corresponding to structure #1 also appeared on the autoradiogram.
It is possible that structure #1, produced by extension of the
detector signal primer, may be captured when hybridized to a Q'-C'
capture signal primer (prior to capture signal primer extension and
generation of structure #2; see the reaction step following
structure #1 in FIG. 1A). Although small amounts of structure # 1
were apparently captured and detected in addition to the predicted
structures #3 and #9, all captured and detected secondary
amplification products were target specific and did not appear in
samples lacking genomic M.tb DNA.
The background radioactivity detected on the beads in the absence
of M.tb DNA (0 initial genome molecules) appears to be due to
nonspecific binding of unreacted detector signal primers.
Electrophoretic analysis of beads from these samples showed that
the only radioactive material present was a very faint band
corresponding to the detector signal primers, even after overnight
exposure of the autoradiogram. However, the secondary amplification
products were clearly detected above these background levels of
signal.
SEQUENCE LISTINGS
1
25124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1caatgttgtt ccttgaggaa gttg 24220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2gttgttcctt gaggaagttg 20317DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3gttccttgag gaagttg
17441DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4cgattccgct ccagacttct cgggaacaaa gaaggcatcg t
41541DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5accgcatcga atgcatgtct cgggtggcag cattgttatt a
41640DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 6acgttagcca ccatacggat acccaaagac cacattggca
40740DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7actgatccgc actaacgact acccaaagac cacacggact
40840DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8acgttagcca ccatacttga acccaaagac cacattggca
409126DNAHuman Severe Acute Respitory Syndrome Coronavirus
9aacaaagaag gcatcgtatg ggttgcaact gagggagcct tgaatacacc caaagaccac
60attggcaccc gcaatcctaa taacaatgct gccaccgtgc tacaacttcc tcaaggaaca
120acattg 12610126RNAHuman Severe Acute Respitory Syndrome
Coronavirus 10aacaaagaag gcaucguaug gguugcaacu gagggagccu
ugaauacacc caaagaccac 60auuggcaccc gcaauccuaa uuacaaugcu gccaccgugc
uacaacuucc ucaaggaaca 120acauug 12611126DNAHuman Severe Acute
Respitory Syndrome Coronavirus 11aacaaagaag gcatcgtatg ggttgcaact
gagggagcct tgaatacacc caaagaccac 60acggactccc gcaatcctaa ttacaatgct
gccaccgtgc tacaacttcc tcaaggaaca 120acattg 12612126RNAHuman Severe
Acute Respitory Syndrome Coronavirus 12aacaaagaag gcaucguaug
gguugcaacu gagggagccu ugaauacacc caaagaccac 60acggacuccc gcaauccuaa
uuacaaugcu gccaccgugc uacaacuucc ucaaggaaca 120acauug
1261328DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 13tcccgagtac gttagccacc atacggat
281428DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 14acccgagtag ctatccgcca taagccat
281529DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 15tccccgagta cgttagccac catacttga
291629DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 16tccccgagta ctgatccgca ctaacgact
291722DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 17atgttcccga aggtgtgact tc 221841DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18cgattccgct ccagacttct cgggacaagg aactgattac a 411941DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
19accgcatcga atgcatgtct cgggtgcgtg acattccaaa g 412040DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20acgttagcca ccatacggat caatttgctc caagtgcctc 402140DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
21acgttagcca ccatacttga caatttgctc caagtgcctc 4022111DNAHuman
Severe Acute Respitory Syndrome Coronavirus 22gacaaggaac tgattacaaa
cattggccgc aaattgcaca atttgctcca agtgcctctg 60cattctttgg aatgtcacgc
attggcatgg aagtcacacc ttcgggaaca t 11123111RNAHuman Severe Acute
Respitory Syndrome Coronavirus 23gacaaggaac ugauuacaaa cauuggccgc
aaauugcaca auuugcucca agugccucug 60cauucuuugg aaugucacgc auuggcaugg
aagucacacc uucgggaaca u 1112415DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 24gcctcttcgc tatta
152540DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25agctatccgc cataagccat acccaaagac cacacggact
40
* * * * *
References