U.S. patent application number 09/335218 was filed with the patent office on 2002-02-28 for methods and oligonucleotides for detecting nucleic acid sequence variations.
Invention is credited to MILLA, MARIA A., NADEAU, JAMES G., WALKER, G. TERRANCE, WRIGHT, DAVID J..
Application Number | 20020025519 09/335218 |
Document ID | / |
Family ID | 23310785 |
Filed Date | 2002-02-28 |
United States Patent
Application |
20020025519 |
Kind Code |
A1 |
WRIGHT, DAVID J. ; et
al. |
February 28, 2002 |
METHODS AND OLIGONUCLEOTIDES FOR DETECTING NUCLEIC ACID SEQUENCE
VARIATIONS
Abstract
The present invention provides methods for detecting and
identifying sequence variations in a nucleic acid sequence of
interest using a detector primer. It has been found that the
reduced efficiency of primer extension by DNA polymerases when the
3' end of a primer does not hybridize perfectly with the target can
be adapted for use as a means for distinguishing or identifying the
nucleotide in the target which is at the site where the diagnostic
mismatch between the detector primer and the target occurs. The
detector primer hybridizes to the sequence of interest and is
extended with polymerase. The efficiency of detector primer
extension is detected as an indication of the presence and/or
identity of the sequence variation in the target. The inventive
methods make use of nucleotide mismatches at or near the 3' end of
the detector primer to discriminate between the nucleotide sequence
of interest and a second nucleotide sequence which may occur at
that same site in the target. The methods are particularly well
suited for detecting and identifying single nucleotide differences
between a target sequence of interest (e.g., a mutant allele of a
gene) and a second nucleic acid sequence (e.g., a wild type allele
for the same gene).
Inventors: |
WRIGHT, DAVID J.; (CHAPEL
HILL, NC) ; MILLA, MARIA A.; (WYNNEWOOD, PA) ;
NADEAU, JAMES G.; (CHAPEL HILL, NC) ; WALKER, G.
TERRANCE; (CHAPEL HILL, NC) |
Correspondence
Address: |
RICHARD J RODRICK
BECTON DICKINSON AND COMPANY
1 BECTON DRIVE
FRANKLIN LAKES
NJ
07417
|
Family ID: |
23310785 |
Appl. No.: |
09/335218 |
Filed: |
June 17, 1999 |
Current U.S.
Class: |
435/6.12 ;
435/91.2; 536/24.3 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 2565/1025 20130101; C12Q 2535/125 20130101; C12Q 2531/101
20130101; C12Q 1/6827 20130101; C12Q 2531/119 20130101 |
Class at
Publication: |
435/6 ; 536/24.3;
435/91.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 019/34 |
Claims
What is claimed is:
1. A method for detecting a single nucleotide polymorphism in a
target comprising: a) hybridizing a detector primer and a second
primer to the target such that extension of the second primer by
polymerase displaces the detector primer from the target sequence,
wherein the detector primer comprises a diagnostic nucleotide for
the single nucleotide polymorphism which is a 3' terminal
nucleotide of the detector primer or about one to four nucleotides
from the 3' terminal nucleotide; b) extending the detector primer
and the second primer with polymerase to produce a displaced
detector primer extension product; c) determining an efficiency of
detector primer extension, and; d) detecting the presence or
absence of the single nucleotide polymorphism based on the
efficiency of detector primer extension.
2. The method of claim 1 wherein the single nucleotide polymorphism
is identified using the detector primer.
3. The method of claim 2 wherein the single nucleotide polymorphism
is identified using multiple detector primers, each comprising a
different diagnostic nucleotide.
4. The method of claim 3 wherein two detector primers are used to
identify which of two possible alleles is present in the target
sequence.
5. The method of claim 3 wherein four detector primers are used to
identify the nucleotide present in the target sequence at the
position of the single nucleotide polymorphism.
6. The method of claim 3 wherein each of the multiple detector
primers has a different 5' tail sequence.
7. The method of claim 1 wherein the detector primer further
comprises a nucleotide which forms a nondiagnostic mismatch with
the target sequence.
8. The method of claim 7 wherein the nondiagnostic nucleotide is
positioned within fifteen nucleotides of the diagnostic nucleotide
in the detector primer.
9. The method of claim 8 wherein the nondiagnostic nucleotide is
positioned 1-5 nucleotides from the diagnostic nucleotide in the
detector primer.
10. The method of claim 9 wherein the nondiagnostic nucleotide is
adjacent to the diagnostic nucleotide in the detector primer.
11. The method of claim 7 wherein the detector primer is about
15-36 nucleotides long.
12. The method of claim 11 wherein the detector primer is about
18-24 nucleotides long.
13. The method of claim 1 wherein the second primer is an
amplification primer.
14. The method of claim 13 wherein the amplification reaction is
selected from the group consisting of SDA, 3SR, NASBA, TMA and
PCR.
15. The method of claim 1 wherein the detector primer is about
12-50 nucleotides long.
16. The method of claim 15 wherein the detector primer is about
12-24 nucleotides long.
17. The method of claim 16 wherein the detector primer is about
12-19 nucleotides long.
18. The method of claim 1 wherein the presence or absence of the
single nucleotide polymorphism is detected by means of a label
associated with the detector primer.
19. The method of claim 18 wherein the label becomes detectable
upon extension of the detector primer or produces a change in
signal upon extension of the detector primer.
20. The method of claim 19 wherein the label is a fluorescent
donor/quencher dye pair and a decrease in donor dye fluorescence is
detected as an indication of the presence of the single nucleotide
polymorphism.
21. The method of claim 19 wherein a change in fluorescence
polarization is detected as an indication of the presence of the
single nucleotide polymorphism.
22. The method of claim 1 wherein a single nucleotide polymorphism
in an HFE gene is detected.
23. The method of claim 22 wherein the single nucleotide
polymorphism is detected in exon 4 or exon 2 of the HFE gene.
24. The method of claim 1 wherein the efficiency of detector primer
extension is determined quantitatively.
25. A method for detecting a single nucleotide polymorphism in a
target comprising, in an isothermal nucleic acid amplification
reaction: a) hybridizing a detector primer to the target, wherein
the detector primer comprises a diagnostic nucleotide for the
single nucleotide polymorphism about one to four nucleotides from a
3' terminal nucleotide of the detector primer which is
complementary to the target sequence; b) amplifying the target by
hybridization and extension of the detector primer; c) determining
an efficiency of detector primer extension, and; d) detecting the
presence or absence of the single nucleotide polymorphism based on
the efficiency of detector primer extension.
26. The method of claim 25 wherein the single nucleotide
polymorphism is identified using the detector primer.
27. The method of claim 26 wherein the single nucleotide
polymorphism is identified using two or more detector primers, each
comprising a different diagnostic nucleotide.
28. The method of claim 27 wherein two detector primers are used to
identify which of two possible alleles is present in the target
sequence.
29. The method of claim 27 wherein four detector primers are used
to identify the nucleotide present in the target sequence at the
position of the single nucleotide polymorphism.
30. The method of claim 27 wherein each of the multiple detector
primers has a different 5' tail sequence.
31. The method of claim 25 wherein the detector primer further
comprises a nucleotide which forms a nondiagnostic mismatch with
the target sequence.
32. The method of claim 31 wherein the nondiagnostic nucleotide is
positioned within fifteen nucleotides of the diagnostic nucleotide
in the detector primer.
33. The method of claim 32 wherein the nondiagnostic nucleotide is
positioned 1-5 nucleotides from the diagnostic nucleotide in the
detector primer.
34. The method of claim 33 wherein the nondiagnostic nucleotide is
adjacent to the diagnostic nucleotide in the detector primer.
35. The method of claim 31 wherein the detector primer is about
15-36 nucleotides long.
36. The method of claim 35 wherein the detector primer is about
18-24 nucleotides long.
37. The method of claim 25 wherein the isothermal amplification
reaction is selected from the group consisting of SDA, 3SR, NASBA
and TMA.
38. The method of claim 25 wherein the detector primer is about
12-50 nucleotides long.
39. The method of claim 38 wherein the detector primer is about
12-24 nucleotides long.
40. The method of claim 39 wherein the detector primer is about
12-19 nucleotides long.
41. The method of claim 25 wherein the presence or absence of the
single nucleotide polymorphism is detected by means of a label
associated with the detector primer.
42. The method of claim 41 wherein the label becomes detectable
upon extension of the detector primer or produces a change in
signal upon extension of the detector primer.
43. The method of claim 42 wherein the label is a fluorescent
donor/quencher dye pair and a decrease in donor dye fluorescence is
detected as an indication of the presence of the single nucleotide
polymorphism.
44. The method of claim 42 wherein a change in fluorescence
polarization is detected as an indication of the presence of the
single nucleotide polymorphism.
45. The method of claim 25 wherein the efficiency of detector
primer extension is determined quantitatively.
46. An oligonucleotide which comprises: a) a nucleotide sequence
which hybridizes to an internal segment of a target nucleic acid
downstream from a hybridization site for a primer such that
extension of the primer displaces the oligonucleotide from the
target sequence, and; b) a 3' terminal nucleotide or a nucleotide
about one to four nucleotides from the 3' terminal nucleotide which
is diagnostic for a single nucleotide polymorphism which may be
present in the target nucleic acid.
47. The oligonucleotide of claim 46 wherein the diagnostic
nucleotide is the 3' terminal nucleotide (N) or N-1.
48. The oligonucleotide of claim 46 further comprising a
nondiagnostic nucleotide within about one to fifteen nucleotides
from the diagnostic nucleotide.
49. The oligonucleotide of claim 48 wherein the nondiagnostic
nucleotide is within about one to five nucleotides from the
diagnostic nucleotide.
50. The oligonucleotide of claim 49 wherein the diagnostic and
nondiagnostic nucleotides, respectively, are selected from the
group consisting of N and N-3, N-1 and N-2, and N-2 and N-3.
51. The oligonucleotide of claim 46 which hybridizes downstream
from an amplification primer for the target nucleic acid.
52. An oligonucleotide which is an amplification primer for an
isothermal nucleic acid amplification reaction, the oligonucleotide
comprising: a) a 3' terminal nucleotide which is complementary to
the target, and; b) about one to four nucleotides from the 3'
terminal nucleotide, a diagnostic nucleotide for a single
nucleotide polymorphism which may be present in a target to be
amplified.
53. The oligonucleotide of claim 52 wherein the diagnostic
nucleotide is at N-1 or N-2.
54. The method of claim 25 further comprising, prior to amplifying,
displacing the hybridized detector primer from the target by
extension of an upstream primer.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for detecting and
identifying sequence variations in nucleic acids.
BACKGROUND OF THE INVENTION
[0002] Detecting and identifying variations in DNA sequences among
individuals and species has provided insights into evolutionary
relationships, inherited disorders, acquired disorders and other
aspects of molecular genetics. Analysis of sequence variation has
routinely been performed by analysis of restriction fragment length
polymorphism (RFLP) which relies on a change in restriction
fragment length as a result of a change in sequence. RFLP analysis
requires size-separation of restriction fragments on a gel and
Southern blotting with an appropriate probe. This technique is slow
and labor intensive and cannot be used if the sequence change does
not result in a new or eliminated restriction site.
[0003] More recently, PCR has been used to facilitate sequence
analysis of DNA. For example, allele-specific oligonucleotides have
been used to probe dot blots of PCR products for disease diagnosis.
If a point mutation creates or eliminates a restriction site,
cleavage of PCR products may be used for genetic diagnosis (e.g.,
sickle cell anemia). General PCR techniques for analysis of
sequence variations have also been reported. S. Kwok, et al. (1990.
Nucl. Acids Res. 18:999-1005) evaluated the effect on PCR of
various primer-template mismatches for the purpose of designing
primers for amplification of HIV which would be tolerant of
sequence variations. The authors also recognized that their studies
could facilitate development of primers for allele-specific
amplification. Kwok, et al. report that a 3' terminal mismatch on
the PCR primer produced variable results. In contrast, with the
exception of a 3' T mismatch, a 3' terminal mismatch accompanied by
a second mismatch within the last four nucleotides of the primer
generally produced a dramatic reduction in amplification product.
The authors report that a single mismatch one nucleotide from the
3' terminus (N-1), two nucleotides from the 3' terminus (N-2) or
three nucleotides from the 3' terminus (N-3) had no effect on the
efficiency of amplification by PCR. C. R. Newton, et al. (1989.
Nucl. Acids Res. 17:2503-2516) report an improvement in PCR for
analysis of any known mutation in genomic DNA. The system is
referred to as Amplification Refractory Mutation System or ARMS and
employs an allele-specific PCR primer. The 3' terminal nucleotide
of the PCR amplification primer is allele specific and therefore
will not function as an amplification primer in PCR if it is
mismatched to the target. The authors also report that in some
cases additional mismatches near the 3' terminus of the
amplification primer improve allele discrimination.
SUMMARY OF THE INVENTION
[0004] The present invention provides methods for detecting and
identifying sequence variations in a nucleic acid sequence of
interest using a detector primer. The detector primer hybridizes to
the sequence of interest and is extended by polymerase if the 3'
end hybridizes efficiently with the target. The methods are
particularly well suited for detecting and identifying single
nucleotide differences between the target sequence being evaluated
(e.g., a mutant allele of a gene) and a second nucleic acid
sequence (e.g., a wild type allele for the same gene), as they make
use of nucleotide mismatches at or near the 3' end of the detector
primer to discriminate between a first nucleotide and a second
nucleotide which may occur at that site in the target. It has been
found that the reduced efficiency of primer extension by DNA
polymerases when one or more nucleotides at or near the 3' terminus
of a primer do not efficiently hybridize with the target can be
adapted for use as a means for distinguishing or identifying a
nucleotide in the target which is at the site where the one or more
nucleotides of the detector primer hybridize. The efficiency of the
extension reaction for a selected detector primer hybridized to a
selected target is monitored by determining the relative amount of
extended detector primer which is produced in the extension
reaction.
DESCRIPTION OF THE DRAWINGS
[0005] FIGS. 1A, 1B, 1C, 1D and 1E show the results of Example 1
for detection and identification of SNP's using a model target
system and detector primers with a diagnostic 3' terminal
nucleotide and a nondiagnostic mismatch at N-3.
[0006] FIGS. 2A, 2B, 2C, 2D and 2E show the results of Example 2
for detection and identification of SNP's using a model target
system and detector primers with a diagnostic nucleotide at N-1 and
no nondiagnostic mismatch.
[0007] FIGS. 3A, 3B and 3C show the results of Example 3 for
real-time simultaneous detection and identification of two alleles
of exon 4 of the HFE gene.
[0008] FIGS. 4A, 4B and 4C show the results of Example 4 for
real-time simultaneous detection and identification of two alleles
of exon 2 of the HFE gene.
[0009] FIG. 5 illustrates a possible mechanism for generation of
false-positive signals when multiple detector primers in an
amplification reaction have the same 5' tail sequence.
[0010] FIG. 6 shows the results of Example 5, comparing the
performance of multiple detector primers in reactions when the
multiple detector primers have the same or different 5' tail
sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The methods of the invention are useful for detecting
variants of a nucleic acid sequence contained in a target nucleic
acid. In particular, the methods of the invention are directed to
detecting single nucleotide polymorphisms (SNPs) in a nucleic acid
sequence of interest (e.g., alleles) and, optionally, to
identifying such SNPs or alleles. Such nucleotide sequence variants
may be detected directly in a sample to be analyzed during
amplification of the target sequence. The inventive methods are
based upon the relative inefficiency of primer extension by DNA
polymerases when there are mismatches at or near the 3' end of a
primer hybridized to an otherwise complementary sequence.
Applicants have found that by selecting nucleotides at or near the
3' end of a detector primer such that one or more mismatches will
occur when the detector primer is hybridized to a first allele of a
target nucleic acid and correct base pairing will occur when the
detector primer is hybridized to a second allele of the target
nucleic acid, the difference in the efficiency of polymerase
extension when the detector primer is hybridized to the two
different alleles may be used to indicate which allele the target
nucleic acid contains. When any one of multiple alleles may be
present, multiple detector primers are employed in the analysis,
each with a different potential mismatch at or near the 3' end. The
detector primer which is most efficiently extended provides the
identity of the allele (i.e., the identity of the nucleotide
present in the target sequence being analyzed). For example, if a
set of detector primers comprising A, G, C and T at the site of the
allele to be identified is hybridized to the target of interest and
extended, the identity of the allele will be the complement of the
nucleotide in the signal primer which was most efficiently extended
by the polymerase. For identification of the allele in a single
reaction, multiple detector primers are present in the reaction and
each of the detector primers has a separately detectable label
associated with it (e.g., different fluorophores which can be
distinguished within the mixture of detector primers).
[0012] More specifically, the detector primers of the invention are
oligonucleotides which hybridize to the target sequence of interest
and are extended by DNA polymerase during the isothermal
amplification reaction. The nucleotide sequence of the detector
primer is selected such that it hybridizes specifically to the
target nucleic acid of interest and the majority of the detector
primer base-pairs correctly in typical Watson-Crick fashion with
the target. However, the nucleotide sequence of the detector primer
at or near the 3' end is selected to discriminate between
different-SNPs or alleles of the target sequence (the diagnostic
nucleotide position). The diagnostic nucleotide is defined as the
nucleotide in the detector primer which allows analysis (e.g.,
presence or identification) of a particular allele in a selected
target. That is, the sequence of the 3' end of the detector primer
is selected such that hybridization with a first single nucleotide
variation of the target sequence (e.g., a wild-type or mutant
allele of a gene) results in correct Watson-Crick base-pairing at
the site of the SNP and hybridization of the detector primer with a
target containing a second single nucleotide variation of the
target sequence at the same site (e.g., a second mutant allele of
the gene) results in a mismatch between the detector primer and the
target. As an example of how mismatches in the primer allow allele
discrimination in amplification reactions, if a detector primer
having a C residue at the diagnostic nucleotide position produces a
high signal indicative of efficient extension of the detector
primer, this indicates that the target allele is G. In contrast,
low signal for the extended detector primer indicates that the
target allele is not G. Use of a single detector primer to make the
analysis allows identification of an allele if only one SNP is
expected to occur in the target. If there may be multiple different
alleles present at the same nucleotide position, a single detector
primer will provide information on the presence or absence of the
allele for which the detector primer is diagnostic. To identify the
allele when multiple SNPs are possible, multiple detector primers
containing A, T and G at the site of the SNP may be used to
identify the allele in the target, i.e., the detector primer which
produces a high signal associated with detector primer extension
product contains the nucleotide which is the complement of the SNP
in the target. In the present invention, the potentially mismatched
nucleotide of the detector primer is placed at the 3' terminus or
about one to four nucleotide residues from the 3' terminus (i.e.,
at the N, N-1, N-2, N-3 or N-4 position).
[0013] It has also unexpectedly been found that in many cases it is
preferable to place a second mismatch in the sequence of the
detector primer which is not directed to detection or
identification of the allele of interest. The second, nondiagnostic
mismatch often improves the level of discrimination between the
SNPs being detected or identified and is preferably selected based
on a region of the target sequence which is not expected to vary so
that the nondiagnostic mismatch will occur regardless of the target
allele being analyzed. The second mismatch may occur at any site
within the detector primer which produces a positive effect on
allele discrimination, but typically produces the greatest
improvement when it is near the diagnostic nucleotide. This is
typically within one to fifteen nucleotides from the diagnostic
nucleotide, but preferably within about 1-5 nucleotides of the
diagnostic nucleotide of the detector primer. Applicants believe
that the second, nondiagnostic mismatch has a positional effect
rather than a general effect on the T.sub.m of the detector primer,
based on the observation that as the nondiagnostic mismatch is
moved away from the diagnostic mismatch its positive effect on
allele discrimination diminishes. Those skilled in the art are
capable of determining through routine experimentation the
appropriate placement of the nondiagnostic mismatch in a detector
primer by evaluating its effect on allele discrimination using the
detector primer.
[0014] Although it is known that a mismatch in a shorter
oligonucleotide will have a greater effect on hybridization than a
mismatch in a longer oligonucleotide, allele discrimination using
the detector primers of the invention cannot be attributed entirely
to a T.sub.m-associated hybridization effect. For example, moving
the position of the diagnostic nucleotide away from the 3' end of
the detector primer toward the center of the molecule substantially
reduces discrimination. If the sole mechanism of discrimination
between alleles was T.sub.m-associated hybridization efficiency,
this repositioning should increase rather than decrease allele
discrimination. We have observed the opposite, i.e., that the best
allele discrimination occurs when the diagnostic nucleotide is near
the 3' end of the detector primer. In addition, we have observed
that detector primers which contain a diagnostic mismatch which is
not at the 3' terminus and an additional nondiagnostic mismatch as
described below provide good allele discrimination in relatively
longer detector primers where simple differences in hybridization
efficiency between matched and mismatched detector primers are
expected to be minimal. The fact that the nondiagnostic nucleotide
improves discrimination when located near the diagnostic nucleotide
and has little effect when placed greater than fifteen nucleotides
away further suggests that factors in addition to modification of
hybridization efficiency are involved in allele discrimination
according to the invention.
[0015] The detector primers of the invention are typically about
12-50 nucleotides in length. When only a diagnostic nucleotide is
present, the detector primer is preferably about 12-24 nucleotides
long, more preferably about 12-19 nucleotides long. For detector
primers containing both a diagnostic and a nondiagnostic
nucleotide, lengths of about 12-50 nucleotide are preferred, 15-36
nucleotides are more preferred and 18-24 nucleotides are most
preferred.
[0016] The detector primers may be employed in a variety of ways in
the isothermal amplification methods of the invention. In a first
embodiment, the detector primer may be an amplification primer for
use in a nucleic acid amplification reaction. That is, the detector
primer may perform two functions in the amplification
reaction--amplification of the target sequence of interest and
detection or identification of SNPs within the target sequence (a
"detector/amplification primer"). The structure and function of
amplification primers for SDA, 3SR, NASBA, TMA and other isothermal
amplification reactions are well known in the art and it is within
the ordinary skill in the art to adapt these amplification primers
for use as detector primers in the present invention by selecting
the 3' nucleotide sequence as taught herein. For PCR, no special
sequences or structures are required in the amplification primer to
drive the amplification reaction. For this reason, amplification
primers for PCR generally consist only of target binding sequences.
In other amplification reactions, however, the amplification
primers comprise specialized sequences and structures necessary for
the amplification reaction to occur. For example, amplification
primers for 3SR and NASBA comprise an RNA polymerase promoter near
the 5' end. The promoter is appended to the target sequence and
serves to drive the amplification reaction by directing
transcription of multiple RNA copies of the target. Amplification
primers for SDA comprise a recognition site for a restriction
endonuclease near the 5' end. The restriction site is appended to
the target sequence and becomes hemimodified and double-stranded
during the amplification reaction. Nicking of the restriction site
once it becomes double stranded drives the SDA reaction by allowing
synthesis and displacement of multiple copies of the target by
polymerase.
[0017] When the detector/amplification primer forms a mismatch with
the target at or near it's 3' end, amplification efficiency is
reduced and the accompanying reduction in signal upon detection of
the extended detector/amplification primer (i.e., the amplification
product or amplicon) indicates the presence or the identity of an
SNP at the nucleotide position in the target where the diagnostic
mismatch with the detector/amplification primer occurred. If the
detector/amplification primer is tagged with a label which produces
a signal change when the detector/amplification primer has been
extended (as discussed below), the extension products may be
detected in real-time as amplification of the target occurs, thus
eliminating the additional steps of post-amplification detection of
extension products. In isothermal amplification reactions such as
SDA, a single mismatch at N-1 or N-2 in the detector/amplification
primer in general may provide more efficient allele discrimination
than a single mismatch at the 3' terminus. Therefore, a 3' terminal
mismatch is not preferred when the detector primer is an
amplification primer for an isothermal amplification reaction. This
is in contrast to the teaching of the prior art for temperature
cycling amplification reactions, where a 3' terminal mismatch on
the PCR amplification primer reportedly gave adequate allele
discrimination (see Kwok, et al., supra). However, also in contrast
to the teaching of the prior art for PCR, in the isothermal
amplification methods of the present invention a mismatch on the
detector/amplification primer at N-1 to N-4 and a complementary 3'
terminal nucleotide results in excellent allele discrimination,
particularly if the optional second nondiagnostic mismatch is
included. This embodiment is therefore preferred for
detector/amplification primers of the invention.
[0018] In an alternative preferred embodiment, the detector primer
is used in an isothermal amplification reaction as a signal primer
(also referred to as a detector probe) as taught in U.S. Pat. No.
5,547,861, the disclosure of which is hereby incorporated by
reference. In the amplification reaction, the signal primer
hybridizes to the target sequence downstream of an amplification
primer such that extension of the amplification primer displaces
the signal primer and its extension product. After extension, the
signal primer includes the downstream sequence which is the
hybridization site for the second amplification primer. The second
amplification primer hybridizes to the extended signal primer and
primes synthesis of its complementary strand. Production of these
double-stranded secondary amplification products may be detected
not only as an indication of the presence of the target sequence,
but in the methods of the invention a signal primer which has the
sequence characteristics of a detector primer (a detector/signal
primer) also facilitates detection and/or identification of SNP's
within the target sequence. In this embodiment, a diagnostic
mismatch at either the 3' terminus (N) or at N-1 to N-4 provides
excellent allele discrimination. Allele discrimination is further
improved with the use of a second nondiagnostic mismatch as
previously described, particularly when using longer detector
primers where the difference in hybridization efficiency between
matched and mismatched primers is small. This finding was
unexpected, as a 3' terminal diagnostic mismatch alone produced
poor allele discrimination in detector/amplification primers. Use
of a detector/signal primer in an isothermal amplification reaction
also allows detection of extension products and analysis of SNPs in
real-time (i.e., concurrently with amplification) when the
detector/signal primer is labeled with a reporter group which
produces a detectable change in the signal when the detector/signal
primer is extended. Alternatively, the detector/signal primer may
be used post-amplification or without amplifying the target for
detection of SNPs. In this embodiment, the detector signal primer
is hybridized to the target downstream from any primer which is
extendible by polymerase such that extension of the second primer
displaces the detector/signal primer and any detector/signal primer
extension products which may be produced.
[0019] Applicants hypothesize that the different results obtained
with a diagnostic mismatch at the 3' terminus of a detector/signal
primer as compared to a diagnostic mismatch at the 3' terminus of a
detector/amplification primer may be at least partially due to a
kinetic effect. If a signal primer is not efficiently extended on a
target to which it is hybridized (e.g., when it contains
mismatches), it will be quickly displaced from the template by
extension of the upstream amplification primer. If the signal
primer is efficiently extended, extension will occur before the
signal primer is displaced from the target. That is, the upstream
amplification primer (which is typically perfectly matched and
efficiently extended) imposes a "time-limit" for extension on the
detector/signal primer. In contrast, the amplification primer in an
isothermal amplification reaction typically does not have a
time-limit for extension imposed upon it by additional components
of the isothermal amplification reaction or by thermocycling.
Therefore, with sufficient time available, a detector/amplification
primer may eventually be extended even when the extension reaction
is inefficient. This phenomenon could reduce discrimination between
alleles when a detector/amplification primer with a 3' terminal
mismatch is employed in isothermal amplification reactions.
However, a time limit may be imposed prior to amplification if a
second primer binds upstream from the amplification primer, as
described in U.S. Pat. No. 5,270,184 (e.g., the "external" or
"bumper" primer). In this case, extension of the upstream primer
places a time limit on extension of the amplification primer. If
extension of the amplification primer is retarded by a mismatch at
or near the 3' end, the amplification primer may be displaced by
elongation of the upstream primer before it is extended. This
kinetic effect is expected to enhance the ability of amplification
primers to discriminate between matched and mismatched targets
prior to amplification when there is an upstream primer to displace
them. If mispriming occurs, however, the ability of amplification
primers to correct a mismatch with the target may result in an
amplification product which is not a faithful copy of the original
target. Amplification primers produce amplicons that are perfectly
matched with the amplification primers which produced them, thus
eliminating the basis of allele discrimination. In contrast, such
"correction" does not occur with signal primers.
[0020] Whether hybridization of the detector primer results in
correct base-pairing or a mismatch at the diagnostic nucleotide
position of the target being analyzed is determined by evaluating
the relative efficiency of detector primer extension by DNA
polymerase. This determination may be quantitative or qualitative.
Detector primer extension is less efficient in the presence of a
mismatch at or near the 3' end and more efficient when the entire
3' end is correctly base-paired with the target. That is,
relatively more extended detector primer product is produced with
correct base-pairing near the 3' terminus. The extended detector
primer is typically detected by means of a label associated with
the detector primer. The label may be directly detectable or
detectable only after subsequent reaction as is known in the art.
Alternatively, the detector primer itself may be unlabeled and the
extension product detected by hybridization to a labeled probe or
in a subsequent reaction such as treatment with ethidium bromide
for visualization on a gel. The relative amount of signal from the
label which is associated with the extended detector primer as
compared to the amount of signal associated with the unextended
primer serves as an indication of the amount of extension product
produced and the efficiency of the extension reaction.
[0021] There are many techniques known in the art for determining
the presence or amount of extended detector primer product produced
in the amplification reaction. First, the extension products of the
detector primer may be detected and/or quantified by their
increased size, for example by separation from unextended detector
primer by gel elecrophoresis or by selectively capturing the
extended detector primer on a solid phase. However, in a preferred
embodiment the detector primers are labeled with a reporter group
which is detectable only when the detector primer has been extended
or a label which produces a change in signal only when the detector
primer has been extended. One example of such labels are
fluorescent dyes which undergo changes in fluorescence polarization
when the oligonucleotides to which they are linked have been
hybridized to and extended on a target sequence. Methods employing
changes in fluorescence polarization to detect hybridization and
extension of a signal primer are described in U.S. Pat. No.
5,800,989; U.S. Pat. No. 5,593,867 and; U.S. Pat. No. 5,641,633.
These patents describe using changes in fluorescence polarization
which occur when the signal primer becomes double-stranded (made
possible by its successful extension on the target sequence) to
detect target amplification. In the methods of the invention,
changes in fluorescence polarization of a fluorescently-labeled
detector primer may be used to evaluate extension efficiency and to
detect or identify an SNP in the target being amplified.
[0022] A second example of labels which undergo a detectable change
in signal indicative of primer extension are fluorescent
donor/quencher dye pairs. The quencher dye may also be fluorescent
but need not be. When the donor and quencher are in close
proximity, fluorescence of the donor is quenched. As the dyes are
moved farther apart, quenching is reduced and donor fluorescence
increases. The use of such donor/quencher dye pairs in a variety of
mechanisms for increasing the distance between the dyes in the
presence of target for detection of target nucleic acids is
described in U.S. Pat. No. 5,846,726; U.S. Pat. No. 5,691,145, and;
EP 0 881 302. Both the use of donor/quencher dye pairs in signal
primer amplification systems and in extendible primer/probes for
detection of unamplified or post-amplification targets are
disclosed. In the present invention, the detector primers of the
invention may be labeled with donor/quencher dye pairs and employed
for detection and/or identification of SNP's in the target as is
known in the art.
[0023] As disclosed in the foregoing references, a variety of
primer extension detection systems are known for use in essentially
any nucleic acid amplification reaction. They are particularly
well-suited to isothermal amplification reactions where they
provide rapid, real-time detection of primer extension. In the
methods of the present invention, detector primers may be labeled
with structures containing fluorescent reporter groups as taught in
the foregoing references and the extension product detected by
changes in fluorescence polarization or fluorescence quenching.
Alternatively the detector primer may be unlabeled and its
extension product detected by hybridization to a labeled
primer/probe with detection of changes in fluorescence polarization
or fluorescence quenching of the primer/probe as taught in these
references.
EXAMPLE 1
[0024] To demonstrate identification of a single nucleotide
polymorphism using labeled detector primers model target
oligonucleotides differing by only a single nucleotide were
prepared as follows: Four oligonucleotides containing identical
sequences except at one position were synthesized. The variant
position of the oligonucleotide contained either adenosine (A),
cytosine (C), guanine (G) or thymine (T). A fifth oligonucleotide
complementary to the 3' termini of the four variant
oligonucleotides was also synthesized. Each of the four variant
oligonucleotides was mixed with the fifth oligonucleotide, heated
for 2 min. in a boiling water bath and equilibrated to 37.degree.
C. in a dry incubator. The annealed variant oligonucleotide and the
fifth oligonucleotide were then extended in a primer extension
reaction comprising 14 mM deoxycytidine
.alpha.-(O-1-thio)-triphosphate, 2 mM deoxyadenosine triphosphate,
2 mM deoxyguanosine triphosphate, 2 mM thymidine triphosphate and
40 units of exonuclease deficient Klenow DNA polymerase. The primer
extension reactions were allowed to proceed for 45 min. at
37.degree. C., following which the Klenow polymerase was
inactivated by incubating the reactions at 70.degree. C. for 10
min. in a dry incubator. This produced four double-stranded DNA
model target sequences differing only at one nucleotide position.
The targets were designated A, C, G and T targets.
[0025] A second set of four oligonucleotides which hybridize to the
model target sequences with their 3' termini at the polymorphic
nucleotide position were also synthesized for use as detector
primers. Each of the four detector primers had one of the four
nucleotide bases (A, C, G or T) at its 3' terminus (N, the
diagnostic nucleotide) and an "A" nucleotide at the position three
bases from the 3' terminus which formed a mismatch with the model
target sequence (N-3, the nondiagnostic nucleotide). The four
detector primers were radiolabeled in 25 .mu.l reactions containing
1 .mu.M detector primer, 25 units of T4 polynucleotide kinase
(PNK), 175 .mu.Ci of .alpha.-[.sup.32P]-adenosine triphosphate
(.sup.32P-ATP), and PNK buffer at 1 .times. concentration. Labeling
reactions were initiated by addition of PNK to a solution
containing the other components. The reactions were incubated for
20 min. at 37.degree. C., than heated in a boiling water bath for 5
min. to inactivate the PNK.
[0026] The detector primers were used as signal primers in the
amplification reaction. A 5 .mu.l aliquot of each of the labeled
detector primer preparation was added to a separate SDA reaction
for each target (50 .mu.l comprising 40 mM
KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 pH 7.6; 10% v/v glycerol; 7.5 mM
magnesium acetate; 0.5 .mu.M each amplification primer; 1.4 mM
deoxycytidine .alpha.-(O-1-thio)-triphosphate; 0.5 mM
deoxyadenosine triphosphate; 0.5 mM deoxyguanosine triphosphate;
0.5 mM thymidine triphosphate; 0.1 mg/ml bovine serum albumin; 0.5
.mu.g human placental DNA, 104 A, C, G or T target DNA molecules;
100 nM radiolabeled detector primer; 160 units BsoBi restriction
endonuclease and 25 units Bst DNA polymerase large fragment). The
SDA reactions were assembled without the BsoBI and Bst, and the
target DNA duplexes were denatured by heating these reaction mixes
for 3 min. in a boiling water bath. The reaction mixtures were
equilibrated to 55.degree. C. for 3 min. in a dry incubator and SDA
was initiated by adding 160 units of BsoBI and 25 units of Bst
polymerase large fragment to each reaction (total volume of enzymes
was 2 .mu.l, adjusted with 50% v/v glycerol). SDA was allowed to
proceed for 30 min. at 55.degree. C. Aliquots of each reaction (5
.mu.l) were removed at 5 min. intervals during the reaction and
added to 5 .mu.l of sequencing stop solution to quench the SDA
reaction. When all samples had been collected, they were incubated
in a boiling water bath for 3 min. and 5 .mu.l of each sample was
loaded onto an 8% polyacrylamide, 7 M urea sequencing gel.
Following electrophoresis at 65 W for 50 min., the radiolabeled
reaction products and unreacted probe on the gel were quantified by
exposing a Molecular Dynamics Phosphorimager.TM. plate to the gel
for 15 min. and reading the number of counts present in the
radiolabeled product and probe bands on the Phosphorimager.TM.
plate using ImageQuant.TM. software.
[0027] FIGS. 1A, 1B, 1C and 1D show the results obtained for
extension of each of the four detector primers on each of the four
different model target sequences. In each case, the detector primer
with the 3' nucleotide that was the correct match for the
polymorphic nucleotide in the target DNA sequence was
preferentially extended during SDA by the DNA polymerase as
compared to the detector primers which did not contain the correct
3' match for the polymorphic nucleotide in the target, as evidenced
by greater amounts of the correct detector primer after
amplification. For example, the 3'A detector primer was
preferentially extended only in the SDA reactions containing the T
target sequence (FIG. 1A). There was essentially no extension of
the 3'A detector primer on the C, G or A target sequences.
Extension products produced during SDA included full-length
extended radiolabeled DNA probes and the nicked extended amplicons
characteristic of SDA. Similarly, the T target supported little or
no extension of the 3C, 3G and 3T detector primers (FIGS. 1B, 1C
and 1D).
[0028] Similar results were seen using each of the other detector
primers in target amplification reactions (3C, 3G and 3T detector
primers shown in FIGS. 1B, 1C and 1D, respectively), i.e., the
detector primer was preferentially extended on the target which
produced the correct 3' match at the polymorphic position in the
target. In contrast, when an SDA amplification primer with a C at
the 3' terminus was used as a detector/amplification primer in the
amplification reactions the allele could not be identified (FIG.
1E). These results illustrate that N/N-3 detector/signal primers
according to the invention can be used in isothermal amplification
reactions to identify the nucleotide present at a selected position
in a target nucleic acid sequence, whereas N/N-3
detector/amplification primers for isothermal amplification
reactions do not effectively discriminate between alleles.
EXAMPLE 2
[0029] Example 1 was repeated except that the detector/signal
primers were synthesized so that the variant, diagnostic nucleotide
was positioned one nucleotide from the 3' terminus of the detector
primer (N-1) and the overall length of the detector primer was
shortened by 4 nucleotides at the 5' end. The detector primers also
made a perfect match with the target DNA at the position three
nucleotides from the 3' terminus of the detector primer. Each of
the four detector primers was added to separate SDA reactions
containing 10.sup.4 molecules of each of the target sequences. The
targets were amplified and detected as previously described. FIGS.
2A (-1A detector primer), 2B (-1C detector primer), 2C (-1G
detector primer) and 2D (-1T detector primer) show the results of
the experiments.
[0030] In every case, during amplification the detector primer was
preferentially extended on the target which contained the perfect
match at the variant position. Signals obtained with the perfectly
matched detector primer and target were 30- to 100-fold higher than
signals obtained with any of the mismatched detector primer/target
pairs. This difference in signal allowed unambiguous identification
of the polymorphic nucleotide in the target and is in contrast to
the results reported for PCR by Kwok, et al., supra, where an N-1
mismatch had no effect on the yield of PCR amplicons.
[0031] Similar results were obtained using a detector/signal primer
having an N-1 diagnostic nucleotide (G) and a second nondiagnostic
mismatch with the targets at N-2 (A). This detector primer was five
nucleotides longer at the 5' end than the detector primers used in
Example 1. The detector primer was added to each of four separate
SDA reactions prepared as in Example 1, and was found to be
preferentially extended on the target which contained the correct
match for the diagnostic nucleotide (C). FIG. 2E shows that the
signal obtained with the singly mismatched target was over
five-fold higher than that obtained with any of the doubly
mismatched targets and that the C allele could be easily
distinguished from the T, G and A alleles of the target.
EXAMPLE 3
[0032] In the following experiments, a single nucleotide
polymorphism in exon 4 of the HFE gene (the gene responsible for
hemochromatosis) and the wild-type allele were detected and
identified simultaneously in real-time during amplification using
the detector primers of the invention. The wild-type allele is a G
at nucleotide 845, whereas the mutant allele is an A at this
position. This results in a cysteine to tyrosine change at amino
acid position 282 in the protein.
[0033] SDA was generally performed as described in U.S. Pat. No.
5,846,726, except that each reaction mixture contained two
detector/signal primers according to the invention (one specific
for the mutant allele and one specific for the wild-type allele)
and BsoBI was substituted for AvaI. The final concentrations of
components in each 100 .mu.L reaction were 50 mM KiPO.sub.4 (pH
7.5), 6.0 mM MgOAc, 0.2 mM each DTTP, dGTP, dATP, 1.4 mM
dCTP.alpha.S, 5 .mu.g/mL acetylated BSA, 15% (v/v) glycerol, 400 ng
salmon sperm DNA, 20 units exo Klenow Bst polymerase, 160 units
BsoBI and either 0 or 10.sup.5 copies of target DNA. In this
example, target DNA consisted of PCR products generated from DNA
cloned from either normal or mutant HFE exon 4 DNA. Normal HFE exon
4 DNA contains a G at nucleotide position 845 of the HFE wild-type
gene and the mutant HFE exon 4 DNA contained an SNP at that
position in which the nucleotide was A. Each sample also contained
two detector/signal primers (SEQ ID NO:1 and SEQ ID NO:2 below),
two unlabeled bumper primers (SEQ ID NO:3 and SEQ ID NO:4 below)
and two unlabeled SDA amplification primers (SEQ ID NO:5 and SEQ ID
NO:6 below). Underlined sequences indicate complementarity to the
target sequence. The base A at position N-3 which is not underlined
is not complementary to the corresponding nucleotide in the target
sequence. This internal mismatch improves the selectivity of this
detector primer for the nucleotide position 845. C* represents the
3' terminal nucleotide (position N of the detector primer) which
pairs with the G at position 845 of the wild-type target. T*
represents the 3' terminal nucleotide which pairs with A at
nucleotide position 845 of the mutant target (the G845A mutation).
Italicized sequences represent restriction enzyme recognition sites
(RERS). In the amplification primers the RERS provides the nicking
site which drives SDA. In the detector primers the RERS is flanked
by a two dyes which form a donor/quencher dye pair. As the
detector/signal primer is extended, displaced and rendered
double-stranded the RERS also becomes double-stranded and cleavable
by the restriction enzyme. To detect the amount of double-stranded
extension product the reaction products are treated with the
appropriate restriction enzyme to cleave the RERS of the detector
primer. Quenching of the fluorescent dye decreases as the
double-stranded products are cleaved and the dye pair is separated.
The increase in fluorescence is an indicator of the amount of
extended, double-stranded detector primer produced. If the detector
primer is not efficiently extended the RERS remains
single-stranded, is not cleaved by the restriction enzyme and the
fluorescent dye remains quenched. Failure to detect an increase in
fluorescence therefore indicates that the detector primer was not
efficiently extended on the target.
[0034] SEQ ID NO:1--Detector primer specific for nucleotide G at
position 845 (wild-type):
[0035] FAM-TC CTCGAGT(dabcyl)AT GGG TGC TCC ACC AGG C* (300 nM)
[0036] SEQ ID NO:2--Detector primer specific for nucleotide A at
position 845 (mutant):
[0037] Rox-TT CTC GAGT(dabcyl)TA CAT GGG TGC TCC ACC AGG T* (300
nM)
[0038] SEQ ID NO:3--first bumper primer for exon 4:
[0039] CGA ACC TAA AGA CGT ATT CGG C (50 nM)
[0040] SEQ ID NO:4--second bumper primer for exon 4:
[0041] CCC CAA TAG ATT TTC TCA GCT CC (50 nM)
[0042] SEQ ID NO:5--first SDA amplification primer for exon 4:
[0043] ACC GCA TCG ATT GCA TGT CTC GGG CTG GAT ACC CTT GGC T
[0044] SEQ ID NO:6--second SDA amplification primer for exon 4:
[0045] CGA TTC CGC TCC AGA CTT CTC GGG AGA TCA CAA TGA GGG GCT
GA
[0046] Each SDA reaction included both detector primers, each
labeled with a different fluorophore as shown above. The reactions
were assembled in microwells to contain all reagents except Bst and
BsoBI and amplification was initiated after heat denaturation and
equilibration to 55.degree. C. by addition of the enzymes. The
microwells were sealed and placed into a Cytofluor II.TM. which had
been modified to permit temperature control. Bandpass filters were
used to limit excitation to one wavelength range characteristic of
fluorescein (475-495 nm) and a second range specific for ROX
(635-655 nm). For each well, one fluorescein and one ROX reading
were made every 45 seconds. Reactions were typically monitored for
90 min. Control reactions contained no target DNA.
[0047] The results are shown in FIGS. 3A, 3B and 3C. In samples
containing only targets derived from normal exon 4 DNA (FIG. 3A),
fluorescence increased strongly with time in the emission
wavelength range characteristic of fluorescein (FAM, 520-540 nm),
indicating that the SEQ ID NO:1 detector primer was efficiently
extended on this target and identifying the presence of the
wild-type allele. In contrast, in the normal exon 4 samples
emission fluorescence characteristic of ROX (635-655 nm) remained
low, indicating that the SEQ ID NO:2 detector primer was not
efficiently extended on the target during amplification and the
confirming the absence of the mutant allele. In contrast, the
fluorescence profile was reversed for samples containing DNA
derived from mutant exon 4 DNA and lacking normal DNA (FIG. 3B). In
these samples, fluorescence increased strongly at the emission
wavelengths of ROX but not at FAM wavelengths, indicating the
presence of the mutant allele and the absence of the wild-type
allele. In the sample containing both wild-type and mutant DNA,
fluorescence increased in both monitored ranges, indicating the
presence of both alleles in the sample (FIG. 3C).
[0048] In a similar experiment, an alternative detector primer
specific for the wild type allele was tested and its performance
compared to the SEQ ID NO:1 detector primer. The alternative
detector primer had a diagnostic nucleotide at N-2 and a second
nondiagnostic nucleotide at N-3 (an N-2/N-3 detector primer; FAM-TC
CTC GAG T(dabcyl)AT GGG TGC TCC ACC TGA C*AC; SEQ ID NO:14). In
addition, amplification primer SEQ ID NO:5 was replaced with SEQ ID
NO:15 (ACG CAG CAG CAC ACA TTC TCG GGG MG AGC AGA GAT ATA CGT) Two
samples were tested using SEQ ID NO:14--one containing only wild
type target and the other containing only mutant target. Each test
reaction contained SEQ ID NO:14 for detection of the wild type
allele and SEQ ID NO:2 for detection of the mutant target. The SEQ
ID NO:1/SEQ ID NO:2 detector primer system served as a control
reaction. Both fluorescein and rhodamine fluorescense were
monitored and the fluorescence readings for each sample were
plotted. In the sample containing the wild-type target, the N-2/N-3
detector primer was converted, resulting in a three-fold increase
in fluorescein emission. The mutant-specific detector primer
remained unconverted and the rhodamine emission was essentially
unchanged. In the sample containing only mutant target, the pattern
was reversed. The N-2/N-3 detector primer was not converted, as
indicated by no change in fluorescein emission, but rhodamine
emission from the mutant-specific detector primer increased about
three-fold. Comparison of the results to the control reaction
demonstrated that the target specificity for SEQ ID NO:14 is
approximately equivalent to the target specificity for SEQ ID
NO:1.
[0049] It has also been found that a wild-type specific detector
primer having the sequence FAM-TA GCA GTC CCG AGA CTG CT(dabcyl)A
TGG GTG CTC CAC CAG GC* (SEQ ID NO:16) provides more sensitive
detection of the wild-type allele than SEQ ID NO:1, although it is
slightly less specific.
EXAMPLE 4
[0050] The experimental protocol of Example 3 was repeated except
that a pair of amplification primers specific for exon 2 of the HFE
gene and detector/signal primers for detection and identification
of wild-type and mutant alleles in exon 2 were used. The wild-type
allele is C at nucleotide 187. The mutant allele has a G in this
position, resulting in a histidine to lysine change at amino acid
63 in the protein. These detector primers were designed to
hybridize to the allele contained in the non-coding strand of exon
2.
[0051] SEQ ID NO:7--Detector primer for wild-type allele at
nucleotide position 187 (C187, i.e., G on the complementary
strand):
[0052] FAM-TC CTC GAGT(dabcyl)TA CCA GCT GTT CGT GTT CTA TGA TC*
(300 nM)
[0053] SEQ ID NO:8--Detector primer for mutant allele at nucleotide
position 187 (G187, i.e., C on the complementary strand):
[0054] Rox-TA CCG CAC T(dabcyl)GA TTA CCA GCT GTT CGT GTT CTA TM
TG* (300 nM)
[0055] SEQ ID NO:9--First bumper primer for exon 2:
[0056] TGA ACA TGT GAT CCC ACC CT (50 nM)
[0057] SEQ ID NO:10--Second bumper primer for exon 2:
[0058] CCC CAA TAG ATT TTC TCA GCT CC (50 nM)
[0059] SEQ ID NO:11--First amplification primer for SDA:
[0060] ACC GCA TCG MT GCA TGT CTC GGG AGC TTT GGG CTA CGT GGA
TG
[0061] SEQ ID NO:12--Second amplification primer for SDA:
[0062] CGA TTC CGC TCC AGA CTT CTC GGG GCT CCA CAC GGC GAC TCT
[0063] SEQ ID NO:7 did not contain a nondiagnostic mismatch with
the target. SEQ ID NO:8 did not contain an RERS, as the ROX/Dabcyl
dye pair dequenches upon formation of the extended, double-stranded
detector primer product. Cleavage with a restriction enzyme is not
necessary to observe the increase in fluorescence.
[0064] The results are shown in FIGS. 4A, 4B and 4C for SDA
reactions containing 10.sup.7 copies of target DNA derived from
either wild-type or mutant exon 2. In samples containing target
derived from normal exon 2 DNA only (FIG. 4A), fluorescence
increased strongly with time in the emission wavelength range for
fluorescein, indicating the presence of the wild-type allele. In
addition, fluorescence in the emission wavelength range for
rhodamine remained low for these samples, indicating the absence of
the mutant allele. In contrast, the fluorescence profile was
reversed for samples containing DNA derived only from mutant exon 2
(FIG. 4B). In this case, ROX fluorescence increased strongly and
FAM fluorescence remained low. In samples containing both wild-type
and mutant DNA (FIG. 4C), fluorescence from both fluorophores
increased strongly, indicating the presence of both alleles in a
single sample.
[0065] In addition, it has been found that substituting an
amplification primer having the sequence CGA TAC GCT CCT GAC TTC
TCG GGA CM ACG GCG ACT CTC AT (SEQ ID NO:17) for SEQ ID NO:12 in
the reaction provides more efficient amplification of the exon 2
target. Further, an N-1 detector primer having the sequence Rox-TA
GCG CCC GAG CGC T(dabcyl)AT GTT CGT GTT CTA TGA TC*A (SEQ ID NO:18)
provides improved allele discrimination when used in combination
with the alternative amplification primer SEQ ID NO:17.
EXAMPLE 5
[0066] This example illustrates the use of 5' tail sequences in
detector primers to modulate cross-reactivity of multiple
allele-specific probes in nucleic acid amplification reactions.
Although this example employed SDA, similar results are expected
for other amplification methods known in the art, including PCR,
NASBA, 3SR, etc., which involve extension of a labeled probe or
primer to discriminate between two alleles.
[0067] Examples 3 and 4 describe SDA reactions which contain two
differentially labeled detector/signal primers, one specific for
the wild-type allele and the other specific for a mutant allele.
Thus, each reaction mixture is capable of detecting either the
mutant allele, the wild-type allele or both alleles simultaneously.
The ability to analyze one sample for either allele is more
convenient and reliable, requires less sample and is less expensive
than the alternative approach of splitting the sample sample and
performing two separate single-detector primer assays. However, it
has been observed that in reaction mixtures containing two or more
differentially-labeled detector primers an increased level of
spurious, cross-reactive signal may be generated by one detector
primer when the target of a second detector primer is present.
Applicants believe that this cross-reactivity is caused or
exacerbated by the simultaneous presence of both detector primers
in the same reaction mixture, as cross-reactivity is diminished or
absent in single-detector primer reaction mixtures. It has been
discovered that the cross-reactivity can be substantially
diminished in such multiple detector primer reaction mixtures by
designing detector primers so that the 5' tail sequences of the two
detector primers are substantially different. This result was
unexpected, as the 5' tails are not complementary to the original
target sequences and the allele-specific nucleotides are located
away from the 5' tails at or near the 3' ends of the detector
primers.
[0068] FIG. 5 illustrates the possible source of this unexpected
cross-reactivity, using SDA as an illustrative example. The
"wild-type" target depicted contains a C at the nucleotide position
to be diagnosed. The detector primer for this allele therefore
contains a 3' terminal G, as shown. For purposes of this
illustration, the "mutant" allele (not shown) contains a T at the
diagnostic position, and its detector primer contains a 3' terminal
A. Both detector primers are present in the amplification reaction.
During amplification, the C-specific detector primer hybridizes to
the target and is extended and converted to the double-stranded
form which is cleavable to separate the donor/quencher dye pair.
The resulting increase in fluorescence indicates the presence of
the C-containing target. The remaining double-stranded species then
undergoes linear amplification, as this species contains a nickable
restriction site. Linear amplification produces a single-stranded
species containing a C at the diagnostic position. Two alternative
reaction pathways are then possible. In one case, the linear
amplification product may hybridize to the appropriate C-specific
detector primer and be converted to cleaved product as before,
further enhancing the C-specific signal. Alternatively, the linear
amplification reaction product may hybridize spuriously to the
T-specific detector primer. Tthe single mismatch at the 3' end of
the T-specific detector primer would not be sufficient to prevent
such errant hybridization. However, if the 5' tail sequences of the
T-specific and C-specific detector primers are identical
hybridization could occur and the spuriously-hybridized T-specific
detector primer would be converted quickly into a cleaved
fluorescent product without the need for extension of the A:C
mismatch. This results in a signal falsely indicative of the
presence of a T nucleotide at the diagnostic position. If, however,
the T-specific and C-specific detector primers contain different 5'
tail sequences, spuriously hybridized T-specific detector primer
will not undergo cleavage, as the 5' tail cannot be converted to
double-stranded form by hybridization to the linear amplification
product. The detector primer would then remain uncleaved even if
errantly hybridized to wild-type target and no false-positive
signal would be produced. In the illustrated example, the fact that
the two detector primers have the same restriction site would not
be sufficient to allow the tails to hybridize provided the rest of
the 5' tail sequence was different.
[0069] Although FIG. 5 illustrates a mechanism for spurious
generation of false-positive signals in SDA, similar reactions will
occur in other amplification methods. Each time a single-stranded
C-containing detector primer extension product is generated in PCR,
NASBA, 3SR, TMA or any other amplification reaction, it may
hybridize to either the C-specific detector primer (correctly) or
to the T-specific detector primer (incorrectly). If the two
detector primers have identical 5' tail sequences the extension
product will be complementary to either one at its 5' end and the
RERS will be double-stranded and cleavable. If the two detector
primers have different 5' tail sequences the double-stranded RERS
will not be generated when the detector primer hybridizes to the
incorrect target and no false-positive signal will be
generated.
[0070] To illustrate this phenomenon, four SDA reactions were
assembled. All reactions contained the mutant-specific detector
primer SEQ ID NO:2 (300 nM). Reactions 1 and 2 contained wild
type-specific SEQ ID NO:13 (FAM-TT CTC GAG T(dabcyl)TA CAT GGG TGC
TCC ACC AGG C* (300 nM), which has a 5' tail sequence identical to
SEQ ID NO:2. Reactions 3 and 4 contained the fluorescein-labeled
wild type-specific detector primer SEQ ID NO:1 (300 nM) in which
the 5' tail sequence differs from SEQ ID NO:2 except for the RERS
(CTCGAG). The reaction mixtures also contained either wild-type
(reactions 1 and 3) or mutant (reactions 2 and 4) target DNA
(10.sup.5 copies per reaction) derived from exon 4 of the HFE gene
(see Example 3). The reactions were carried out as in Example 3
except that only fluorescein fluorescence emissions were
detected.
[0071] The results are shown in FIG. 6. Reaction 1 produced a
strong increase in fluorescein fluorescence, indicative of the
presence of the wild-type allele in the target. Reaction 2, which
contained mutant DNA only, produced a diminished but substantial
fluorescence increase even though no wild-type DNA was present.
This signal represented spurious conversion of the wild-type
"specific" detector primer, possibly through the mechanism
illustrated in FIG. 5, as the 5' tail sequences of the two detector
primers present in the reaction were identical. When the 5' tail
sequence of the wild-type detector primer was changed (SEQ ID
NO:1), the cross-reacting signal was suppressed (reaction 4)
without substantially affecting the target-specific signal
(reaction 3).
Sequence CWU 1
1
18 1 27 DNA Homo sapiens 1 tcctcgagta tgggtgctcc accaggc 27 2 30
DNA Homo sapiens 2 ttctcgagtt acatgggtgc tccaccaggt 30 3 22 DNA
Homo sapiens 3 cgaacctaaa gacgtattcg gc 22 4 23 DNA Homo sapiens 4
ccccaataga ttttctcagc tcc 23 5 40 DNA Homo sapiens 5 accgcatcga
ttgcatgtct cgggctggat acccttggct 40 6 44 DNA Homo sapiens 6
cgattccgct ccagacttct cgggagatca caatgagggg ctga 44 7 34 DNA Homo
sapiens 7 tcctcgagtt accagctgtt cgtgttctat gatc 34 8 37 DNA Homo
sapiens 8 taccgcactg attaccagct gttcgtgttc tataatg 37 9 20 DNA Homo
sapiens 9 tgaacatgtg atcccaccct 20 10 23 DNA Homo sapiens 10
ccccaataga ttttctcagc tcc 23 11 44 DNA Homo sapiens 11 accgcatcga
atgcatgtct cgggagcttt gggctacgtg gatg 44 12 42 DNA Homo sapiens 12
cgattccgct ccagacttct cggggctcca cacggcgact ct 42 13 30 DNA Homo
sapiens 13 ttctcgagtt acatgggtgc tccaccaggc 30 14 29 DNA Homo
sapiens 14 tcctcgagta tgggtgctcc acctgacac 29 15 42 DNA Homo
sapiens 15 acgcagcagc acacattctc ggggaagagc agagatatac gt 42 16 37
DNA Homo sapiens 16 tagcagtccc gagactgcta tgggtgctcc accaggc 37 17
41 DNA Homo sapiens 17 cgatacgctc ctgacttctc gggacaaacg gcgactctca
t 41 18 35 DNA Homo sapiens 18 tagcgcccga gcgctatgtt cgtgttctat
gatca 35
* * * * *