U.S. patent application number 11/087920 was filed with the patent office on 2006-02-23 for oligonucleotide sizing using cleavable primers.
This patent application is currently assigned to Sequenom, Inc.. Invention is credited to Christopher Hank Becker, Joseph Albert Monforte, Daniel Joseph Pollart, Thomas Andrew Shaler.
Application Number | 20060040282 11/087920 |
Document ID | / |
Family ID | 27034396 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060040282 |
Kind Code |
A1 |
Monforte; Joseph Albert ; et
al. |
February 23, 2006 |
Oligonucleotide sizing using cleavable primers
Abstract
The present invention provides modified oligonucleotide primers
designed to incorporate a cleavable moiety so that a 3' portion of
the primer (linked to an extension product) can be released from an
upstream 5' portion of the primer. Upon selective cleavage of the
cleavable site, primer extension products that contain about five
or fewer base pairs of the primer sequence are released, to provide
more useful sizing and sequence information per fragment than
extension products containing the entire primer.
Inventors: |
Monforte; Joseph Albert;
(Kensington, CA) ; Becker; Christopher Hank;
(Mountain View, CA) ; Shaler; Thomas Andrew;
(Menlo Park, CA) ; Pollart; Daniel Joseph;
(Alameda, CA) |
Correspondence
Address: |
BIOTECHNOLOGY LAW GROUP
c/o PORTFOLIO IP
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Sequenom, Inc.
San Diego
CA
|
Family ID: |
27034396 |
Appl. No.: |
11/087920 |
Filed: |
March 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09139386 |
Aug 25, 1998 |
6949633 |
|
|
11087920 |
Mar 22, 2005 |
|
|
|
08639363 |
Apr 26, 1996 |
5830655 |
|
|
09139386 |
Aug 25, 1998 |
|
|
|
08445751 |
May 22, 1995 |
5700642 |
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08639363 |
Apr 26, 1996 |
|
|
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 1/6853 20130101;
C12Q 1/6816 20130101; Y10T 436/255 20150115; C12Q 1/6872 20130101;
C12Q 1/6858 20130101; Y02A 50/30 20180101; C12Q 1/6869 20130101;
Y02A 50/54 20180101; C12Q 1/6853 20130101; C12Q 2563/167 20130101;
C12Q 2525/131 20130101; C12Q 1/6869 20130101; C12Q 2563/167
20130101; C12Q 2533/101 20130101; C12Q 2525/131 20130101; C12Q
1/6872 20130101; C12Q 2533/101 20130101; C12Q 2525/131 20130101;
C12Q 1/6816 20130101; C12Q 2565/518 20130101; C12Q 2563/167
20130101; C12Q 2521/301 20130101; C12Q 1/6872 20130101; C12Q
2565/518 20130101; C12Q 2563/167 20130101; C12Q 2521/301 20130101;
C12Q 1/6858 20130101; C12Q 2533/101 20130101; C12Q 2565/627
20130101; C12Q 2525/131 20130101; C12Q 1/6858 20130101; C12Q
2533/101 20130101; C12Q 2565/627 20130101; C12Q 2523/319 20130101;
C12Q 1/6872 20130101; C12Q 2533/101 20130101; C12Q 2523/319
20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for determining presence of a polymorphism, comprising:
(a) hybridizing a primer, having a 5' end and a 3' end, with a
target nucleic acid suspected of containing a polymorphism, wherein
said primer has a first region containing the 5' end of the primer
and a second region containing the 3' end of the primer and a
cleavable site; (b) extending the 3' end of the primer with a
polymerase in the presence of a nucleotide to generate an extension
product; (c) cleaving said extension product at the cleavable site
to release an extension segment; sizing the extension segment by
mass spectrometry, whereby said cleaving is effective to increase
the read length of the extension segment relative to the read
length of the product of step (b); and identifying any added
nucleotides.
2. The method of claim 1, wherein the first region of the primer
further comprises an immobilization attachment site and the
extension product is immobilized onto a solid support at the
immobilization attachment site prior to the cleaving step.
3. The method of claim 2, further comprising washing the extension
product after said immobilizing and prior to said cleaving.
4. The method of claim 1, wherein the nucleotide is selected from
the group consisting of a deoxynucleotide, a chain-terminating
nucleotide and a derivative thereof.
5. The method of claim 3, wherein the nucleotide is a
chain-terminating nucleotide.
6. The method of claim 5, wherein the chain-terminating nucleotide
is a dideoxynucleotide.
7. The method of claim 6, wherein the dideoxynucleotide is selected
from the group consisting of ddATP, ddTTP, ddUTP, ddGTP, ddITP and
ddCTP.
8. The method of claim 5, wherein the chain-terminating nucleotide
is mass modified.
9. The method of claim 1, wherein the target nucleic acid is
DNA.
10. The method of claim 1, wherein the target nucleic acid is
RNA.
11. The method of claim 1, wherein the target nucleic acid is
immobilized.
12. The method of claim 11, wherein the target nucleic acid is
immobilized prior to said extending.
13. The method of claim 11, wherein the target nucleic acid is
immobilized after said extending.
14. The method of claim 1, wherein the cleavable site is a blocking
nucleotide capable of blocking 5' to 3' enzyme-promoted digestion,
and where said cleaving is carried out by digesting the first
region of the primer with an enzyme having a 5' to 3' exonuclease
activity.
15. The method of claim 14, wherein the enzyme is selected from the
group consisting of T7 gene 6 exonuclease and
phosphodiesterase.
16. The method of claim 14, wherein the blocking nucleotide is
selected from the group consisting of phosphorothioate, methyl
phosphonate, phosphotriester, and peptide nucleic acid.
17. The method of claim 1, where the cleavable site is selected
from the group consisting of dialkoxysilane,
3'-(S)-phosphorothioate, 5'-(S)-phosphorothioate,
3'-(N)-hosphoramidate, 5'-(N)phosphoramidate, uracil, and
ribose.
18. The method of claim 1, wherein said sizing is by time-of-flight
mass spectrometry.
19. The method of claim 1, wherein a plurality of primers are
hybridized to more than one target nucleic acid and the location of
the cleavable site contained within the primers is varied to
increase the mass difference between their respective extension
segments.
Description
[0001] The present application is a divisional of U.S. application
Ser. No. 09/139,386 filed Aug. 25, 1998, allowed, which is a
continuation-in-part of U.S. patent application Ser. No. 08/639,363
filed Apr. 26, 1996, which has since issued as U.S. Pat. No.
5,830,655, which is a continuation-in-part of U.S. patent
application Ser. No. 08/445,751 filed May 22, 1995 which has since
issued as U.S. Pat. No. 5,700,642. The entire text of each of the
above-referenced disclosures is specifically incorporated by
reference herein without disclaimer.
1.0 BACKGROUND OF THE INVENTION
[0002] 1.1 Filed of the Invention
[0003] The present invention relates to oligonucleotide
compositions containing cleavable primers and diagnostic and
analytical methods employing such primers.
[0004] 1.2 Description of Related Art
[0005] DNA, the primary genetic material, is a complex molecule
consisting of two intertwined polynucleotide chains, each
nucleotide containing a deoxyribose unit, a phosphate group and a
nitrogenous heterocyclic base. The two polynucleotide strands are
held together via hydrogen bonding interactions between
complementary base pairs. A Normal human being possesses 23 pairs
of chromosomes containing a total of about 100,000 genes. The
length of DNA contained within the human chromosomes totals about
3.3 billion base pairs, with a typical gene containing about 30,000
base pairs.
[0006] Approximately 4,000 human disorders are attributed to
genetic causes. Hundreds of genes responsible for various disorders
have been mapped, and sequence information is being accumulated
rapidly. A principal goal of the Human Genome Project is to find
all genes associate with each disorder. The definitive diagnostic
test for any specific genetic disease (or predisposition to
disease) will be the identification of polymorphic variations in
the DNA sequence of affected cells that result in alterations of
gene function. Furthermore, response to specific medications may
depend on the presence of polymorphisms. Developing DNA (or RNA)
screening as a practical tool for medical diagnostics requires a
method that is inexpensive, accurate, expeditious, and robust.
[0007] Due to the vast amount of genetic information yet to be
gathered in both human and non-human genomes, intense efforts are
underway to develop new and faster methods of DNA analysis,
including DNA detection, sizing, quantification, sequencing, gene
identification, and the mapping of human disease genes. Efforts to
analyze DNA have been greatly aided by the development of a process
for in vitro application of DNA, namely, the polymerase chain
reaction (PCR.RTM.). PCR.RTM. provides the ability to amplify and
obtain direct sequence information from as little as one copy of a
target DNA sequence.
[0008] Typically, PCR.RTM. application is carried out by placing a
mixture of target double-stranded DNA, mixture of deoxynucleotide
triphosphates, buffer, two primers (one phosphate-labeled) and DNA
polymerase (e.g., heat stable Taq polymerase) in a thermocycler
which cycles between temperatures for denaturation, annealing, and
synthesis. The selection of primers defines the region to be
amplified. In the first stage of the cycle, the temperature is
raised to separate the double stranded DNA strands to form the
single-stranded templates for application. The temperature is then
lowered to generate the primed templates for DNA polymerase. In a
third stage, the temperature is raised to promote Taq-promoted DNA
synthesis, and the cycle of strand separation, annealing of
primers, and synthesis is repeated for about as any as 30-60
cycles. Standard detection, sizing, DNA sequencing methods as
described above, while providing useful information, are often
tedious and costly. Many of the commonly employed techniques
involve multiple handling steps. Further, the most common method of
fragment analysis--gel electrophoresis--is a relatively
time-consuming process.
[0009] Oligonucleotide sizing and sequence analysis is typically
carried out by first utilizing either the enzymatic method
developed by Sanger and Coulson, or by chemical degradation,
developed by Maxam and Gilbert. The Sanger method uses enzymatic
chain extension coupled with chain-terminating dideoxy-precursors
to produce randomly terminated DNA fragments. The Maxam and Gilbert
Technique involves four different base-specific reactions carried
out on portions of the DNA target to produce four sets of
radiolabeled fragments. Both techniques utilize gel electrophoresis
to separate resultant DNA fragments of differing lengths.
[0010] In conventional DNA analysis, the DNA fragments are labeled
with radioisotopes. After separation on sequencing gels, the
fragments are visualized by the image they generate upon a piece of
film applied the gel.
[0011] Other methods of DNA analysis have been described which
eliminate the use of radioisotopes. One example of such a method
uses fluorophores or fluorescent tags. In general four different
fluorophores, each having a different absorption and emission
spectrum, are attached to the DNA primers using chemical DNA
synthesis techniques. Primers with different fluorescent labels are
used in each of the four enzymatic sequencing reactions. In an
alternate approach to the four dye fluorescence-based detection, a
dye is chemically attached to a chain-terminating base analog after
enzymatic extension. In this approach, synthesis of the different
dye-primers is avoided. Mono- and poly- functional intercalator
compounds have also been developed as reagents for high-sensitivity
fluorescence detection (Glazer et al., 1992). These planar aromatic
fluorophores (e.g., ethidium homodimer, thiazole orange homodimer,
oxazole yellow homodimer) insert between adjacent base pairs of
double stranded DNA.
[0012] Although the efficiency of these processes has been improved
by automation, faster and cheaper methods must still be developed
to efficiently carry out large-scale DNA analyses. The advantages
of using mass spectrometry for analyzing DNA included a dramatic
increase in both the speed of analysis (a few seconds per sample)
and the accuracy of direct mass measurements. In contrast,
electrophoretic methods require significantly longer lengths of
time (minutes to hours) and can only measure the size of DNA
fragments as a function of relative mobility to comigrating
standards. Gel-based separation systems also suffer from a number
of artifacts that reduce the accuracy of size measurements. These
mobility artifacts are related the specific sequences of DNA
fragments and the persistence of secondary and tertiary structural
elements even under highly denaturing conditions.
[0013] The inventors have performed significant work in developing
time-of-flight mass spectrometry ("TOF-MS") as a means for
separating and sizing DNA molecules, although other forms of mass
spectrometry can be used and are within the scope of this
invention. Balancing the throughput and high mass accuracy
advantages of TOF-MS is the limited size range for which the
accuracy and resolution necessary for characterizing DNA by mass
spectrometry is available. Current state of the art for TOF-MS
offers single nucleotide resolution up to .about.100 nucleotides in
size and four nucleotide resolution up to .about.160 nucleotides in
size. These numbers are expected to grow as new improvements are
developed in the mass spectrometric field.
[0014] Existing gel-based protocols for the analysis of DNA often
do not work with TOF-MS because the PCR.RTM. product size range,
typically between 100 and 800 nucleotides, is outside the current
resolution capabilities of TOF-MS. Application of DNA analysis to
TOF-MS requires the development of new primer sets that produce
small PCR.RTM. products 50 to 160 nucleotides in size, preferably
50 to 100 nucleotides in size.
[0015] Gel-based systems are capable of multiplexing the analysis
of 2 or more DNA sequences contained at multiple loci using two
approaches. The first approach is to size partition the different
PCR.RTM. product loci. Size partitioning involves designing the
PCR.RTM. primers used to amplify different loci so that the allele
PCR.RTM. product size range for each locus covers a different and
separable part of the gel size spectrum, as an example, the
PCR.RTM. primers for Locus A might be designed so that the allele
size range is from 250 to 300 nucleotides, while the primers for
Locus B are designed to produce an allele size range from 340 to
410 nucleotides.
[0016] The second approach to multiplexing 2 or more DNA sequences
contained at different loci on gel-based systems is the use of
spectroscopic partitioning. Current state of the art for gel-based
systems involves the use of fluorescent dyes as specific
spectroscopic markers for different PCR.RTM. amplified loci.
Different chromophores that emit light at different color
wavelengths provide the means for differential detection of two
different PCR.RTM. products even if they are exactly the same size,
thus 2 or more loci can produce PCR.RTM. products with allele size
range from 250 to 300 nucleotides, while Locus B with a red
fluorescent tag produces an allele size range of 270 to 330
nucleotides. A scanning, laser-excited fluorescence detection
device monitors the wavelength of emissions and assigns different
PCR.RTM. product sizes, and their corresponding allele values, to
their specific based on their fluorescent color.
[0017] In contrast, mass spectrometry directly detects the molecule
eliminating the need for optical spectroscopic partitioning as a
means for multiplexing. Because of this direct detection, MS also
allows for improved accuracy in DNA analysis. MS also lends itself
to high-throughput, highly-automated processes for analyzing DNA.
Therefore, new methods and primers are needed in order to employ
mass spectrometry for the analysis of DNA and for multiplexed
analysis.
2.0 SUMMARY OF THE INVENTION
[0018] The present invention provides an oligonucleotide
composition containing a modified primer having a 5' end and a 3'
end and containing at least one selectively cleavable site.
Preferably, the cleavable site is located at or within about five
nucleotides from the 3' end of the primer.
[0019] The modified oligonucleotide primer which has a 5' end and a
3' end, is composed of two separate nucleotide regions. The first
region contains the 5' end, while the second region contains the 3'
end of the primer, where the 3' end is capable of serving as a
priming site for enzymatic extension, typically by a polymerase or
ligase. The second region also contains a cleavable site which
connects the first and second primer regions. In a preferred
embodiment, the first region of the primer contains at least three
nucleotides. The first primer region may optionally contain one or
more secondary cleavable sites, located between the 5' end of the
primer and the second region cleavable site (e.g., to breakdown the
first region into smaller fragments). For a modified primer
containing a secondary cleavable site, the furthest downstream
cleavable site should ideally be cleaved with near 100% efficiency,
to avoid the formation of secondary or shadow products with
additional bases.
[0020] In one embodiment of the invention, the cleavable site is
located at or within about five nucleotides from the 3' end of the
primer. In an alternate embodiment, the second primer region
consists of a single nucleotide that also contains the cleavable
site, such as a ribonucleotide. The second region may alternatively
be composed of only the cleavable site.
[0021] The chemically cleavable site may be a modified based, a
modified sugar, or a chemically cleavable group incorporated into
the phosphate backbone of the primer, such as phosphorothioate or
other chemically cleavable linkages. Cleavable sites contained
within the modified primer composition include chemically cleavable
groups such as dialkoxysilane, 3'(S)-phosphorothioate,
5'(S)-phosphorothioate, 3'(N)-phosphoramidate,
5'-(N)-phosphoramidate, and ribose.
[0022] Additional cleavable sites include sites include nucleotides
cleavable by an enzyme such as a nuclease. In one embodiment, the
cleavable site within the modified primer composition is a single
uracil incorporated to replace a thymine, where the uracil is cut
site-specifically by treatment with uracil DNA-glycosylase,
followed by alkali treatment. In another embodiment, the cleavable
site is a restriction endonuclease cleavable site, where the
recognition sequence is located in the first primer region (i.e.,
upstream of the cleavage site). In a preferred embodiment, the
restriction endonuclease cleavable site is located at or within
about five nucleotides from the 3' end of the primer. It is also
possible, such as with class IIs restriction enzymes, to position
the cleavable site or cut site within the extension product.
Restriction endonucleases for use in cleaving the modified primers
of the invention include class IIs restriction endonucleases such
as BpmI, BsgI, BseRI, BsmFI, and FokI. A modified primer including
a BpmI or BsgI recognition site contains (i) a first region
containing the recognition site, 5'-CTGGAG-3' or 5'-GTGCAG-3',
respectively, and (ii) located 16 bases downstream from the last
nucleotide of the recognition sequence, in the second primer
region, is the cleavable site. A modified primer containing a BseRI
or BsmFI recognition site, e.g. 5'-CTGGAG-3' or 5'-GTGCAG-3',
respectively, and (ii) located 16 bases downstream from the last
nucleotide of the recognition sequence, in the second primer
region, is the cleavable site. A modified primer containing a BseRI
or BsmFI recognition site, e.g., 5'-GAGGAG-3', or 5'-GGGAC-3',
respectively, contains a cleavable site located 10 bases downstream
from the list nucleotide of the recognition sequence, while a
primer containing a FokI recognition sequence (e.g., 5'-GGATG-3')
possesses a cleavable site 9 bases downstream from the last
nucleotide of the recognition sequence.
[0023] In a yet another embodiment, the cleavable site is a
restriction endonuclease cleavable site, where the recognition
sequence is located in the first primer region (i.e., upstream of
the cleavage site), and the first primer region contains a 5'
hairpin-type (self-complementary double stranded) domain. The 5'
hairpin domain includes the double stranded recognition site for
the restriction enzyme. The second (single stranded) primer region
contains (i) the cleavable site (i.e., restriction endonuclease cut
site, and (ii) is composed of nucleotides complementary to a single
stranded target, thus serving a priming site for enzymatic
extension. Following enzymatic extension of the primer, the product
is cleaved by treatment with a suitable class IIS restriction
endonuclease, followed any denaturation to release the single
stranded extension segment.
[0024] In another embodiment, the cleavable site is a nucleotide or
series of nucleotides capable of blocking or terminating 5' to 3'
enzyme-promoted digestion by an enzyme having 5' to 3' exonuclease
activity, such a T7 Gene 6 Exonuclease or phosphodiesterase such as
that derived from calf spleen. Blocking nucleotides include peptide
nucleic acids and nucleotides containing a phosphorothioate, methyl
phosphonate or borano-phosphate group. Modifications at the 3'
position of the sugar also may block the digestion by an
exonuclease. In a primer extension reaction utilizing a modified
primer containing a blocking nucleotide as the cleavable site,
following a primer extension reaction, the resulting product,
composed of (i) a modified primer containing a blocking nucleotide,
and (ii) and extension segment, is treated with a nuclease such as
an exonuclease having a 5' to 3' exonuclease activity. Nuclease
treatment typically results in digestion of the first region of the
primer to generate an extension segment composed of nucleotides
downstream (i.e., 3') of the cleavable site. Preferably, the
blocking group does not inhibit enzymatic extension of the
primer.
[0025] The modified primer may further include an immobilization
attachment site for binding to a solid support. The immobilization
attachment site may be located either upstream (i.e., 5' to) or
downstream (i.e., 3') of the cleavable site. Preferably, the
blocking group does not inhibit enzymatic extension of the
primer.
[0026] The modified primer may further include an immobilization
attachment site for binding to a solid support. The immobilization
attachment site may be located either upstream (i.e., 5' to) or
downstream (i.e., 3' to) of the cleavable site. In one embodiment,
the immobilization attachment site is located at the 5' end or 5'
relative to the cleavable site (i.e., upstream of the cleavable
site) of the modified primer. In another embodiment, the
immobilization attachment site is located at the 3' end or 3'
relative to the cleavable site (i.e., downstream of the cleavable
site). Alternatively, the immobilization attachment site may be
contained within the extension segment resulting from an enzymatic
extension reaction, or, may be contained within a target nucleic
acid.
[0027] For modified primers including an immobilization attachment
site, the primer is attachable to a solid support by either a
covalent or non-covalent linkage between the solid support and the
primer immobilization attachment site to provide an immobilized
modified oligonucleotide composition. Solid supports for use in the
present invention include glass, silicon, polystyrene, cellulose,
teflon, polystyrene divinyl benzene, aluminum, steel, iron, copper,
nickel, silver and gold.
[0028] In one embodiment, the primer is attachable to a solid
support via an intervening spacer arm, with a typical spacer giving
six or more atoms in length.
[0029] In another embodiment, the modified primer containing an
immobilization attachment site in the first primer region composed
of a series of a bases complementary to an intermediary
oligonucleotide. The modified primer is immobilized by specific
hybridization of the immobilization attachment site to the
intermediary oligonucleotide, which is bound to a solid support.
The intermediary oligonucleotide can be complementary to all or a
portion of the sequence of the modified primer. The intermediary
nucleotide is typically composed of 6 or more bases, and preferably
more than 8 bases. Additionally, the intermediary oligonucleotide
may also be homologous to a region within target nucleic acid
(template) molecule.
[0030] In one embodiment, the modified primers of the present
invention ar oligonucleotide, such as DNA or RNA, having
phosphodiester internucleotide linkages. In another embodiment, the
modified primers are oligonucleotide analogues composed of
alternative backbone structures containing internucleotide linkages
such as methylphosphonate, phosphotriester, phosphorothioate,
peptide, and the like. Primers for use in the invention should be
capable of hydrogen bonding in a sequence-specific manner to a
target sequence.
[0031] The invention also provides a method for determining the
size of a primer extension product using the modified primers of
the present invention. In employing the method, oligonucleotide
size analysis is carried out by firs contacting modified primer of
the present invention with a target nucleic acid molecule (e.g. DNA
or RNA) to effect hybridization of the primer with the single
stranded target. The modified primer is complementary to the target
and contains a first region containing the 5' end of the primer,
and a second region containing the 3' end of the primer, where the
3' end is capable of serving as a priming site for enzymatic
extension. The second region of the primer also contains a
cleavable site.
[0032] In determining the size of a primer extension product, the
primer is extended by enzymatic means, typically by action of a
polymerase or ligase, to generate a mixture containing a product
composed of the primer and one more extension segments. The
resulting product is cleaved at the cleavable site and the
resulting extension segment is then sized by any of a number of
suitable analytical techniques, preferably mass spectrometry. In
accordance with the invention, the mass of the extension segment is
decreased and the read length of the extension segment is increased
relative to the read length of the product composed of the modified
primer and the extension segment.
[0033] In a preferred embodiment for determining the size of an
extension product, the modified primer (first or second region) or
template is immobilized by attachment to a solid support.
Immobilization may be via a covalent or non-covalent linkage.
Exemplary non-covalent linkages include ligand-protein interactions
and base-specific hydrogen bonding. The extension segment from an
enzymatic extension reaction can also be immobilized by attachment
to a solid support, and is released from the solid support prior to
sizing or sequence determination. In the latter embodiment,
enzymatic extension is typically carried out in the presence of a
nucleotide containing (i) an immobilization attachment site (and
(ii) a releasable site, such as the exemplary nucleotide,
biotinylated-disulfide-dideoxynucleotide. Following enzymatic
extension, for example, with a ligase or polymerase, the extension
product is immobilized, denatured, and cleaved at the cleavable
site, leaving the extension segment affixed to the solid support.
Released primer (the portion upstream of the cleavable site),
template and additional mixture components are typically removed by
washing, and the immobilized extension segment is then cleaved at
the releasable site, to release the extension segment for
sizing.
[0034] In one embodiment of the method, the modified primer is
immobilized via specific hybridization of the immobilization
attachment site to an intermediary oligonucleotide which is bound
to a solid support (solid phase bound intermediary oligonucleotide
which is bound to a solid support (solid phase bound intermediary
oligonucleotide, SPBIO). In one particular embodiment, the
immobilization attachment site is located in the first primer
region and is composed of a series of bases complementary to the
intermediary oligonucleotide. Alternatively, if a portion of the
sequence of the extension product is known, the immobilization
attachment sight may be contained within a region of the extension
segment.
[0035] In yet another embodiment, the SPBIO is homologous to a
target nucleic acid molecule, resulting in a competition between
the SPBIO and the target for hybridizing to the modified primer.
Following enzymatic extension, the product (composed of the primer
and an extension segment) is attached to a solid support via
hybridization to the SPBIO. To promote hybridization of the SPBIO
to the product, the amount of target molecule may be reduced either
by (i) carrying out a target-selective digestion which leaves the
primer and extension product intact, or (ii) reducing the amount of
target molecule. Template specific digestion may be chemical or
enzymatic.
[0036] In a related embodiment of the method, the modified primer
is immobilized via hybridization to a SPBIO, where the first primer
region contains a first portion which is complementary to the
SPBIO, and downstream or 3'of this portion of the first region is a
second portion of the first primer region which is complementary to
the target molecule but is not complementary to the SPBIO. The
first portion of the first region of the modified primer will
typically be composed of at least six or more nucleotides which are
complementary to the SPBIO.
[0037] Alternatively, indirect immobilization of the modified
primer can be accomplished by carrying out an enzymatic extension
reaction with a target nucleic acid which is attached to a solid
support. The target molecule can be immobilized either prior to or
subsequent to primer extension.
[0038] Optionally, the reaction mixture containing immobilized and
non-immobilized species is washed prior to cleavage at the
cleavable site allowing ready separation of immobilized versus
non-immobilized species prior to sizing of the extension
segments.
[0039] The primer can be immobilized at the immobilization
attachment site either prior to or after enzymatic extension,
depending upon the nature of immobilization. Generally, when the
immobilization attachment site is contained within the first primer
region, the immobilization attachment remains intact under the
selected cleavage conditions to retain a significant portion of
nucleotides from the modified primer (e.g., those comprising the
first primer region) in immobilized form. In accordance with the
present method, the read length of the extension segment resulting
from cleavage at the cleavable site is increased relative to the
read length of the product composed of the primer and extension
segment.
[0040] In one embodiment of the invention, the extension segments,
typically containing no more than about five nucleotides derived
from the modified oligonucleotide primer, are sized using mass
spectrometry. Such sizing may utilize matrix-assisted laser
desorption ionization mass spectrometry, and more particularly, may
be accomplished using time-of-flight mass spectrometry.
[0041] The sizing method may also be coupled with amplification of
a target nucleic acid.
[0042] In one embodiment of this aspect of the invention, first and
second primers are combined with a target nucleic acid under
conditions effective to promote the hybridization of the primers to
the nucleic acid to generate primer/nucleic acid complexes. One of
the primers (e.g., the first primer) is complementary to the target
nucleic acid and has a first region containing the 5' end of the
primer and an immobilization attachment site. The first primer
further contains a second region containing the 3' end of the
primer, where the 3' end is capable of serving as a priming site
for enzymatic extension. The second region of the first primer
further contains a cleavable site. The second primer is homologous
to the target nucleic acid.
[0043] The primer/nucleic acid complexes are converted to double
strand fragments in the presence of a suitable polymerase and all
four deoxynucleotide triphosphates (dNTPs) or modified versions
thereof. The number of primer-containing fragments is amplified by
successively repeating the steps of: (i) denaturing the double
strand fragments to produce single strand fragments, (ii)
hybridizing the single strands with the primers to form
strand/primer complexes, (iii) generating double strand fragments
from the strand/primer complexes in the presence of a polymerase
and all four dNTPs, and (iv) repeating steps (i) to (iii) until a
desired degree of amplification has been achieved.
[0044] The amplified fragments are then denatured to generate a
mixture including a product composed of the first primer and an
extension segment. In one embodiment of this aspect of the
invention, the amplified fragments containing the first primer are
immobilized at the immobilization attachment site and the
non-immobilized amplified fragments are removed, typically by
washing. The first primer is then cleaved from the immobilized
product at the cleavable site, causing the release of the extension
segment from the support.
[0045] In an alternate embodiment, the amplified fragments may be
immobilized prior to denaturing. Generally, the amplified fragments
are immobilized prior to cleaving at the cleavable site, to enable
release and subsequent analysis of the extension segments resulting
from such cleavage, in the absence of other species (e.g., primers,
reactants, excess dNTPs).
[0046] The extension segments are then sized by mass spectrometry.
The read length of the extension segment is increased relative to
the read length of the product composed of the first primer and the
extension segment by cleavage at the cleavable site.
[0047] Another embodiment of the sizing method provides first and
second primers, where one of the primers, i.e., the first primer,
contains a cleavable site, and another primer, i.e., the second
primer, contains an immobilization attachment site for binding to a
solid support. The second primer is composed of a 5' end and 3'
end, is homologous to the target nucleic acid, and includes a first
segment containing the 3' end of the second primer, and a second
segment containing the 5' end of the primer and an immobilization
attachment site.
[0048] These first and second primers are combined with a target
nucleic acid to generate primer/nucleic acid complexes and
converted to double stranded fragments in the presence of
polymerase and deoxynucleoside-triphosphates. The sizing method can
be carried out using a high concentration of target nucleic acid to
generate substantial amounts of primer extension product, or
alternatively, may be coupled with various rounds of amplification.
Upon achieving a desired amount of product, extension products
containing the second primer are immobilized by attachment at the
immobilization attachment site. The extension product is the
cleaved at the cleavable site to generate a mixture which includes
a double stranded product. Non-immobilized cleaved fragments are
removed, preferably by washing, and the double stranded product is
denatured to release the extension segment, which is sized by mass
spectrometry, where the mass of the extension segment is decreased
and the read length of the extension segment is increased relative
to the read length of the primer/nucleic acid double stranded
fragments. Immobilization of the extension product may occur either
before or after cleavage at the cleavable site.
[0049] As will be appreciated the cleavable site of the first
primer and the immobilization attachment site of the second primer
include those of the type described above.
[0050] In one embodiment, the first primer contains a class IIs
restriction enzyme recognition site in the first primer region, and
a cleavable site in the second primer region, and the second primer
contains an immobilization attachment site for attachment to a
solid support. Cleavage at the cleavable site is carried out by
addition of a restriction endonuclease selective for the
recognition site contained in the first primer region to provide
(i) released fragments containing the first region of the first
primer and (ii) a double stranded product, which is immobilized
prior to denaturing to release the desired extension segment.
[0051] Also encompassed is a method for determining the size of
more than one primer extension product including the steps of: (a)
hybridizing a plurality of primers with more than one target
nucleic acid, wherein each of said primers (i) is complementary to
at least one target nucleic acid; (ii) has a first region
containing the 5'end of the primer, and (iii) has a second region,
containing the 3' end of the primer and a cleavable site, wherein
the 3' end is cable of being extended by an enzyme; (b) extending
the primers with the enzyme to generate a polynucleotide mixture
containing more than one extension product; (c) cleaving more than
one extension product at its respective cleavable site to release
more than one extension segment, wherein the location of the
cleavable site of at least two primers is selected to increase the
mass difference between their respective extension segments; and
(d) sizing the released extension segments by mass spectrometry
(such as TOF MS), whereby said cleaving is effective to increase
the read length of the extension segments relative to the read
length of the products of step (b). This method may also be coupled
with amplification of a target nucleic acid.
[0052] In some embodiments, the first region of at least one of
said primers comprises an immobilization attachment site which may
be used to immobilize one or more extension products onto a solid
support. In these cases, it may be preferable to wash the extension
product after said immobilizing and prior to said cleaving
step.
[0053] This method may be used to size extension products of DNA or
RNA target nucleic acids as well as mixtures thereof. The target
nucleic acids may also be immobilized, either prior to or after
said extending step.
[0054] At least one cleavable site may be a blocking nucleotide
capable of blocking a 5' to 3' enzyme-promoted digestion, and
wherein said cleaving is carried out by digesting the first region
of at least one primer with an enzyme haying a 5' to 3' exonuclease
activity. At least one cleavable site may also comprise a modified
base, a modified sugar, or a chemically cleavable group
incorporated into the phosphate backbone. Exemplary cleavable sites
include dialkoxysilane, 3'S)-phosphorothioate,5
'-(S)-phosphorothioate,3 '-(N)-phosphoramidate,
5'-(N)phosphoramidate, uracil, and ribose.
[0055] Another aspect of this invention is a kit for analyzing at
least one target nucleic acid. This kit typically includes a first
primer complementary to a first target nucleic acid and a second
primer complementary to an extension product of the first primer.
The first primer typically has a first region containing the 5' end
of the first primer and a second region containing the 3' end of
the first primer and chemically cleavable site.
[0056] These kits may be used to analyze single- and
double-stranded target nucleic acids. The use of polymerase or
ligase to extend nucleic acids are known in the art and one of
skill in the art in light of the present disclosure would be able
to select suitable primer pairs (i.e., of first and second primers)
to analyze single- and double-stranded targets. For example, where
double-stranded target nucleic acids are being analyzed, the first
primer may typically be complementary to one strand of the target
nucleic acid adjacent and upstream to the sequence one is
interested in analyzing while the second primer may be
complementary to the other strand adjacent and upstream from the
sequence of interest. Where the target nucleic acid is
single-stranded, the first and second primers may both be
complementary to portions of the strand, for example, the portions
flanking the sequence of interest.
[0057] Either the first or second primer or both primers may also
contain an immobilization attachment site. This site is usually
upstream of the chemically cleavable site and may preferably be
located in the first region of the first primer. A suitable
immobilization attachment site is any site capable of being
attached to a group on a solid support. These sites may be a
substituent on a base or sugar of the primer. An IAS may be, for
example, an antigen, biotin, or digoxigenin.
[0058] The kit may also include a solid support. The solid support
be is capable of being attached to the immobilization attachment
site may be supplied as a separate component or may be supplied
already attached to the IAS. Exemplary solid supports include
glass, silicon, polystyrene, aluminum, steel, iron, copper, nickel,
silver and gold. These supports may also contain groups capable of
being attached to the IAS and the selection of the solid support
may be determined by the IAS. For example, where the IAS is biotin,
the solid support may contain an avidin or streptavidin
functionality or molecule attached thereto. The solid support may
also contain an antibody. If a digoxigenin IAS is being employed,
the solid support may contain the antibody capable of binding
digoxigening, such as anti-digoxigenin.
[0059] In some aspects the immobilization attachment site may also
be capable of being attached to an intervening spacer arm bound to
the solid support, where the intervening spacer arm may be of any
length but is usually six or more atoms in length. The
immobilization attachment site may also be a single stranded
nucleic acid complementary to an intermediary oligonucleotide bound
to the solid support.
[0060] Any chemically cleavable site may be employed, such as a
modified base, a modified sugar (ribose), or a chemically cleavable
group incorporated into the phosphate backbone. Exemplary
chemically cleavable sites include dialkoxysilane,
3'-(S)-phosphorothioate, 5'-(S)-phosphorothioate,
3'(N)-phosphoramidate, 5'(N)phosphoramidate, and ribose.
[0061] This kit may also include an enzyme for extending the first
and second primers, such as a DNA polymerase or a ligase. Also
included may be a reagent capable of cleaving the chemically
cleavable site. Some exemplary chemical reagents included
2-iodoethanol, 2,3-epoxy-1-propanol, silver, and fluoride.
[0062] This kit may be further adapted for analyzing more than one
target nucleic acid simultaneously or sequentially. These adapted
kits may typically further include a third primer complementary to
a second target nucleic acid having a first region containing the
5' end of the third primer and an immobilization attachment site
and a second region containing the 3' end of the third primer and a
chemically cleavable site; and a fourth primer complementary to an
extension product of the third primer. In this embodiment, the
first and third primers should be selected such that the masses of
the extension segments generated by extending the primers in the
presence of the target nucleic acids and cleaving at the cleavable
sites of the first and third primers are distinguishable by mass
spectrometry. One way of accomplishing that, particularly where the
masses of the sequences added by extending the first and third
primers are similar, is to locate the cleavable sites of the first
and third primer at differing locations relative to the 3' ends of
the first and primers.
[0063] Also encompassed are kits for simultaneously analyzing more
than one target nucleic acid. These kits include a plurality of
first primers complementary to a plurality of target nucleic acids
each having a first region containing the 5' end, and a second
region containing the 3' end of the respective first primer and a
chemically cleavable site; and a plurality of second primers
complementary to a plurality of extension products of the plurality
of first primers. In these kits, the various first primers are
selected such that the masses of extension segments generated by
extending the primers in the presence of the target nucleic acids
and cleaving at the cleavable sites of the first primers are
distinguishable by mass spectrometry. For example, the cleavable
sites of at least two of the first primers may be located at
differing locations relative to their respective ends. Thus, the
location of the cleavable sites of the first primers relative to
their respective 3' ends may be selected to distinguish the masses
of extension segments generated by extending the first and second
primers in the presence of the plurality of target nucleic acids
and cleaving at the cleavable sites of the first primers.
[0064] This kit may be used to analyze double-stranded and
single-stranded nucleic acids simultaneously or sequentially and
may further contain an enzyme for extending the first and second
primers or a solid support.
[0065] At least one first or second primer may optionally include
an immobilization attachment site for binding to a solid
support.
[0066] In a related aspect, a method of sequencing is provided that
utilizes the modified primers of the invention for determining the
sequence of a target molecule by mass spectrometry. In one
embodiment of this aspect of the invention, the sequence of a
target nucleic acid is determined by hybridizing a modified
immobilizable primer of the present invention with a target nucleic
acid, such as DNA or RNA, followed by enzymatically extending the
primer in the presence of a first of four different dideoxy
nucleotides (chain terminators) to generate a mixture of primer
extension products. The primer extension products each contain a
primer and an extension segment. The extension products are
denatured, immobilized, and washed to remove non-immobilized
species present in the reaction. As in the embodiments described
above, immobilization can occur before or after enzymatic
extension, and is typically carried out prior to cleavage at the
cleavable site. Subsequent to immobilization and removal of
non-immobilized species, the primer extension products are cleaved
to release the extension segments. The extension segments are sized
by mass spectrometry, and the above steps are repeated with each of
the three remaining different dideoxy nucleotides. The sequence of
the target is then determined by comparing the sizes of the
extension segments obtained from each of the four extension
reactions. In a variation of the above, a single primer extension
reaction is carried out using a mixture composed of more than one
chain-terminating nucleotide, up to all four (e.g., subsets of the
following containing at least one dideoxynucleotide: dTTP, ddTTP,
dATP, ddATP, dCTP, ddCTP, dGTP, and ddGTP). The resulting reaction
mixture, containing up to all four-base specifically terminated
products, is then analyzed using the mass data and known mass
values for the four bases. Optionally, mass modified nucleotides
may be utilized to enhance the resolution of the product mixture.
Sequencing can also be carried out using the modified primers of
the invention coupled with alternate sequencing methodologies which
do not employ dideoxynucleosides.
[0067] Similarly, methods of analyzing single nucleotide
polymorphisms (SNPs) are provided that utilize the modified primers
of the invention of determining the presence, nature and location
of an SNP by mass spectrometry. In one embodiment, the SNP is
analyzed by hybridizing a modified immobilizable primer of the
present invention with a target nucleic acid, such as DNA or RNA,
followed by enzymatically extending the primer in the presence of
only the four dideoxy nucleotides (chain terminators, i.e., ddTTP,
ddUTP, ddATP, ddCTP, ddITP and ddGTP) to generate a primer
extension product. Thus, the primers are only extended by one
base--the base complementary to the site of the suspected
polymorphism.
[0068] The primer extension product for these SNP analysis methods
each contain a primer and a single added dideoxy nucleotide. The
extension products are denatured, immobilized, and washed to remove
non-immobilized species present in the reaction. As in the
embodiments described above, immobilization can occur before or
after enzymatic extension, and is typically carried out prior to
cleavage at the cleavable site. Subsequent immobilization and
removal of non-immobilized species, the primer extension products
are cleaved to release the added dideoxynucleotide attached to a
portion of the primer. The released fragment is sized by mass
spectrometry and the mass of the added dideoxynucleotide determined
by subtracting the known mass of the portion of the primer 3' of
the cleavable site. The presence and nature of the SNP is then
determined by comparing the identity of the added base to that
found in the wild-type (nonpolymorphic) nucleic acid. This method
may also be employed with multiple primers ending at different
locations to determine the location of possible SNPs. Optionally,
mass modified nucleotides may be utilized to enhance the resolution
of the product mixture.
[0069] Methods for detecting SNPs can be expanded to the analysis
of polymorphisms in general. Polymorphisms include both naturally
occurring, somatic sequence variations and those arising from
mutation. Polymorphisms include but are not limited to: sequence
microvariants where one or more nucleotides in a localized region
vary from individual to individual, insertions and deletions which
can vary in size from one nucleotides to millions of bases, and
microsatellite or nucleotide repeats which vary by numbers of
repeats. Nucleotide repeats include homogeneous repeats such as
dinucleotide, trinucleotide, tetranucleotide or larger repeats,
where the same sequence in repeated multiple times, and also
heteronucleotide repeats where sequence motifs are found to repeat.
For a given locus the number of nucleotide repeats may vary
depending on the individual. Through the use of strategic placement
of the cleavable primer and a combination of deoxy and dideoxy or
other chain-terminating nucleotides one may be able to span the
region of a polymorphism, e.g., placement of the cleavable primer
adjacent and upstream from a polymorphism, and extension of the
primer by polymerase, wherein a dideoxy nucleotide is added
downstream from the polymorphism to terminate extension. Variations
in the size of the chain terminated extension products can be used
to identify the type and variance with a given polymorphism.
[0070] An aspect thus includes methods for determining the presence
of a polymorphism in a target nucleic acid. These methods typically
include the steps of: hybridizing a primer with a target nucleic
acid suspected of containing a polymorphism, where the primer has a
first region containing the 5' end of the primer and a second
region containing the 3' end of the primer and a cleavable site;
extending the 3' end of the primer with a polymerase in the
presence of a nucleotide to generate an extension product; cleaving
the extension product at the cleavable site to release an extension
segment; sizing the extension segment by mass spectrometry (such as
by TOF MS), whereby said cleaving is effective to increase the read
length of the extension product; and identifying any added
nucleotides. This method may be employed to analyze many types of
nucleic acids, such as RNA and DNA, both single- and
double-stranded.
[0071] The extension step may be carried out in the presence of one
or more nucleotides, including, for example, deoxynucleotides,
chain terminating nucleotides and derivatives thereof, depending on
the nature of the suspected polymorphism. For example, if one is
interested in identifying an SNP, the extension step may be carried
out in the presence of only chain-terminating nucleotides, such
dideoxynucleotides (e.g. ddATP, ddTTP, ddUTP, ddGTP, ddITP and
ddCTP) and mass-modified chain-terminating nucleotides, and then
identification of the added dideoxynucleotide determines the
presence and identify of the single nucleotide polymorphism.
Mass-modified are well known in the art and may bae used to, for
example, increase the mass difference of certain nucleotides (such
as deoxy- and dideoxy- adenine and thymine).
[0072] The first region of the primer may contain an immobilization
attachment site and the extension product may be immobilized onto a
solid support at the immobilization attachment site prior to the
cleaving step. In this embodiment, it may also be desirable to wash
the extension product after said immobilizing and prior to said
cleaving.
[0073] In some embodiments, it may be desirable to immobilize the
target nucleic acid. Under these circumstances, the target nucleic
acid may be immobilized prior to or after said extending.
[0074] Cleavable sites for these methods include blocking
nucleotides capable of blocking 5' to 3' enzyme-promoted digestion.
In these instances, the cleaving may be carried out by digesting
the first region of the primer with an enzyme with an enzyme having
a 5' to 3' exonuclease activity, such as T7 gene 6 exonuclease and
phosphodiesterase. Such blocking nucleotides include
phosphorothioates, methyl phosphonates, phosphotriesters, and
peptide nucleic acids.
[0075] Cleavable sites may also be chemically cleavable sites, such
as modified sugar, modified bases, and groups incorporating a
chemically cleavable group into the phosphate backbone of the
primer. Some exemplary cleavable sites include dialkoxysilane,
3'(S)-phosphorothioate, 5 '-(S)-phosphorothioate,3
'-(N)-phosphoramidate, 5'-(N)phosphoramidate, uracil, and
ribose.
[0076] This method may also be coupled with amplification of the
target nucleic acid.
[0077] This method may also be adapted for analyzing a plurality of
target nucleic acids. In these embodiments, typically a plurality
of primers are hybridized to more than one target nucleic acid and
the location of the cleavable site contained within the primers is
varied to increase the mass difference between the respective
extension segments.
[0078] In another embodiment, the sequence of a target nucleic
acid, such as DNA or RNA, is determined by first hybridizing a
primer with a target CAN, where the primer (i) is complementary to
the target DNA; (ii) has a first region containing the 5' end of
the primer and an immobilization attachment site; and (iii) has a
second region containing the 3' end of the primer and a cleavable
site. The 3' end of the primer is also capable of serving as a
priming site for enzymatic extension.
[0079] The primer is then extended with an enzyme in the presence
of a first of four different deoxynucleotide alpha-thiotriphosphate
analogs (dNTPalphaS) to generate a mixture of primer extension
products containing phosphorothioate linkages. The
phosphorothioate-containing extension products are then treated
with a reagent that cleaves specifically at the phosphorothioate
positions. Suitable reagents for promoting
phosphorothioate-specific cleavage include 3' to 5' exonuclease,
2-iodoethanol, and 2,3-epoxy-1-propanol. Treating of the extension
products is typically carried out under conditions that produce
limited cleavage of the phosphorothioate linkages, resulting in the
production of a group of primer extension degradation products.
Alternatively, the primer extension reaction can be carried out
using a limited amount of the alpha-thio-deoxynucleoside
triphosphate analog along with the corresponding conventional
deoxynucleoside triphosphate (dNTP). The resulting extension
products are then treated with a phosphorothioate-selective reagent
as described above, under conditions effective to cleave all of the
phosphorothioate groups incorporated into the extension product
(complete cleavage).
[0080] The primer extension degradation products are immobilized at
the immobilization attachment sites to produce immobilized primer
extension degradation products (i.e., a nested set of fragments
specific to the 5' end containing the primer), each containing a
primer and an extension segment. In alternative embodiments of this
aspect of the invention, immobilization may be carried out either
(i) prior to enzymatic extension, (ii) after enzymatic extension,
(iii) prior to treating the phosphorothioate-containing primer
extension products with a phosphorothioate-specific cleaving
reagent, or (iv) or after such treating.
[0081] Subsequent to immobilization, the primer extension
degradation products are washed to remove non-immobilized species.
Cleavage at the cleavable site results in the release of extension
segments, which are then sized by mass spectrometry. Using the
sequencing method of this aspect of the invention., the read length
of any given extension segment is increased relative to the read
lengthy of its corresponding primer extension degradation
product.
[0082] The steps of hybridization, enzymatic extension, treatment
with a phosphorothioate-cleaving reagent, immobilization, washing,
cleaving, sand sizing are then repeated with a second, third, and
fourth of the four different dNTPalphaS analogs to determine the
sequence of the target DNA by comparison of the sizes of the
extension segments obtained from each of the four extension
reactions. Optionally, the steps of hybridization, enzymatic
extension, treatment with a phosphorothioate-cleaving reagent,
immobilization, washing, and cleaving can be carried out in the
presence of from 2-4 different dNTPalphaS analogs, followed by
sizing of the resulting extension segments by mass
spectrometry.
[0083] In all aspects the invention, the extension segments from
multiple primers may be analyzed simultaneously. In cases where the
different primer extension products are all of similar size and
mass, one may opt to vary the position of the cleavage site in the
different primers such that the fragments released after cleavage
are distinguishable, or more easily distinguishable by MS analysis.
Thus, another embodiments encompasses the creation and use of
mixtures of cleavable primers for multiplexed analysis where the
cleavage site is located at different sites along the backbone for
different primers relative to the segment added by extension such
that the masses of the cleaved and released fragments are
distinguishable by mass. In cases where each of the primers in a
multiplex analysis is likely to be extended to create
similarly-sized extension products (by mass), for example, single
base dideoxynucleotide additions and LCR or PCR amplifications, the
placement of the cleavage site at different locations within the
cleavable primers will result in cleaved and released extension
products that will vary by one or more nucleotides. By
differentiating the masses of the cleaved products, one may observe
multiple primer extension products simultaneously. An example for
single nucleotide extensions is shown in FIG. 17.
[0084] In these aspects, the initial primers may have very
different masses but still result in extension products which would
have similar masses. In these embodiments, it is the masses of the
cleaved fragments that should be distinguished relative to each
other by moving the cleavable site within the various primers as
opposed to the masses of the primers or uncleaved extension
products.
[0085] According to yet another aspect, a method of fingerprinting
is provided which utilizes the modified primers of the invention of
the invention for obtaining a fingerprint of a target
oligonucleotide.
[0086] These and other objects and features of the invention will
become more fully apparent when the following detailed description
is read in conjunction with the accompanying figures and
examples.
3.0 BRIEF DESCRIPTION OF THE DRAWINGS
[0087] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0088] FIGS. 1A-1W show a native phosphodiester internucleotide
linkage (FIG. 1A) and exemplary internucleoside cleavable sites for
use in the oligonucleotide composition of the present invention:
dialkoxysilane (FIG. 1B); beta-cyano ether (FIG. 1C);
5'-deoxy-5'-aminocarbamate (FIG. 1D); 3'-deoxy-3'-aminocarbamate
(FIG. 1E); urea (FIG. 1F); 2'-cyano-3',5'-phosphodiester (FIG. 1G);
3'-(S)-phosphorothioate (FIG. 1H); 5'-(S)-phosphorothioate (FIG.
1I); 3'-(N)-phosphoroamidate (FIG. 1J); 5'-(N)-phosphoroamidate
(FIG. 1K); alpha-amino amide (FIG. 1L); vicinal diol (FIG. 1M);
ribonucleoside insertion (FIG. 1N); 2'-amino-3',5'-phosphodiester
(FIG. 1O); allylic sulfoxide (FIG. 1P); ester (FIG. 1Q); silyl
ether (FIG. 1R); dithioacetal (FIG. 1S); 5'-thio-formal (FIG. 1T);
alpha-hycroxy-methyl-phosphonic bisamide (FIG. 1U); acetal (FIG.
1V); and 3'-thio-formal (FIG. 1W).
[0089] FIGS. 2A-2M include a number of exemplary immobilization
attachment linkages for use in immobilizing the first region of a
modified oligonucleotide primer: functionalized maleimide (FIG. 2A,
where Y equals sulfur, oxygen, or nitrogen); carbamate (FIG. 2B);
ester FIGS. 2C, 2I and 2K, where Y equals nitrogen); thiolester
(FIGS. 2C, 2I and 2K, where Y equals sulfur); (N)-functionalized
thiourea (FIG. 2D); mercuric-sulfide (FIG. 2E); amino (FIG. 2F);
disulfide (FIG. 2G); amide (FIG. 2H); hydrazone (FIG. 2J);
streptavidin or avidin/biotin (FIG. 2L); and gold-sulfide (FIG.
2M).
[0090] FIGS. 3A-3B illustrate time-of-flight mass spectra for
samples of a modified oligonucleotide primer containing a cleavable
ribose desorbed from a solid matrix of 3-hydroxypicolinic acid both
before (FIG. 3A) and after (FIG. 3B) selective cleavage.
[0091] FIG. 4 is a time-of-flight mass spectrum of the cleavage
product of an immobilized 18-mer containing a cleavable ribose at
the 10-position.
[0092] FIGS. 5A-5E illustrate four alternate embodiments of an
immobilized cleavable primer in accordance with the invention. FIG.
5A presents an immobilized modified primer having two regions
connected by a cleavable site, X. FIG. 5B shows a biotin molecule
connected to the 5' end of a modified primer. FIG. 5C depicts the
capture of a modified primer prior to enzymatic extension on an
avidin-functionalized solid support. FIG. 5D represents a modified
primer affixed to a solid support through a 5-allylamino
substituent at the 5 position of a uracil. FIG. 5D represents a
modified primer affixed to a solid support through a 5-allylamino
substituent at the 5 position of a uracil. FIG. 5E presents a
modified primer containing a terminal ribose cleavage site, X.
[0093] FIGS. 6A and 6B illustrate an exemplary method of
determining the sequence of a target DNA molecule using the
immobilizable, cleavable primers of the present invention.
[0094] FIGS. 7A and 7B illustrate the respective single gene
mutation sites identified for two distinct genetic disorders,
sickle cell anemia (FIG. 7A) and alpha.sub.1-antitrypsin deficiency
(FIG. 7B), suitable for detection using the modified primers of the
present invention.
[0095] FIG. 8 illustrates the immobilization of a modified primer
via competitive hybridization of the first primer region to both a
target molecule and a solid phase bound intermediary
oligonucleotide, SPBIO.
[0096] FIG. 9 illustrates the immobilization of a modified primer
via hybridization to a SPBIO, where the modified primer contains a
first primer region composed of a first portion complementary to
the SPBIO and a downstream second portion complementary to a target
molecule but not complementary to the SPBIO.
[0097] FIG. 10 illustrates immobilization of an enzymatic extension
product (composed of a modified primer and an extension segment)
via base-pairing interaction to a target molecule bound to a solid
support.
[0098] FIG. 11 illustrates sequence-specific cleavage of an
enzymatic extension product, where the modified primer contains a
restriction recognition site in the first primer region and a
cleavable site in the second primer region.
[0099] FIG. 12 illustrates an exemplary sizing method of the
present invention using first and second modified primers, where
the first primer contains an enzyme-cleavable site and the second
primer contains an immobilization attachment site.
[0100] FIG. 13 is a MALDI time-of-flight mass spectrum of primer
extension products obtained from an extension reaction using a
primer containing a 5-thiol thymidine cleavable site and a 10-fold
excess of primer. Primer extension products were immobilized via
hybridization to a complementary biotinylated intermediary
oligonucleotide bound to streptavidin coated beads and released by
chemical cleavage for subsequent size analysis.
[0101] FIGS. 14A and 14B are MALDI time-of-flight mass spectra
illustrating the utility of the present method in detecting single
base substitutions (point mutations) between oligonucleotides,
using base-specific digestion. FIG. 14A corresponds to reaction
product(s) derived from template 16-c/19-G, SEQ ID NO:17 and FIG.
14B corresponds to reaction product(s) derived from template
16-A/19-T, SEQ ID NO:18.
[0102] FIGS. 15A and 15B are MALDI time-of-flight mass spectra of
primer extension products obtained using (i) a cleavable primer
according to the present invention (FIG. 15B) versus (ii) a full
length primer (FIG. 15A), illustrating the difference in both
resolution and read length of the resulting spectra.
[0103] FIG. 16 illustrates sequence-specific cleavage of an
enzymatic extension product, where the modified primer contains a
class IIs restriction enzyme recognition site in the first primer
region, composed of a 5' hairpin-type (self-complementary double
stranded) domain, and a cleavable site in the second primer
region.
[0104] FIG. 17 illustrates multiplexed analysis by moving the
cleavable site along the primer. Primer 1 contains a cleavable site
(o) between bases 3 and 4 while primer 2 has a cleavable site
between bases 5 and 6. Single base extension (step a) by a dideoxy
chain terminating nucleotide ( ?) yields two products having
similar masses. However, cleavage (step b) of the two primers
(preferably after immobilization and purification) results in two
released fragments more easily distinguishable by mass: primer
product 1 having 4 bases and primer product 2 having 6 bases.
4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0105] 4.1 DEFINITIONS
[0106] The following terms, as used herein, have the meanings as
indicated:
[0107] In referring to a position within a single stranded
oligonucleotide, a position that is "upstream" from a specified
position is located 5' to this position, while a position that is
"downstream" is located 3' to the point of reference.
[0108] An "immobilization attachment site" or IAS is a site which
may be present within an oligonucleotide primer for binding to a
solid support material directly, through an intervening spacer arm,
or by specific hybridization to an intermediary oligonucleotide
which is bound to a solid support. The immobilization attachment
site may be located either upstream (i.e., 5' to) or downstream
(i.e., 3' to) of the cleavable site and may require chemical
modification prior to binding to the solid support. The IAS may
also be flanked both upstream and downstream by more than one
cleavable sites. Alternatively, the immobilization attachment site
may be contained within a target nucleic acid. The immobilization
attachment site can be a select functional group for covalent
bonding to a solid support, such as those representative functional
groups shown in FIGS. 2A-2K, and FIG. 2M. The immobilization
attachment site can also be a ligand such as biotin, for attachment
via a high-affinity non-covalent interaction with a solid support.
Further for immobilization attachment site can also be composed of
a series of bases complementary to an intermediary oligonucleotide.
Immobilization of the modified primer is effected, for example, by
the specific hybridization of the immobilization attachment site to
an intermediary oligonucleotide, which is bound to a solid support.
The intermediary oligonucleotide may also act as the template. The
immobilization attachment site may be attached to the solid support
by either chemical or enzymatic means. Upon attachment of the
immobilization attachment site to a solid support, the resulting
immobilization linkage is one which remains stable under the
conditions employed for cleaving the cleavable site and does not
inhibit base pair hybridization nor block the ability to extend the
primer from its 3' end.
[0109] Two nucleic acid fragments are considered to be "selectively
hybridizable" if they are capable of specifically hybridizing to
one another but not to other polynucleotides, Under typical
hybridization and wash conditions, as described, for example in
Maniatis et al., 1982.
[0110] Two nucleic acid fragments are considered to be
"complementary" if they are capable of specifically hybridizing to
one another (i) under typical hybridization and wash conditions, as
described, for example, in Maniatis et al. (1982) or (ii) using
reduced stringency wash conditions that allow at most about 25-30%
base pair mismatches, for example: 2.times.SSC, 0.1% SDS, room
temperature twice, 30 min each; then 2.times.SSC, 0.1% SDS,
37.degree. C. once, 30 min; then 2.times.SSC room temperature
twice, 10 min each.
[0111] "Cleavable site" as used herein is a reactive moiety
typically (i) located at or within about five nucleotides from the
3' end of a primer, and (ii) selectively cleavable by appropriate
non-enzymatic or enzymatic means including chemical, thermal, or
photolytic, to enable release of primer extension products that
typically contain none or a relatively small number of base pairs
of the modified primer. Cleavable site refers both to the
selectively cleavable functional group as described above and also
to protected forms thereof. The cleavable site may, for example, be
(i) located along the polymer backbone (i.e., a modified 3'-5'
internucleotide linkage in place of one of the phosphodiester
groups), (ii) as a substituent on or replacement of one of the
bases or sugars of the oligonucleotide primer, or (iii) as the 3'
terminal residue (e.g., a ribonucleotide at the 3' end of the
oligodeoxyribonucleotide primer). The cleavable site is stable
under standard solid phase DNA synthesis conditions, during primer
immobilization, hybridization, primer extension, and washing
conditions.
[0112] As used herein, a cleavable site may also be a nucleotide
cleavable by an enzyme such as a nuclease. For example, the
cleavable site may be a restriction endonuclease cleavable site,
where the recognition sequence is located in the first primer
region (i.e., upstream of the cleavage site). Exemplary restriction
endonucleases for use in cleaving at the cleavable site include
BpmI, BsgI, BseRI, BsmFI, and FokI.
[0113] A cleavable site may also be a nucleotide or series of
nucleotides capable of blocking or terminating 5' to 3'
enzyme-promoted digestion by an enzyme having 5' to 3' exonuclease
activity, such as T7 gene 6 exonuclease (Amersham Life Sciences,
Arlington Heights, Ill.). Representative blocking nucleotides are
those containing a phosphorothioate, borano-phosphate, or peptide
group. The blocking nucleotide/cleavable site should be one which
does not inhibit enzymatic extension of the primer.
[0114] As described herein, "fingerprinting" refers to a method of
determining the positions of no more than two different bases in a
target oligonucleotide strand, as opposed to "sequencing", which
refers to a determination of the complete nucleotide sequence of
(and also the corresponding amino acid sequence encoded by ) a
target oligonucleotide, including the identity and position of each
nucleotide present in the target strand or its complement.
[0115] "Non-covalent linkage" refers to any type of non-covalent
bonding interaction, and is used herein primarily to describe
different types of immobilization attachment sites. A non-covalent
linkage includes base-specific hydrogen bonding interactions, such
as those occurring between complementary nucleotide base pairs, or
may refer to a high affinity ligand-protein interaction, such as
the biotin/avidin or biotin/streptavidin interaction
(K.sub.a=10.sup.15 M.sup.-1).
[0116] "Extension segment" refers to the product resulting from in
vitro enzymatic extension at the 3' end of a primer, excluding the
portion of nucleotides originally present in the primer prior to
extension.
[0117] As used herein "a" will be understood to mean one or more.
Thus, "a nucleotide" may refer, for example, to one, two, three,
four, five or more nucleotides.
[0118] As used herein, "read length" refers to the number of
nucleotides of a given target sequence for which new analytical
data (e.g., sizing, quantification, sequencing) can be obtained.
New data refers to fragment information for primer extension
products which excludes data derived from portions of the target
DNA complementary to the primer(s) employed (e.g. regions for which
sequence information is already known).
[0119] Read length is typically method dependent (i.e., a function
of the detection method being employed). In some analytical methods
size resolution may have an essentially finite upper limit, (e.g.,
up to 100 nucleotides. One advantage of the present invention is
the ability to improve the amount of new or useful information
about a target DNA sequence that can be obtained from a primer
extension product, when the products are analyzed using such a
method.
[0120] For example, using the modified primers of the present
invention, the read length of an exemplary extension segment would
be determined as follows. A modified primer composed of nucleotides
complementary to a DNA target and having a cleavable linkage
between nucleotides 17 and 18 (e.g., with a polymerase or ligase),
and the resulting primer extension product (subsequent to
immobilization) is cleaved at the cleavable site to produce an
extension segment containing only one nucleotide derived from the
primer. In carrying out sizing of the extension products, the read
length is equal to the total number of nucleotides detected (X)
minus the one nucleotide derived from the second region of the
primer, or X-1.
[0121] In contrast, prior to cleavage at the cleavable site, the
product composed of the primer and the same set of extension
segments would have a read length of X-18, where 18 equals the
number of bases in the primer. Thus the amount of new or useful
sequencing or size information for primer extension products
obtained using the modified primers of the present invention is
improved.
4.2 Oligonucleotide Compositions: Synthesis of Modified Primers
4.2.1 Feature of the Modified Primer
[0122] The oligonucleotide primers of the present invention (i) are
designed for optional attachment to a solid support in a manner
that does not block the ability to extend the primer from its 3'
end, and (ii) incorporate a cleavable moiety so that a 3' portion
of the primer linked to an extension segment can be released from
an upstream portion of the primer, 5' to the cleavable site. The
upstream portion of the timer, referred to herein as the first
primer region, typically contains a sigNificant number of the total
number of nucleotides present in the primer, so that upon cleavage
at the cleavable site, the amount of new fragment information for
primer extension products is maximized.
[0123] The modified primers of the invention preferable contain an
immobilization attachment site for attaching the primer to a solid
support. For modified primers containing an internal immobilization
attachment site (i.e., a site contained with the primer itself),
the immobilization attachment site or IAS is generally separated
from the cleavable site by at least three nucleotides. Upon
selective cleavage of the cleavable site, a large portion of the
primer fragment remains affixed to the solid support. This enables
the release of primer extension products that contain about five or
fewer base pairs of the primer sequence, to extend the useful size
analysis range (e.g., increased read lengths), as illustrated in
FIG. 15A and FIG. 15B.
[0124] FIG. 15A and FIG. 15B are mass spectra of products from
primer extension reactions, illustrating the difference in fragment
information obtained for cleaved primer extension segments
according to the invention (FIG. 15B) versus non-cleaved full
primer-extension segments (FIG. 15A). The details of the primer
extension reactions and primer cleavable are described in Example
8. For sequencing applications, the modified primers can also
provide more useful sequence information per fragment than
extension products containing the entire primer.
[0125] Exemplary oligonucleotide sequences for use as primers or
probes in the oligonucleotide compositions of the present invention
typically have lengths ranging from about eight to thirty
nucleotides, preferably between about ten or fifteen nucleotides to
about twenty or twenty five nucleotides. Typically, the
oligonucleotide sequences are complementary to a site upstream,
relative to the 5' end, of the target sequence of interest, based
on known sequence information of the target molecule. The
oligonucleotide sequence may additionally contain a label in the
releasable primer fragment (e.g., the second region), such as a
radioactive or fluorescent tag, depending upon the method of
sequence analysis employed.
[0126] The modified primers of the present invention, having a 5'
end and a 3' end, are generally composed of two separate nucleotide
regions. In one embodiment of the invention as illustrated in FIG.
5A, the two regions are connected by a cleavable site, as indicated
by "X". The heterocyclic bases, adenine, thymine, guanine, and
cytosine are commonly indicated in the figures by the symbol "B".
The first region containing the 5' end of the primer, contains an
immobilization attachment site, "I", for attachment to a solid
support. In the embodiment shown in FIG. 5A, the modified primer is
in immobilized form. The immobilization site may optionally be
separated from the 5' end of the primer by a spacer arm, as
indicated. Spacer arms for use in the present invention are
generally six or more atoms in length.
[0127] The number of nucleotides in the first region will vary, but
typically will be at least about three nucleotides. In a preferred
embodiment, the first primer region contains a significant portion
of the nucleotides (e.g., typically from about 3-20 nucleotides)
composing the modified primer. As shown, the cleavable linkage,
"X", is a 3'-5'-internucleotide cleavable site which connects the
first region to the second region. the second region, which
contains the 3' end of the primer, is composed of as few
nucleotides as is feasible, although the number will vary depending
on the primer employed. Preferably, the second region contains from
zero to five nucleotides and the total number of nucleotides in the
modified primer will be between about eight and thirty, and
preferably between ten and twenty five. The 3' end of the modified
primer serves as a priming site of enzymatic extension.
[0128] FIG. 5B illustrates an alternate embodiment of the present
invention in which a biotin molecule is connected to the 5' end of
a modified primer. The biotin is attached to the 5' end of the
primer through an extended spacer arm which serves to reduce steric
hindrance. Biotin, a relatively small vitamin molecule that binds
with high affinity to both avidin and streptavidin, is one
exemplary immobilization attachment site for use in the present
invention. Biotinylated primers are available from commercial
sources or may be synthesized by methods commonly employed in the
art, typically utilizing a functionalized biotin reagent containing
a reactive group suitable for coupling. As in FIG. 5A above, an
internucleotide cleavable linkage separates the two regions of the
modified primer. The second region contains a 3' end suitable for
enzymatic extension, which may take place either prior to or after
immobilization to a solid support.
[0129] FIG. 5C illustrates capture of the modified primer of FIG.
5B prior to enzymatic extension on an avidin-functionalized solid
support. In this embodiment of the invention, the modified primer
is immobilized via a high affinity interaction between avidin and
biotin, as indicated by "I".
[0130] FIG. 5D illustrates an alternate embodiment of the invention
in which the modified primer is attached to a solid support through
an immobilization attachment site present as a substituent on one
of the heterocyclic bases. As shown in FIG. 5D, the site for
immobilization is an amino residue substituted at the position of a
uracil (Dattagupta, 1989), and more specifically, is a 5-allylamino
substituent. The amino group may be in protected form (e.g.,
trifluoroacetamido) prior to attachment to the solid support. As
indicated, immobilization to the solid support is through an amide
linkage, although any of a number of immobilization attachment
linkages may be used, as will be described in more detail below.
Coupling of the amino residue to a solid support is generally
carried out by using an activated support material, such as an
N-hydroxysuccinimide (NHS) ester functionalized support.
[0131] In the embodiment shown in FIG. 5E, the immobilized primer
is in a branched or "T"-configuration. As in the above-embodiments,
the modified primer contains two regions separated by a cleavable
linkage indicated by "X". The first region contains the 5' end of
the primer and an immobilization attachment site as described
above. Referring to the design of a "T"-configured primer as shown
in FIG. 5D, generally, a large portion of the nucleotides composing
the modified primer and required the sequence specific target
binding are located 5' of the "central" deoxyribose, indicated by
an arrow. The second region contains the 3' end of the primer which
serves as a priming site for enzymatic extension. In the exemplary
modified primer shown, following hybridization to a target DNA, and
enzymatic extension followed by denaturing and washing, selective
cleavage of the cleavable site "X" releases the second region of
the modified primer along with the extension product (e.g., the
extension segment), while the first region containing a large
portion of the nucleotides required for sequence specific target
binding remains immobilized.
[0132] FIG. 5E illustrates an exemplary modified primer containing
a terminal cleavage site, as indicated by an (X). In this
embodiment of the invention, the cleavable linkage is represented
by a ribose moiety, although any of a number of terminal cleavable
sites may be employed. As shown, the modified primer is in
immobilized form and contains an immobilization attachment site 5'
of the cleavable site. The first region, contains the
immobilization attachment site and the portion of the primer up to,
but not including, the ribose. The ribose, or, alternatively, the
cleavable site, represents the second primer region and also serves
as a priming site for enzymatic extension.
[0133] Additional cleavable sites according to the invention
include nucleotides cleavable by an enzyme such as a nuclease or
glycosylase. FIG. 11 illustrates an exemplary cleavable primer
containing a restriction recognition site in the first primer
region and a cleavable site in the second primer region. As can be
seen in FIG. 11, the primer 75 contains two regions, a first 5'
region containing a restriction endonuclease recognition sequence
73 and a second region 76 containing a cleavable site 77,
downstream or 3' to the recognition sequence. Preferably, the
restriction endonuclease cleavable site is located at or within
about five nucleotides from the 3' end of the primer.
[0134] Representative restriction endonucleases for use in cleaving
the modified primers of the invention include BpmI, BsgI, BseRI,
BsmFI, and FokI, all of which make staggered cuts. A modified
primer including a BpmI or BsgI recognition site contains (i) a
first region which includes the recognition sequence, 5'-CTGGAG-3'
or 5'-GTGCAG-3', respectively, and (ii) located 16 bases downstream
from the last nucleotide of the recognition sequence, in the second
primer region, is the cleavable site. A modified primer containing
a BseRI or BsmFI recognition site, 5'GAGGAG-3' or 5'-GGGAC-3',
respectively, contains a cleavable site located 10 bases downstream
from the last nucleotide of the recognition sequence, while a
primer containing a FokI recognition sequence (5'-GGATG-3')
possesses a cleavable site 9 bases downstream from the last
nucleotide of the recognition sequence.
[0135] Returning now to FIG. 11, the first region of the primer
contains two separate domains. The first domain, 73, is composed of
a series of bases recognizable by a restriction endonuclease as
described above. The second domain of the first primer region, 76,
is 3' to the restriction endonuclease recognition sequence and
contains nucleotides complimentary to a target DNA molecule 81
which acts as a template for enzymatic extension of the primer. The
first domain of the first primer region 73 may optionally hybridize
to the target molecule.
[0136] After carrying out a primer extension reaction to form a
primer extension product 79, as will be described in more detail
below, the double stranded product is denatured 85, and an
oligonucleotide 83 complementary to both first 73 and second 76
domains within the first primer region is added to the reaction
mixture, preferably in an excess amount. Typically, the
complementary oligonucleotide 83 contains about 15-25 nucleotides,
sufficient to allow restriction enzyme recognition and cleavage at
the cleavable site. Preferably, the restriction endonuclease
cleavable site is at or near the 3' end of the primer.
[0137] The reaction mixture is then allow to cool and reanneal 86.
Due to the excess of complementary oligonucleotides 83 present,
hybridization of the primer-extension product to the
oligonucleotide compliment is favored, as indicated at 87.
Restriction endonuclease is then added to the mixture, as shown at
89, to promote cleavage at the cleavable site to release an
extension segment 91, a small fragment of the complimentary
oligonucleotide 3' to the cleavable site 93, and a larger
primer/complementary oligonucleotide fragment 5' of the cleavable
site 95.
[0138] A primer of the type described above may also contain an
immobilization attachment site (IAS) downstream from the cleavable
site, to enable immobilization of the extension segment.
Introduction of an IAS should not adversely affect (i)
sequence-specific binding of the template to the modified primer,
(ii) sequence-specific binding of the primer to the complimentary
oligonucleotide 83, (iii) enzymatic extension of the primer, or
(iv) the cutting ability of the restriction enzyme. Generally, the
extension product is immobilized and washed to remove reaction
products (salts, enzymes, nucleotide fragments, reagents) prior to
release and subsequent size and sequence analysis. Other approaches
include (i) the use of a primer or extension segment containing an
immobilization attachment site, where, after enzymatically
extending the primer and denaturing the double stranded product,
the single stranded primer-extension product is captured via
binding at the immobilization attachment site, followed by removal
of the template and addition of complementary oligonucleotide 83,
as described above, or (ii) the use of a template modified to
contain an immobilization attachment site, for capturing the
template either prior to or after enzymatic extension, prior to
addition of oligonucleotide 83.
[0139] A variation of a cleavable primer of the type illustrated in
FIG. 11 is shown in FIG. 16, where the first primer region contains
a universal restriction recognition site within a hairpin
(Szybalski, 1985). Referring now to FIG. 16, the cleavable site 127
is a class IIs restriction endonuclease cleavable site, where the
double stranded enzyme recognition sequence 129 is located in the
first primer region (i.e. upstream of the cleavage site), and the
first primer region contains a 5' hairpin-type (self-complimentary
double stranded) domain. The 5' hairpin domain 131 includes the
double stranded recognition site 129 for the restriction enzyme.
The second (single stranded) primer region (i) the cleavable site
(i.e. restriction endonuclease cut site), and (ii) is composed of
nucleotides complimentary to a single stranded target 133, thus
serving as a priming site for enzymatic extension. Following
enzymatic extension of the primer (shown at 135), the product 137
is cleaved 139 by treatment with a suitable class IIs restriction
endonuclease to release fragments 141 and 143, followed by
denaturation to release the single stranded extension segment for
subsequent analysis, i.e. by mass spectrometry. As indicated in the
figure at 145, the template may optionally be attached to a solid
phase support at any stage during the process.
[0140] In some instances, the cleavable site is a nucleotide
capable of blocking or terminating 5' to 3' enzyme-promoted
digestion by an enzyme having 5' to 3.varies.exonuclease activity,
such as T7 gene 6 exonuclease, ExoVIII, RecJ, and spleen
phosphodiesterase II. Such "blocking" nucleotides include
nucleotides containing phosphorothioate, borano-phosphate, or
peptide group as will be described below. In a prior extension
reaction utilizing a modified primer containing a blocking
nucleotide as the cleavable site, following a primer extension
reaction, the resulting product, composed of (i)a modified primer
containing a blocking nucleotide, and (ii)an extension segment, is
treated with a release such as an exonuclease having a 5' to 3'
exonuclease activity. Nuclease treatment typically results in
digestion of the first region of the primer to generate an
extension segment composed of nucleotides downstream (i.e. 3') of
the cleavable site.
[0141] In all of the exemplary embodiments described above,
cleavage of the cleavable site results in the release of newly
synthesized primer extension products containing little or none of
the nucleotide bases originally present in the modified primer.
[0142] 4.2.2 Introduction of the Cleavable Site
[0143] The cleavable site (composed of a modified nucleotide) is
typically introduced into an oligonucleotide probe by using one of
following synthetic approaches although many other approaches as
may be contemplated by those of skill in the art are also
encompassed by the present invention. Depending upon the choice of
cleavable site to be introduced, either a functionalized nucleotide
or a modified nucleotide dimer (or larger multimer) may first be
prepared, and then selectively introduced into a growing
oligonucleotide fragment during the course of primer synthesis. The
primer containing the cleavable site may be prepared using solution
synthesis, or preferably, employing automated solid phase synthesis
conditions using a DNA synthesizer.
[0144] In forming a modified dimer, two suitably protected
nucleotides are coupled to each other to form a modified
3'-5'-internucleotide linkage. The dimer containing the cleavable
site (or a protected form thereof) is then incorporated into the
oligonucleotide primer during synthesis to form a modified
oligonucleotide containing a cleavable site. The cleavable site is
chemically cleavable under select conditions but is stable under
standard solid phase DNA synthesis, solid support attachment,
primer extension and hybridization conditions.
[0145] Alternatively, funtionalization is carried out on a single
nucleotide to introduce a reactive group suitable for forming a
cleavable site upon reaction with a second nucleotide molecule or
during primer synthesis.
[0146] Although funtionalization may take place at sites within the
base or the sugar of the nucleotide, typically, modification will
be carried out to result in an oligonucleotide primer containing a
specific cleavable site in place of one of the phosphodiester
linkages of the resulting polymer or oligonucleotide. Preferred
non-internucleotide locations for modification or introduction of a
cleavable site include C(5) of thymine and N(4) of cytosine, as
these two base sites are readily chemically manipulated without
preventing base pairing.
[0147] A number of exemplary internucleoside cleavable sites for
use in the oligonucleotide composition of the present invention are
illustrated in FIGS. 1B-1W. FIG. 1A is an illustration of an
unmodified, native 3,-5,-phosphodiester linkage. A cleavable site
or linkage for use in the invention is one which may be introduced
at a specific position within the oligonucleotide sequence,
preferable at or within about five nucleotides from the 3' end of
the primer, and is selectively cleaved under conditions which do
not permit cleavage of the immobilization attachment site. In one
preferred embodiment, the cleavable site is located at the 3' end
of the primer. The cleavable linkage should also be one that is
chemically accessible.
[0148] Chemically cleavable internucleotide linkages for use in the
present invention include but are not limited to the following, as
illustrated in FIGS. 1B-1W, respectively: dialkoxysilane, FIG. 1B;
beta-cyano ether, FIG. 1C; 5'-deoxy-5'-aminocarbamate, FIG. 1D;
3'deoxy-3'aminocarbamate, FIG. 1E; urea, FIG. 1F;
2'cyano-3',5'-phosphodiester, FIG. 1G; 3'-(S)-phosphorothioate,
FIG. 1H; 5'-(S)-phosphorothioate, FIG. 1I; 3'-(N)-phosphoramidate,
FIG. 1J; 5'-(N)-phosphoramidate, FIG. 1K; alpha-amino amide, FIG.
1L; vicinal diol, FIG. 1M; ribonucleoside insertion, FIG. 1N.;
2'-amino-3',5'-phosphodiester, FIG. 1O; allylic sulfoxide, FIG. 1P;
ester, FIG. 1Q; silyl ether, FIG. 1R; dithioacetal, FIG. 1S;
5'-thio-formal, FIG. 1T; alpha-hydroxy-methyl-phosphonic bisamide,
FIG. 1U; acetal, FIG. 1V; and 3'-thio-formal, FIG. 1W. Other
chemically cleavable linkages include methylphosphonate and
phosphotriester. Cleavable linkages suitable for non-chemical
cleavage methods such as photolysis or thermolysis include
nitrobenzyl ether (NBE), cis-syn thymidine dimer (Nadji et al.,
1992), and cyclohexene.
[0149] Nucleoside dimers containing the cleavable linkages
illustrated in FIGS 1B-1W are synthesized using standard nucleic
acid chemistry known to one of skill in the art (Hobbs, 1990;
Townsend et al., 1986). Alternatively, one may directly synthesize
a modified nucleoside containing either a 5'- or 3'-reactive group
(or protected form thereof) for use in standard solid phase
synthesis to introduce the desired cleavable linkage.
2'-Functionalized nucleosides are typically prepared from the
corresponding ribonucleoside starting materials. An internucleotide
beta-cyano ether linkage, as shown in FIG. 1C, may be formed by
reaction of a suitably protected nucleoside with a
5'-(2-cyanoallyl) functionalized 3'-phosphoramidite. Selective
cleavage is effected by a beta-elimination reaction upon treatment
with base, promoted by the presence of the beta-cyano substituent.
A nucleoside dimer containing a 3'-(O)-carbamate internucleoside
bond is prepared by any of a number of synthetic approaches
including reaction between the corresponding 3'-acyl chloride and a
5'-amino-modified nucleoside. Alternatively, a 3'-modified
isocyanate nucleoside is prepared and subsequently reacted with the
5'-hydroxyl of a suitably protected nucleoside. A nucleoside dimer
containing a 5'-(O)-carbamate cleavable linkage is prepared from
the imadazole carbamate precursor.
[0150] Oligonucleosides containing methyl phosphonate linkages are
prepared using solid support based synthesis with phosphonamidite
reagents used in place of the standard phosphonamidites (Agrawal
and Goodchild, 1987). Phosphotriesters are some labile under basic
deblocking conditions, however, this cleavable group may be
introduced into an oligonucleotide backbone by using mild reaction
conditions or more labile amine protecting groups (MIller et al.,
1971). Methanol or ethanol in the presence of tosyl chloride is
used to esterify the internucleoside phosphate group (Moody et al.,
1989); methyl methanesulfonate may also be used as a methylating
agent (Koole et al., 1987).
[0151] Preferred cleavable sites for use in the modified
oligonucleotide composition include dialkoxysilane, ribose, 3'-and
5'-phosphoramidate, and 3'-and 5'-phosphorothioate.
[0152] In one embodiment of the present invention, the cleavable
site contained in the modified oligonucleotide primer is
dialkoxysilane (Ogilvie et al., 1986; Seliger et al., 1987; Cormier
et al., 1988). Synthesis of a primer containing a dialkoxysilane
internucleotide linkage is described in Example 1A. Although the
preparation of a diisopropylsilyl-linked dinucleoside is described
in Example 1A, alkyl groups for use as substituents on silicon are
not limited to isopropyl and may be either straight chain or
branched alky groups. Further, the two alkyl substituents on
silicon may be identical, as in the case of diisopropylsilyl, or
may be different. A variety of dialkylsilylating reagents are
available from Petrarch Systems, Bertram, P. A.
[0153] In the synthetic approach outlined in Example 1A, a reactive
3'-0-silyl ether intermediate is first prepared, followed by
formation of a nucleoside dimer containing a 3'-5'-diisopropylsilyl
internucleoside bridging group. Formation of a 3'-silyl triflate
intermediate is carried out by reacting a
5'-(O)-dimethosytrityl(DMT)-protected nucleoside, such as
5'-(O)-DMT0-thymidine or the N-protected nucleosides
N6-benzoyl-2'-deoxy-5'-(0)-DMT-adenosine, N4-benzoyl-2'-deoxy-5
'-(O)-DMT-cytidine, or N2-isobutryl-2'-deoxy-5'(O)-DMT-guanosine,
with an O-protected silane reagent.
[0154] In Example 1A, the protected nucleoside is treated with the
reactive silane, bis(trifluromethane-sulfonyl(diisopropylsilane, in
the presence of the sterically hindered base,
2,6-di-tert-butyl-4-methylprydine, to promote formation of the
desired 3'-(O)-diisopropylsilyl triflate intermediate. Use of a
bulky base such as the tri-substituted pyridine reagent helps to
prevent formation of the undesired symmetrical nucleoside dimer
formed by condensation of unreacted nucleoside with the triflate
intermediate (Saha et al., 1993).
[0155] Following introduction of the desire 3'-O-silyl ether group,
the 3'-O-diisopropylsilyl triflate intermediate is reacted with
unprotected nucleoside to form the desired nucleoside dimer
containing a 3'(O),5'(O)-dialkoxysilane cleavable site. The
protected dimer may then be further functionalized, for instance,
by conversion of the 3'-hydroxyl to the corresponding
2-cyanoethyl-N,N-diisopropylphosphoramidite for use in automated
solid phase synthesis utilizing standard phosphoramidite chemistry
to provide the desired primer sequence. Selective cleavage of the
dialkoxysilane site is effected by treatment with fluoride ion
(Corey and Snider, 1972).
[0156] Another preferred selectively cleavable functionality for
use in the invention is phosphorothioate. The preparation of
primers containing a 3'(S)-phosphorothioate or a
5'(S)-phosphorothioate internucleotide linkage is described in
Examples 1B and 1C, respectively. In accordance with the modified
oligonucleotide composition of the invention, the phosphorothioate
internucleotide linkage is selectively cleaved under mild oxidative
conditions (Cosstick et al., 1989).
[0157] In one synthetic approach for preparing primers containing a
3'(S)-phosphorothioate cleavable site as described in Example 1B, a
protected 3'-thio-substituted nucleoside starting materials, such
as 5-O-MMT-3'-S-benzoyl-3'-thymidine (Cosstick et al., 1988), is
first deprotected by treatment with base to form the debenzoylated
thiol, 5'-(O)-MMT-3'-thiotymidine, followed by conversion to the
corresponding reactive thiophosphoramidite by reaction with
2-cyanoethyl-N,N-diisopropylaminophosphormonochloridite. The
reactive thiophosphoramidite is coupled to a second nucleoside
molecule to form the corresponding thiophosphoramidite dimer,
followed by oxidation of the phosphorus center to form the fully
protected 3 '(S)-phosphorothioate-linked dimer.
[0158] In order to promote coupling of the thiophosphoramidite to a
second nucleoside molecule such as 3'-O-acetylthymidine and prevent
undesired self-condensation side reactions, an acidic activating
agent, 5-(para-nitrophenyl)tetrazole, is used. The thiophosphite
dimer is oxidized with a suitable oxidant such as
tetrabutylammonium oxone or tetrabutylammonium periodate to form
the fully protected (P--(O)-2-cyanoethyl-3'-acetyl) dimer
containing a protected form of the desired internucleoside linkage.
Deprotection is readily carried out under standard conditions as
described in Example 1B. As discussed above, the nucleoside dimer,
containing a 3'-(S)phosphorothioate cleavable linkage may be
readily incorporated into an oligonucleotide primer using standard
solid phase phosphoramidite chemistry.
[0159] Alternatively, one may use the reactive thiophosphoramidite
directly to introduce the desired 3'-(S)-phosphorothioate linkage
into an oligonucleotide primer during solid phase synthesis. For
introduction of a functionalized nucleoside containing a
3'-(S)-thiophosphoramidite during solid phase synthesis on
controlled pore glass, during the coupling cycle for introducing
the thio-modified nucleoside, the thio-modified nucleoside,
dissolved in acetonitrile saturated with
5-(para-nitrophenyl)tetrazole, is injected into the column
containing the solid support, and the coupling efficiency is
monitored by release of trityl cations.
[0160] After preparing the desired immobilized, cleavable primer in
accordance with the present invention, and carrying out the desired
hybridization and primer extension reactions, the phosphorothioate
internucleotide site is cleaved by treatment with a mild oxidizing
agent such as aqueous silver nitrate.
[0161] Preparation of the corresponding 5-(S)-phosphorothioate
modified oligonucleotide is carried out in a somewhat different
fashion than that described above for the 3'-(S)-phosphorothioate
and is described in detail in Example 1C. The approach makes use of
a key 5-thio-modified nucleoside intermediate for incorporation of
the desired 5'-(S)-phosphorothioate cleavable linkage during solid
phase oligonucleotide synthesis (Mag et al., 1991; Sproat et al.,
1987).
[0162] Synthesis of the nucleoside building block containing a
protected 5'thio group is carried out by first preparing the
5'-tosylate of thymidine by treatment with tosyl chloride, followed
by conversion of the 5'-tosylate to
5'-(S-trityl)-mercapto-5'-deoxythymidine. 5'-Tosyl-thymidine is
converted to 5'-(S-trityl)-mercapto-5'-deoxythymidine by treatment
with a five-fold excess of sodium tritylthiolate, which is prepared
in-situ by deprotonation of tritylmercaptan with sodium hydroxide.
In the above synthetic step, a sulfur atom is introduced into the
5'-position of a nucleoside, forming the S-trityl precursor of the
desired key intermediate. Subsequent phosphitylation at the
3'-position with 2-cyanoethoxy-bix-(N,N-diisopropylamino)phosphine
in the presence of tetrazole results in the desired functionalized
nucleoside,
5'-(S-trityl)-mercapto-5'-deoxythymidine-3'-O-(2-cyanoethyl-N,N-diisoprop-
ylamino)phosphite.
[0163] The 5'-(S)-protected nucleoside intermediate is introduced
into an oligonucleotide primer using standard solid-phase
phosphoramidite chemistry by first coupling it to a deprotected
polymer-bound oligonucleotide. The phosphite linkage is then
oxidized with aqueous I.sub.2, and the S-trityl group is cleaved
with silver nitrate and reduced with dithiothreitol to form a
reactive thiol. The thiol is then coupled to a
2'-deoxynucleoside-3'-phosphoramidite, followed by oxidation of the
thiophosphite linkage to yield the desired 5'-phosphorothioate
cleavable site.
[0164] Selective cleavage of the phosphorothioate site may also be
effected by treatment with an aqueous solution of either silver
nitrate (AgNO.sub.3) or mercuric chloride (HgCl.sub.2).
[0165] Another functional group for use as a cleavable site in the
modified oligonucleotide composition of the invention is
phosphoramidate. Oligonucleotides bearing phosphoramidate
internucleotide linkages can be prepared chemically by standard,
solid phase DNA synthesis (Bannwarth, 1988). Preparation of primers
containing a 5'-(N)-phosphoramidate internucleotide linkage is
described in Example 1D. In the synthetic approach described in
Example 1D, a 5'-amino-modified nucleoside is either purchase
commercially or synthesized by conversion of the 5'-hydroxyl group
to the corresponding azide, followed by reduction over a
palladium/carbon catalyst (Yamamote et al., 1980).
[0166] The 5'-amino group is then protected by treatment with
4-methoxytritychloride, followed by reaction with
bis(diisopropylammonium)tetrazolide and
(2-cyanoethoxy)bis(diisopropylamino)phosphine to form the
corresponding
3'-(2-cyanoethyl)-N,N-diisopropylphosphoramidite-functionalized
nucleoside. This reactive nucleoside, containing a 5'-protected
amino function, is then selectively introduced into an
oligonucleotide fragment during standard solid phase DNA synthesis
utilizing phosphoramidite chemistry, to form the desired
5'-phosphoramidate bond. Selective cleavage of the phosphoramidate
bond is carried out under mild acidic conditions, such as by
treatment with 80% acetic acid. Phosphoramidate linkages are more
labile in the ribo series than in the deoxyribo series (Tomasz et
al., 1981).
[0167] Another functional group for use a cleavable site in the
present oligonucleotide composition is ribose. Modified primers
containing a cleavable ribose are described in Examples 3-5.
Ribose, containing suitable O-protecting groups, is selectively
introduced into a growing oligomer fragment during automated solid
phase synthesis using standard phosphoramidite chemistry. Selective
cleavage is carried out by treatment with dilute ammonium
hydroxide, as described in Examples 3 and 5.
[0168] 4.2.3 Attachment to Solid Support
[0169] In accordance with one aspect of the invention, the
oligonucleotide primers (i) may be designed for attachment to a
solid support in a manner that does not block the ability to extend
the primer from its 3' end, and (ii) incorporate a cleavable moiety
so that a 3' portion of the primer (linked to an extension product)
can be released from an optionally immobilized 5' portion.
[0170] The oligonucleotide primers of the invention are preferably
designed for binding to a solid support material directly, through
an intervening spacer arm, or by specific hybridization to an
intermediary oligonucleotide which is bound to a solid support
(SPBIO). Immobilization may occur at a location upstream (i.e. 5'
to) or downstream (i.e. 3' to) of the cleavable site.
[0171] Attachment to a solid phase may also take place via an
attachment site (i) contained within a nucleic acid extension
segment resulting from an enzymatic extension reaction, or, (ii)
contained within a target nucleic acid.
[0172] The immobilization attachment site can be a select
functional group for covalent bonding to a solid support, such as
those representative functional groups shown in FIGS. 2A-2K, and
FIG. 2M. The immobilization attachment site can also be a ligand
such as biotin, for attachment via a high-affinity non-covalent
interaction with a solid support.
[0173] Further, the immobilization attachment site can also be
composed of a series of bases complementary to an intermediary
oligonucleotide bound to a solid support, as illustrated in FIG. 8
and FIG. 9.
[0174] Referring now to FIG. 8, a primer having a cleavable site 17
as described above is (i) hybridized to a single stranded, target
DNA sequence 21 utilizing conditions under which the target will
anneal stably to the primer, and (ii) enzymatically extended to
form an extension segment 19. The extension product is then exposed
to an intermediary oligonucleotide which is bound to a solid
support 23. The intermediary oligonucleotide is complementary to
all or at least the first region of the primer.
[0175] In the embodiment illustrated in FIG. 8, the sequence of the
intermediary oligonucleotide is homologous with at least a portion
of the target molecule 21, so that both the intermediary
oligonucleotide and the target are competing to hybridize to an
overlapping region of the primer. In instances in which the
sequence of the extension product is known, the sequence of the
intermediary oligonucleotide may be designed to be complementary to
a portion of the extension segment rather than to the primer, or,
to a region containing portions of both the primer and the
extension segment.
[0176] In employing this approach for immobilization, the
concentration of target molecule relative to intermediary
oligonucleotide is preferably reduced in order to favor
hybridization of the primer extension product to the intermediary
oligonucleotide. Hybridization of the extension product to template
is thermodynamically favored, since the template is capable of
hybridizing to the full length of the extension product. The
concentration of target nucleic acid can be reduced by a number of
methods, including specific chemical or enzymatic digestion which
leaves the primer extension product intact.
[0177] Selective digestion of the template may be effected by any
of a number of methods, including the following: (i) use of a
deoxyuridine-containing template; (ii) use of an RNA template to
provide DNA extension products; (iii) use of a template containing
modified internucleoside linkages; or (iv) exonuclease-promoted
digestion of template. Each is described in greater detail
below.
[0178] In employing the first approach, a nucleic acid fragment
which has been site selectively modified (Longo et al., 1990) to
contain deoxyuridine in place of deoxythymidine is used as a
template for the primer extension reaction. Following enzymatic
extension, the template-containing reaction mixture is treated with
uracil DNA glycosylase (Amersham Life Sciences, Arlington Heights,
Ill.) to fragment the template molecule at positions modified to
contain deoxyuridine. Uracil DNA glycosylase excises deoxyuracil
from dU-containing DNA by cleaving the N-glycosidic bond between
the uracil base and the sugar phosphate backbone.
[0179] In the second approach, an RNA template is used to provide
DNA extension products. The template is then selectively removed by
digestion using RNase, such as RNase A.
[0180] Alternatively, as indicated by (iii) above, a template
molecule containing modified internucleoside linkages such as a
phosphoramidate or phosphorothioate is used. Following extension of
the primer, the template is digested by chemically-promoted
cleavage at the modified linkage positions. The choice of
template-modified internucleoside linkage and template-digestion
reagent will depend upon the type of cleavable site present in the
primer. Digestion of template is typically carried out under
conditions which leave the primer cleavable site intact. Cleavage
of phosphorothioate linkage (5'-(O)--P(S)O.sub.2) can be effected
by treatment with glycidol or iodoethanol (Olsen, 1992), while
selective cleavage of a phosphoramidate bond is typically carried
out under mild acidic conditions, such as by treatment with 80%
acetic acid.
[0181] In utilizing exonuclease-promoted digestion of template (as
indicated by (iv) above), the primer extension product is modified
to contain a suitable exonuclease-resistant blocking group as
described previously. Upon exonuclease treatment with either a
3'-5' specific or a 5'-3' specific exonuclease, the "protected"
primer extension product then remains intact, due to the presence
of the blocking group. In utilizing a 3'-5' exonuclease (e.g. snake
venom phosphodiesterase or exonuclease III), a suitable blocking
group is placed at the 3' terminus of the extension product to
prevent enzyme-product to prevent enzyme-promoted degradation.
[0182] The relative concentration of template to solid-bound
intermediary oligonucleotide, can also be minimized, for example
(i) by performing cycle synthesis procedures with limited amounts
of template (e.g., cycle sequencing or strand displacement
amplification), or (ii) adding a large excess (e.g., 10 to
100-fold) of intermediary oligonucleotide to the reaction
mixture.
[0183] Returning now to FIG. 8, under conditions which favor
hybridization of the primer extension product to the solid phase
bound intermediary oligonucleotide, the primer extension product is
immobilized by hybridization to the solid phase bound intermediary
oligonucleotide 23 to form captured product 27 and free (i.e.
single stranded template). The 5' end of the solid phase bound
intermediary oligonucleotide may terminate before or after the
cleavable site of the modified primer. After immobilization as
described above, excess reaction products are removed by washing 29
to provide a purified immobilized product 31. The immobilized
product is cleaved 33 to release the extension segment 35 for
subsequent analysis.
[0184] In accordance with the present invention, immobilization of
a cleavable extended primer by hybridization to an intermediary
solid phase bound oligonucleotide is described in Example 6.
[0185] Briefly, a modified M13 reverse primer containing a
5'-(S)-thymidine cleavable group (SEQ ID NO:12) was (i) hybridized
to a single stranded target, and (ii) enzymatically extended in the
presence of dideoxythymidine to produce a set of
dideoxythymidine-terminated extension fragments at a 8:1 ratio of
primer to template. The single stranded extension products were
then annealed to an intermediary oligonucleotide (SEQ ID NO:13)
complementary to the M13 reverse primer and biotinylated at the 3'
end. The extension product-intermediary oligonucleotide hybrids
were then immobilized by addition of streptavidin-coated magnetic
beads, washed, and the extension product release by silver-nitrate
promoted cleavage of the 5'-(S)-thymidine cleavable group. The
extension segments were analyzed by MALDI time-of-flight mass
spectrometry, as shown in FIG. 13. As can be seen, extension
segments with read lengths up to at least about 33 base pairs can
be detected with good resolution.
[0186] In a variation of the above approach as shown in FIG. 9, the
primer 39 is designed to contain an immobilization attachment site
38 contained in the first primer region composed of a series of
bases complementary to an intermediary oligonucleotide bound to a
solid support. However, in this embodiment, the intermediary
oligonucleotide does not share homology with the template molecule
45, so that the intermediary oligonucleotide and template do not
compete with one another for hybridization to the primer. As
illustrated in FIG. 9, following enzymatic extension to form
extension segment 43, the primer is immobilized by specific
hybridization to the solid phase bond intermediary oligonucleotide
37. This design of primer, template, and intermediary
oligonucleotide allows for simultaneous, non-competitive
hybridization of the primer to both the template and intermediary
oligonucleotide. The solid phase bound extension product is washed
and the template is eliminated 47 to provide a purified immobilized
product 49, which is then cleaved 51 at cleavable site 41 to
release extension segment 53 for subsequent size and/or sequence
analysis as described for FIG. 8 above.
[0187] Alternatively, a primer of the present invention may be
bound to a solid phase via hybridization to a target nucleic acid
which is immobilized, as shown in FIG. 10. In utilizing this
approach, the target molecule 61 acts as both a template for
enzymatic extension of the primer 55 and as an intermediary for
solid phase binding of the primer. The template can be attached to
the solid phase either before or after carrying out enzymatic
extension of the primer to form the extended primer segment 59. As
has been described, immobilization of the primer extension product
allows of ready removal of excess enzyme, salts, etc., 63, to
provide a purified immobilized primer extension product 65. Prior
to analysis, the extended primer is denatured from the template 67
and released into solution. Cleavage at the cleavable site 57
promotes release of an extension segment 71 and a fragment composed
of the first primer region 69. Depending upon the design of the
primer and the mode of product analysis, the presence of primer
fragment 69 may adversely impact the quality of the subsequent
product analysis. In these instances, the same methods used to
eliminate template, as described above, may be used to eliminate
fragment 69. In cases in which the extension segment is sized by
mass spectrometry, the mass of fragment 69 can, in some instances,
be selected to avoid interference with product peaks in the
resulting mass spectra, and may also be used to provide an internal
mass standard.
[0188] Upon attachment of the immobilization attachment site to a
solid support, the resulting immobilization linkage is generally
one which remains stable under the conditions employed for cleaving
the cleavable site and does not inhibit base pair hybridization nor
block the ability to extend the primer from its 3' end. In the
modified primer of the invention, the immobilization attachment
site is typically separated from the cleavable site by at least
three nucleotides. In a preferred embodiment, upon selective
cleavage of the cleavable site, a large portion of the primer
fragment remains affixed to the solid support. This enables the
release of primer extension products that typically contain about
five or fewer base pairs of the primer sequence, to provide more
useful sequence information per fragment than extension products
containing the entire primer.
[0189] The modified primers of the present invention may, for
example, be used for detecting a genetic disorder for which the
nucleotide sequence of both the wild type and mutant alleles are
known. A modified primer for this purpose will have a 5' end and a
3' end and contain from about 8-30 base pairs complementary to the
gene sequence upstream from the known mutation site. Preferably,
the 3' end of the primer is complementary to a site upstream from
the known mutation region by at least about ten base pairs, to
provide verifying sequence information on either side of the
mutation region.
[0190] In accordance with one aspect of the invention, the modified
primer also contains (i) an immobilization site for attachment to a
solid support and (ii) a cleavable site. One primer design
according to the present invention is one in which the
immobilization site is located 5' of the cleavable site which is
preferably located at or within about five base pairs from the 3'
end of the primer.
[0191] This modified primer is then used as a probe to distinguish
the presence of DNA containing the mutant sequence of interest. The
primer is (i) hybridized to an unknown, single stranded, target DNA
sequence utilizing conditions under which both the mutant and the
normal sequences will anneal stably to the primer, and (ii)
enzymatically extended. Primer immobilization may optionally take
place either before or after chain extension. Following chain
extension and release of the immobilized primer extension products
by selective cleavage of the cleavable linkage, the primer
extension products are analyzed to determine the sequence across
the known mutation region and identification of the genetic
disorder, if present.
[0192] An exemplary modified primer containing 20 deoxynucleotide
residues and specific for its ability to detect a know genetic
disorder is shown in FIG. 6A and FIG. 6B. As indicated, the
modified primer contains a first region containing an
immobilization attachment site, "I", that is 5' of the cleavable
site, "X", and consists of a total of 16 nucleotide residues. The
second region contains the 3' end of the primer and contains 4
nucleotides (C-T-G-C). The cleavable linkage, X, connects the first
and second regions.
[0193] In illustrating this aspect of the invention, the modified
primer as shown in FIG. 6A (top) and having the sequence presented
as SEQ ID NO:2, is first hybridized to a single stranded DNA target
having the sequence presented SEQ ID NO:3, as shown in FIG. 6A.
Typically, the hybridization medium contains (i) denatured, unknown
(target) DNA from a human or other biological source, (ii) the
modified probe, and (iii) annealing buffer, such as 5.times.
"SEQUENASE" Buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl.sub.2, 250
mM NaCl) (United States Biochemical Corporation, Cleveland, Ohio).
The annealing reaction is carried out by warming the above mixture
to 65.degree. C. for two minutes, and then allowing the mixture to
cool slowly to room temperature over a period of about thirty
minutes (Maniatis et al., 1982; Ausubel et al., 1989).
[0194] Following hybridization, the modified primer is extended on
the single stranded template with deoxynucleotides and randomly
terminated with dideoxynucleosides using DNA polymerase (e.g.,
"SEQUENASE" DNA Polymerase, Version 1.0 or 2.) to perform DNA
synthesis (Primings et al., 1980; Sanger, 1975). As indicated in
FIG. 6A, extension occurs from the 3' end of the modified primer.
The primer extension products are denatured from the target,
typically using heat or a chemical denaturant such as formamide, to
provide a mixture of both primer extension products and target DNA.
(See, for example, "PROTOCOLS FOR DNA SEQUENCING WITH SEQUENASE T7
DNA POLYMERASE", Version 1.0 or 2.0,4th ed., United States
Biochemical, or "CIRCUMVENT THERMAL CYCLE DIDEOXY DNA SEQUENCING
KIT INSTRUCTION MANUAL", New England Biolabs, Inc., Beverly
Mass.).
[0195] As shown in FIG. 6B, the primer extension products are then
bound to the solid support at the immobilization attachment site,
although immobilization may optionally be carried out prior to
enzymatic extension and/or denaturation. By immobilizing the
extended primers, the target DNA strands which remain free in
solution are readily removed, along with excess reagents, ions,
enzymes and like, in a series of wash steps. Generally, the sold
substrate is washed with large volumes of a wash solution (e.g., 10
mM Tris HCl, 1 mM EDTA; or pure water) at room temperature.
[0196] The solid particles, containing immobilized primer extension
products and free of impurities, are then submitted to conditions
effective to selectively cleave the cleavable site while
maintaining the first primer region having the sequence presented
as SEQ ID NO:4 affixed to the solid support as shown in FIG. 6B. As
indicated in the particular embodiment illustrated in FIG. 6B,
selective cleavage results in release of primer extension products
containing only four nucleotides from the original modified primer.
The supernatant containing the released extension segments is
suitable for subsequent analysis.
[0197] Immobilization simplifies purification of the primer
extension products for subsequent analysis. As discussed above,
undesirable enzymes, salts, reagents, and sequencing targets are
washed away prior to selective cleavage of the extension
product.
[0198] In immobilizing the modified primers of the present
invention, the first region of the primer may be attached to the
solid support material either prior to or after introduction of the
cleavable site. Any of a number of methods commonly employed in the
art may be utilized to immobilize an oligonucleotide on a solid
support (Saiki et al., 1989; Zhang et al., 1991; Kremsky et al.,
1987; Van Ness et al., 1991). Solid support materials for use in
the invention include cellulose, nitrocellulose, nylon membranes,
controlled-pore glass beads, acrylamide gels, polystyrene matrices,
activated dextran, avidin/streptavidin-coated polystyrene beads,
agarose, polyethylene, functionalized plastics, glass, silicon,
aluminum, steel, iron, copper, nickel, silver and gold.
[0199] Some substrates may require functionalization prior to
attachment of an oligonucleotide. Solid substrates that may require
such surface modification include aluminum, steel, iron, copper,
nickel, gold, and silicon. In one approach, the solid substrate
material is functionalized by reaction with a coupling agent, such
as zircoaluminate.
[0200] Zircoaluminates generally contain both oxo and hydroxy
bridges and are characterized by high thermal and hydrolytic
stability. Such compounds, due to their highly metallic nature, are
particularly reactive with metal surfaces such as the metallic
solid supports described above. Bi-functional zircoaluminates
containing a variety of organofunctional groups are commercially
available (e.g., "MANCHEM" Zircoaluminates, Rhone-Poulenc Latex
& Specialty Polymers, Cranbury, N.J.).
[0201] Upon attachment to a solid support, the oligonucleotide,
typically DNA, should couple efficiently to the solid support
material. Further, the immobilized DNA should be both stable upon
immobilization and accessible for base hybridization and other
potential derivatization reactions. The immobilization attachment
site should remain stable under the conditions employed for
selectively cleaving the cleavable site in the modified
oligonucleotide composition of the invention.
[0202] Coupling of an oligonucleotide to solid support may be
carried out through a variety of immobilization attachment
functional groups. Immobilization attachment sites for use in the
present invention include those illustrated in FIGS. 1A-2M.
Attachment of the support material to the oligonucleotide may occur
by reaction between the reactive site on the support and a reactive
site contained within the oligonucleotide or via an intervening
linker or spacer molecule.
[0203] Although any suitable functional group fulfilling the
desired criteria above may be used to attach the oligonucleotide to
the support, preferred linkages include disulfide (FIG. 2G),
carbamate (FIG. 2B), hydrazone (FIG. 2J), ester (FIG. 2C, FIG. 2I
and FIG. 2K, where Y equals oxygen), (N)-functionalized thiourea
(FIG. 2D), functionalized maleimide (FIG. 2A, where Y equals
sulfur, oxygen, or nitrogen), streptavidin or avidin/biotin (FIG.
2L), mercuric-sulfide (FIG. 2E), gold-sulfide (FIG. 2M), amide
(FIG. 2C, FIG. 2L and FIG. 2K, where Y equals nitrogen), thiolester
(FIG. 2C, FIG. 2L and FIG. 2K, where Y equals sulfur). Other
suitable functionalities for attaching to a solid support material
include azo, ether, and amino.
[0204] The immobilization attachment site may be located (i) as a
substituent along the modified primer backbone (e.g.,
derivatization occurring at a terminal 5'-hydroxyl position), (ii)
as a substituent on one of the bases or sugars of the modified
primer, (iii) in the first region of the primer, composed of a
series of bases complementary to a solid phase bound intermediary
oligonucleotide, (iv) within a nucleic acid extension segment
resulting from an enzymatic extension reaction, or (v) contained
within a target nucleic acid.
[0205] Immobilization via a base pairing interaction between the
primer and a solid phase bound intermediary oligonucleotide (SPBIO)
is shown in FIG. 8 and FIG. 9. Indirect immobilization of the
primer extension product via a base pairing interaction to solid
phase bound template is shown in FIG. 10.
[0206] Solid support materials for use in coupling to an
oligonucleotide include functionalized supports such as the
1,1'-carbonyldiimidazole activated supports available from Pierce
(Rockford, Ill.) or functionalized supports such as those
commercially available from Chiron Corp. (Emeryville, Calif.).
Solid supports for use in the present invention include matrix
materials such as 6% cross-linked agarose, Trisacryl GF-2000 (a
hydrophilic matrix material) and TSK HW-65F, all activated with
1,1'-carbonyldiimidazone (Pierce). Immobilization is typically
carried out by reacting a free amino group of an amino-modified
oligonucleotide with the reactive imidazole carbamate of the solid
support. Displacement of the imidazole group results in formation
of a stable N-alkyl carbamate linkage between oligonucleotide and
the support as shown in FIG. 2B. Coupling is usually carried out at
pHs ranging from 9-11 although a pH range from 9.5-10 is
preferable. Coupling to pH sensitive materials may be carried out
in buffer at pHs around 8.5.
[0207] Amino-modified oligonucleotides for use in attaching to a
solid support may be synthesized using standard solid phase DNA
synthesis methodologies employing, for example, the modified
nucleoside phosphoramidite Amino-Modifier-dT (Glen Research,
Sterling, Va.), which contains a base labile trifluoroacetyl group
protecting a primary amine attached to thymine via a 10-atom spacer
arm, phosphoramidite 5'-Amino-Modifier C6 (Glen Research, Sterling,
Va.), which contains a primary amino group protected with an acid
labile monomethoxytrityl group, or
N-trifluoroacetyl-6-aminohexyl-2-cyanoethylN',N'-isopropylphosp-
oramidite (Applied Biosystems, Foster City, Calif.). Although
amino-containing oligonucleotides are most commonly prepared using
phosphoramidite chemistry, any other method which leads to
oligonucleotides containing primary amine groups may also be
used.
[0208] Amino-modified oligonucleotides are readily transformed to
the corresponding thiol or carboxyl-terminated derivatives for use
in immobilization or spacer arm attachment reactions requiring
5-functionalities other than amino. Amino-modified oligonucleotides
may be converted to the corresponding carboxyl derivatives by
reaction with succinic anhydride (Bischoff et al., 1987). If
desired, the carboxyl-derivatized primer may be coupled to a
bifunctional linker such as 1,6-diaminohexane prior to attachment
to the solid support by carrying out the coupling reaction in the
presence of an activating agent such as a water soluble
carbodiimide.
[0209] Thiol-modified oligonucleotides may be prepared by treating
the deprotected 5'amino group of a functionalized oligonucleotide
with dithiobis(succinimidylpriopionate), followed by sulfhydryl
deprotection with dithioerythritol (Bischoff et al., 1987).
[0210] Oligonucleotides containing free amino, thiol, and hydroxyl
functions may also be coupled to supports by utilizing epoxide
ring-opening reactions (Maskos et al., 1992). One such exemplary
epoxy-activated solid support is available from Pierce (Rockford,
Ill.) and contains 1,4-butanediol diglycidyl ether-activated
agarose. Coupling reactions are typically carried out at pHs from
7.5-13, depending upon the stability of the molecule to be
immobilized. In immobilization reactions carried out with the above
Pierce support, the resulting immobilized oligonucleotide is
separated from the solid support by a 13-atom hydrophilic spacer
arm.
[0211] In another immobilization approach, aldehyde groups of a
modified oligonucleotide are coupled to hydrazide groups on a solid
matrix as shown in FIG. 2J. A primary hydroxyl group on an
oligonucleotide is first oxidized to the corresponding aldehyde,
typically with a mild oxidant such as sodium periodate. The
oligonucleotide is then coupled to a hydrazide-containing matrix
such as Pierce's CarboLink.TM. Hydrazide. The coupling reaction is
performed at neutral pH.
[0212] Alternatively, the immobilization reaction is carried out
using a thiol-derivatized oligonucleotide which is coupled to a
functionalized matrix such as Pierce's Immobilized
p-Chloromercuribenzoate (FIG. 2E). The support is a cross-linked
agarose containing an ethylene diamine spacer and coupling takes
place via affinity binding between the mercury and sulfur atoms.
Similarly, as shown in FIG. 2M, immobilization may be carried out
by anchoring a 5'-thiolated primer to a gold surface (Hegner et
al., 1993a). Using this approach, the modified primer is
chemisorbed onto a gold surface e.g., the solid support) via
thiolate bonding. The preparation of polycrystalline gold surface
has been previously described (Hegner et al., 1993b).
[0213] Funtionalization may also be carried out using a homo- or
hetero-bifunctional cross-linker, such as Pierce's Sulfo-SMCC.
Cross-linkers for use in the present invention will typically
contain spacer arms between about 3-20 angstroms in length.
Cross-linkers for use in coupling an oligonucleotide to a solid
support will typically contain functional groups for targeting
reactive primary amines, sulfhydryls, carbonyls, and carboxyls.
Cross-linking agents for reaction with primary amino groups will
typically contain terminal amidoester groups or
N-hydroxysuccinimidyl esters. An exemplary linker such as Pierce's
Sulfo-SMCC contains a reactive carboxyl group at one end for
coupling to amine-derivatized solid supports such as
hexylamine-derivatized polystyrene beads. The other end of the
linker molecule contains a reactive maleimide molecule which
readily reacts with oligonucleotides containing nucleophilic groups
such as hydroxy, thio, or amino. Cross-linkers for reaction with
sulfhydryl groups typically contain terminal maleimide groups,
alkyl or aryl halides, alpha-haloacyls or pyridyl disulfides. A
variety of chemical cross-linking agents are available from Pierce
(Rockford, Ill.).
[0214] Alternatively, coated plates, such as those available from
Pierce, may be used to immobilize the modified oligonucleotide.
Examples of plates for use in immobilizing the oligonucleotides of
the invention include activated plates such as Pierce's
Reacti-Bind.TM. Streptavidin Coated Polystyrene Plates. A primary
amino-containing oligonucleotide is immobilized on the former plate
surface by covalent attachment through a stable amide bond formed
by reaction between the free amino group of the oligonucleotide and
the reactive anhydride (FIG. 2H). The latter plates are effective
for affinity binding of biotinylated oligonucleotides. Gold-coated
plates may also be utilized for binding to thiol-derivatized
primers.
[0215] Biotinylated oligonucleotides for use in immobilization to
streptavidin or avidin-coated solid supports are prepared as
described in Example 2A and shown in FIG. 2L. A variety of
biotinylation reagents are commercially available (e.g., Pierce)
which are functionalized to react with molecules such as modified
oligonucleotides containing primary amino, sulfhydryl, or
carbohydrate groups.
[0216] Returning to Example 2A, an amino-modified primer is treated
with biotin or with a modified form of biotin containing an
intervening spacer arm, such as NHS-SS-Biotin (Pierce), or
NHS-LC-Biotin (Pierce), a biotin derivative containing an eleven
carbon spacer arm between biotin and a terminal
N-hydroxylsuccinimide activated carboxyl group. The biotinylated
primer is then immobilized by attachment to a streptavidin-coated
support. Due to the strong non-covalent biotin/streptavidin
interaction, the immobilized primer is considered to be essentially
irreversibly bound to the solid support. This is one preferred
immobilization attachment for use in the present invention, as the
resulting immobilized complex is unaffected by most extremes of pH,
organic solvents, and other denaturing agents (Green, 1975). An
alternative to avidin(streptavidin)-biotin immobilization is
incorporation of a digoxigenin molecule (Signa, St. Louis, Mo.) in
the modified primer with subsequent capture using anti-digoxigenin
antibodies.
[0217] Enzymatic methods may also be utilized for coupling an
oligonucleotide to a solid support (Goldkorn et al., 1986). In one
exemplary embodiment, a poly(dA) tail is added to the 3' ends of a
double stranded DNA using 3' terminal transferase. The (dA)-tailed
DNA is then hybridized to oligo(dT)-cellulose. To covalently link
the DNA to the solid support, the hybridized sample is first
reacted with a Klenow fragment of DNA polyermase I, followed by
treatment with T4 DNA ligase. The unligated strand of DNA is
separated from the immobilized strand by heating followed by
extensive washing. The method results in ssDNA covalently linked by
its 5' end to a solid support.
[0218] The modified primers of the present invention may also be
affixed onto a gold surface. In utilizing this immobilization
approach, oligonucleotides modified at the 5' end with a linker arm
terminating in a thiol group are chemisorbed with high affinity
onto gold surfaces (Hegner et al., 1993a). Thilated primers,
available through post solid-phase synthesis modification using
commercially available reagents (Pierce, Rockford Ill.), are
immobilized on a thin layer of gold either prior to or following
enzymatic extension. The gold layer is deposited onto the sample
stage for direct analysis by mass spectrometry following internal
cleavage and evaporation. Alternatively, the resulting extension
segments may be transferred onto an alternate surface prior to
analysis.
[0219] 4.3 Reactions Employing the Immobilized Cleavable
Oligonucleotide Composition
[0220] 4.3.1 Hybridization and Extension
[0221] The methods employed for determining the sequence of a
target oligonucleotide strand will often involve Sanger-type
sequencing using the modified cleavable primers of the present
invention. Immobilization of the modified primer on the solid
support may take place either before or after the enzymatic
extension reactions.
[0222] Utilizing the Sanger DNA sequencing procedure,
dideoxynucleosides of each of the four bases are obtained for
inclusion into the reaction mixture. The dideoxy nucleotides are
incorporated into DNA by, for example, E. coli DNA polymerase since
they have a normal .sub.5' triphosphate group. Once incorporated
into the growing DNA strand, the dideoxynucleoside triphosphate
(ddNTP) cannot form a phosphodiester bond with the next incoming
dNTP and growth of the DNA chain is terminated.
[0223] A typical DNA sequencing reaction using the Sanger method
proceeds as follows. The reaction consists of a target DNA strand
be sequenced, a modified primer containing a cleavable site in
accordance with the invention, and that is complementary to the end
of the target strand, a carefully controlled ration of one
particular dideoxynucleoside with its normal deoxynucleotide
counterpart, and the three other deoxynucleoside triphosphates. The
modified primer may or may not be immobilized to solid support at
this point. (Immobilization may occur either before or after the
enzymatic extension reactions, depending on a number of
experimental factors).
[0224] DNA polymerase is added and normal polymerization begins
from the primer. Upon incorporation of a ddNTP, the growth of the
chain is stopped. A series of different strands results, the
lengths of which are dependent on the location of a particular base
relative to the end of the DNA. The target strand is usually
distributed into four DNA polymerase reactions, each containing one
of the four ddNTPs and a modified primer of the present invention.
The extension reaction is then carried out as described above.
Sanger-type DNA sequencing is generally carried out using a DNA
sequencing kit such as "SEQUENASE" Version 1.0 or Version 2.0 T7
DNA Polymerase (United States Biochemical, Cleveland, Ohio). The
"SEQUENASE" Version 1.0 kit uses a chemically-modified DNA
polymerase derived from bacteriophage T7 DNA polymerase in which
the high 3'-5' exonuclease activity of the native T7 DNA polymerase
is inactivated.
[0225] In using the USB "SEQUENASE" kit, double stranded templates
are first denatured (if one is using double stranded template), and
the primer is then hybridized or annealed to the target by heating
for 2 min at 65.degree. C., followed by low cooling to less than
35.degree. C. over about 15-30 min. Supercoiled plasmid DNAs are
denatured by treatment with sodium hydroxide, neutralized, and
ethanol-precipitated in order to anneal the primer for
sequencing.
[0226] Termination mixtures containing the different ddNTPs are
prepared for use in the termination reactions. The annealed DNA
mixture is optionally labeled, and the labeled reaction mixture is
added to each of the four termination tubes. In the present
invention, extension reactions are carried out to produce a
distribution of products ranging from near zero to several hundreds
of base pairs in length. Optionally, a stop solution (containing
formamide, EDTA, bromophenol blue, and xylene cyanol FF) is added
to stop the reactions prior to analysis of the resultant
samples.
[0227] For reactions in which the modified primers were not
immobilized to a solid support prior to enzymatic extension,
immobilization is carried out as described in Section 4.2.2
above.
[0228] The immobilized extended primers are then washed to remove
excess enzymes, ions, salts, and other impurities. In one
embodiment, the extended primers are immobilized onto the surface
of microtiter wells. Immobilization to the solid support
facilitates product purification and subsequent isolation by
cleavage of the timer at the cleavage site followed by removal in
the supernatant.
[0229] Alternatively, DNA sequencing may be carried out using
deoxynucleotide alpha-thiotriphosphates, dNTPalphaSs (available
from United States Biochemical, Cleveland, Ohio), followed by
limited exonuclease-promoted base-specific digestion, such as with
Exonuclease III (New England BioLabs, Beverly, Mass.) or snake
venom phosphodiesterase (Boehringer Mannheim, Mannheim, Germany)
(Olsen et al., 1993). Cleavage of DNA fragments specifically at
positions of incorporated phosphorothioate groups may also be
carried out using chemical reagents such as 2-iodoethanol or
2,3-epoxy-I-propanol (Nakamaye et al., 1988).
[0230] Briefly, the sequencing of a target DNA sequence using the
modified primers of the present invention via the incorporation of
phosphorothioate nucleosides is carried out as follows. A target
DNA sequence is hybridized with modified primer as described above.
The primer is then extended enzymatically in the presence of one
deoxynucleotide alpha-thiotriphosphate (dNTPalphaS) to generate a
mixture of prime extension products containing phosphorotioate
linkages. The primer extension products are then treated with a
reagent that (i) cleaves specifically at the phosphorothioate
linkages such as 2-iodoethanol or 2,3-epoxy-1-propanol, or (ii)
digests DNA downstream from the phosphorothioate linkage, such as a
3'5' exonuclease, under conditions resulting in the production of a
nested set of base-specific primer extension degradation
products.
[0231] Optionally, the primer extension degradation products are
immobilized at the immobilization attachment sites to produce
immobilized primer extension degradation products, each containing
a primer and an extension segment. Alternatively, immobilization
may be carried out (i) prior to enzymatic extension, (ii) after
enzymatic extension, or (iii) prior to treating the
phosphorothioate-containing primer extension products with a
phosphorothioate-specific cleaving reagent.
[0232] Subsequent to immobilization, the primer extension
degradation products are washed to remove non-immobilized species.
Cleavage at the cleavable site results in the release of extension
segments, which are then sized by mass spectrometry. Using the
sequencing method of this aspect of the invention, the read length
of any given extension segment is increased relative to the read
length of its corresponding primer extension degradation
product.
[0233] The methods and modified primers described herein may also
be used for obtaining a fingerprint of a target oligonucleotide. As
described herein, fingerprinting refers to a method of determining
the positions of no more than two different bases in a target
oligonucleotide strand, as opposed to sequencing, which refers to a
determination of the complete nucleotide sequence of (and also, in
the case of a gene exon, the corresponding amino acid sequence
encoded by) a target nucleic acid, including the identity and
position of each nucleotide present in the target strand or its
complement. DNA or RNA fingerprinting, which requires less
reagents, can provide a rapid and cost effective alternative to
sequencing, and may be used in a number of different applications,
e.g. identification of one or more infectious agents in a genomic
sample from a subject, screening cDNA libraries, screening genes
from human or non-human genomes to detect mutations and
polymorphisms and for forensic applications. One preferred method
for determining a single 2-base fingerprint of an oligonucleotide
extension segment, generated using the modified primers of the
present invention, is massed spectrometry.
[0234] In determining a fingerprint of a target oligonucleotide, a
base-specific nested fragment set, containing base-specific
terminated fragments derived from the target molecule, is produced
for subsequent analysis. As referred to herein, a nested set is
defined as a mixture of biopolymers (e.g. DNA, RNA, peptides, or
carbohydrates) for which all of the components of the mixture have
a common terminus and are produced from a single polymeric
sequence. The base-specific nested fragment set may be produced,
for example, by base-specific chain termination using the Sanger
method or by selective chemical cleavage, to be described below. In
instances where amplification of a target molecule is desired,
amplification is carried out using any of a number of conventional
amplification methods including PCR.TM., SPA (single primer
amplification, NASBA (nucleic acid sequence-based amplification),
TMA (transcription-mediated amplification), and SDA
(strand-displacement amplification).
[0235] In fingerprinting methods employing a size fractionating
device for product analysis, such as a mass spectrometer, the
resulting single-base fingerprint can often provide indirect
information regarding the other bases present in the target
sequence. For example, the difference in mass (Am) corresponding to
the positions of two peaks in a mass spectrum may also reveal the
composition of the intervening bases, to be described as
follows.
[0236] To determine a single base fingerprint of a target
oligonucleotide, such as a thymidine fingerprint, a
thymidine-specific nested fragment set is produced as described
above. The resulting fragment family, containing a number of
thymidine-terminated nucleotide fragments, is typically purified
and analyzed by a size fractionation method, such as mass
spectrometry. To increase mass spectrometric performance,
mass-modified nucleotides can be utilized. The resulting mass
spectrum contains a number of peaks, each corresponding to the mass
of a particular thymidine-terminated fragment present in the
product mixture. The differences in mass between the various
thymidine-terminated fragments is then correlated with the
calculated mass of various combinations of nucleotides, preferably
with the assistance of a computer program containing the molecular
weights of the modified primer, the portion of the primer 3' of the
cleavable site, and each of the various nucleotides, to identify
the combination of nucleotides intervening between the known
thymidine positions.
[0237] As an illustration, in considering the difference in mass
between two given fragment peaks, an exemplary mass difference of
1,276 mass units corresponds uniquely to the following base
composition: (two G)+A+T. A single base fingerprint can thus be
used to (i) identify the locations of a particular base within a
target sequence and (ii) determine the base composition within
localized regions of a target sequence.
[0238] For applications requiring a greater level of detail, a
second base-specific nested set is produced and analyzed to produce
a second fingerprint as described above. Different base-specific
nested sets can be produced in single reaction vessel, or in
separate reactions, depending upon the method utilized and the
corresponding reagents required. The different base-specific nested
sets (e.g., thymidine-terminate fragments and cytidine-terminated
fragments) may be analyzed separately, or as a mixture.
[0239] Use of a single base fingerprint for detection of a point
mutation is illustrated in Example 7. Briefly, the cytosine
fingerprints of two distinct single stranded DNA targets (SEQ ID
NO:14 and SEQ ID NO:15) having sequences differing only at
positions 16 and 19, relative to the 3' end (counting upstream
after the priming region), were determined using dideoxycytosine
triphosphate to produce a family of cytosine-terminated nucleotide
fragments, followed by analysis of the resulting reaction product
mixtures by mass spectrometry (FIG. 14A and FIG. 14B). The exact
mass values corresponding to the differences between select peaks
in each of the spectra were calculated, confirming the presence of
two single nucleotide substitutions (point mutations) at positions
16 and 19.
[0240] In an alternative approach, a base-specific nested fragment
set is produced by selectively cleaving a DNA or RNA molecule
modified to contain selectively cleavable groups (e.g. dUTP or
amino functionalized nucleoside triphosphates) at positions
corresponding to a particular base. The resulting uridine-modified
oligonucleotide is then treated with uracil DNA glycosylase to form
a set of fragments, preferably a nested set captured onto a solid
phase. Similarly, a 5'-amino-modified target molecule is cleaved by
treatment with acid.
[0241] Preferably, the above fingerprinting method employs the
cleavable primers of the invention to remove the majority of the
primer from the primer extension fragments. By reducing the mass of
the analyte fragments, the distribution of products is shifted into
the region where mass spectrometry has higher mass accuracy and
resolution. Additionally, it may be useful to mass modify the
different nucleotides by introducing any of a number of mass
modifying functionalities (e.g., by replacing thymidine with
5'-bromouridine, or utilizing an immobilization attachment site,
spacer arm, or alternative internucleotide linkage, as described
above) to enhance the mass difference between the different
nucleotides.
[0242] In a second fingerprinting approach, a restriction
endonuclease is used to generate a non-random fragmentation
pattern, useful, for example, for detection mutations.
[0243] In an alternate embodiment of the invention, target DNA for
sequencing or fingerprinting is amplified using the polymerase
chain reaction or PCR.TM. (Mullis 1987; Mullis et al., 1987;
Kusukawa et al., 1990; Gyllensten, 1989). Briefly, PCR.TM.
amplification is typically carried out by thermal cycling a
cocktail containing the target DNA of interest, a mixture of
deoxynucleotide triphosphates (dNTPs), a reaction buffer, each of
two primers, and an extension enzyme such as Taq DNA polymerase
(United States Biochemical, Cleveland, Ohio) (Erlich, 1989; Innis,
1990). A PCR.TM. run profile typically consists of a 5-min
denaturation step at 94.degree. C., 15 sec at the annealing
temperature, and 1 min at 72.degree. C. Following thermal cycling,
the samples can maintained at 4.degree. C. until removal from the
thermal cycler. Annealing temperatures range from out 55.degree. C.
to 65.degree. C., although most target sequences amplify well at
60.degree. C.
[0244] Amplification is followed by hybridization with the modified
primers of the present invention, enzymatic extension and
sequencing of the products as described above.
[0245] FIG. 12 illustrates PCR.TM. amplification of template
molecule using the modified primers of the present invention.
[0246] As an alternative to using dideoxy chain terminators,
PCR.TM. can be combined with dUTP incorporation to produce a nested
set terminated at the sites of dUTP incorporation by treatment with
uracil DNA-glycosylase. PCR.TM. can also be combined with
phosphorothioate methods, as described above.
[0247] In accordance with the present invention, amplification can
be carried out using first and second primers, where one of the
primers, i.e. the first primer 95, contains a cleavable site 99,
and another primer, i.e. the second primer 105, contains an
immobilization attachment site for binding to a solid support. The
second primer is composed of a 5' end and a 3' end, is homologous
to the target nucleic acid, and includes a first segment containing
the 3' end of the second primer, and a second segment containing
the 5' end of the primer and an immobilization attachment site.
[0248] These first and second primers are combined with a target
nucleic acid 103 to generate primer/nucleic acid complexes and are
converted to double stranded fragments 109 in the presence of a
polymerase and deoxynucleoside triphosphates, as indicated at 107.
The sizing method can be carried out using a large excess of target
nucleic acid to generate substantial amounts of primer extension
product, or alternatively, may be coupled with various rounds of
amplification. Upon achieving a desired amount of product 109,
extension products containing the second primer are immobilized 117
by attachment the immobilization attachment site, either before or
after cleavage at the cleavable site. The extension product is then
cleaved 111 at the cleavable site to generate a mixture which
includes double stranded product 115. Non-immobilized cleaved
fragments are removed 119, preferably by washing, and the purified
double stranded product 121 is denatured 123 to release the
extension segment 125, which is sized by mass spectrometry, where
the read length of the extension segment is increased relative to
the read length of the primer/nucleic double stranded
fragments.
[0249] As will be appreciated, the cleavable site of the first
primer and the immobilization attachment site of the second primer
include those the types described above.
[0250] In the exemplary embodiment illustrated in FIG. 12, the
first primer 95 contains a restriction enzyme recognition site 97
in the first primer region 101, and a cleavable site 99 in the
second primer region, and the second primer contains an
immobilization attachment site for attachment to a solid support.
Cleavage at the cleavable site is carried out by addition of a
restriction endonuclease selective for the recognition site
contained in the first primer region to provide (i) released
fragments containing the first region of the first primer and (ii)
a double stranded product, which is immobilized prior to denaturing
for release of the desired extension segment. As illustrated in
FIG. 12, restriction endonuclease-promoted cleavage results in the
release of a double stranded product 115 and a short double
stranded fragment 113 containing the first region of the
primer.
[0251] 4.3.2 Cleavage
[0252] Cleavage of the selectively cleavable site may be carried
out as described in Section 4.2.1 and in Examples 1A-D and Example
3. Returning to this aspect of the invention, internucleoside silyl
groups such as trialkylsilyl ether and dialkoxysilane are cleaved
by treatment with fluoride ion. Base-cleavable sites for use in the
present invention include beta-cyano ether
5'-deoxy-5'-aminocarbamate, 3'-deoxy-3'-aminocarbamate, urea,
2'-cyano-3',5'-phosphodiester, 2'-amino-3',5'-phosphodiester, ester
and ribose. Thio-containing internucleotide bonds such as
3'-(S)-phosphorothioate and 5'-(S)-phosphorothioate are cleaved by
treatment with silver nitrate or mercuric chloride. Acid cleavable
sites for use in the present invention include
3'-(N)-phosphoramidate, 5'-(N)-phosphoramidate, dithioacetal,
acetal and phosphonic bisamide. An alpha-aminoamide internucleoside
bond is cleavable by treatment with isothicyanate, and titanium is
used to cleave a 2'-amino-3',5'-phosphodiester-O-ortho-benzyl
internucleoside bond. Vicinal diol linkages are cleavable by
treatment with periodate. Thermally cleavable groups include
allylic sulfoxide and cyclohexene while photo-labile linkages
include nitrobenzylether and thymidine dimer.
[0253] Cleavage conditions are utilized which leave the
immobilization attachment site intact, so that a major portion of
the primer remains affixed to the solid support. Preferably,
cleavage of the cleavable site results in primer extension products
containing five or fewer base pairs from the primer sequence. This
maximizes the amount of sequence information provided upon
subsequent analysis.
[0254] 4.3.3 Analysis
[0255] Any of a number of size fractionating devices may be used to
determine the sequence of a target oligonucleotide fragment. Size
fractionation methods for use in the present invention include gel
electrophoresis, such as polyacrylamide or agarose gel
electrophoresis, capillary electrophoresis, mass spectrometry, and
HPLC.
[0256] In methods employing gel electrophoresis sizing and
analysis, the DNA fragments are typically labeled with either
radioisotopes or with attached fluorophores, and visualized using
autoradiography or fluorescence detection, respectively.
[0257] The modified primers of the present invention are
particularly advantageous when they are used to generate
oligonucleotide fragments whose sizes are to be resolved using
technologies that currently have difficulty resolving fragments of
over about 100 base pairs differing by one nucleotide in length,
such as mass spectrometry.
[0258] One preferred method for oligonucleotide analysis using the
modified oligonucleotide composition of the invention is mass
spectrometry, and particularly matrix-assisted laser desorption
ionization (MALDI) mass spectrometry, preferably carried out on a
time-of-flight (TOF) mass spectrometer (Wu et al., 1993). MALDI
mass spectrometry provides a rapid and efficient method for
oligonucleotide sequencing.
[0259] MALDI-TOF mass spectrometry may be used to provide
unfragmented mass spectra of mixed-base oligonucleotides containing
more than 100 base pairs. Moreover, mass spectral resolution of
sequences currently to at least about 40 base pairs in length may
be attained.
[0260] In this method, pulsed ultraviolet laser light is used to
desorb an oligonucleotide out of an absorbing solid matrix, which
causes creation of free, unfragmented, charged oligomers. Mass
analysis is done in a time-of-flight mass spectrometer. Singly
charged molecular ions are typically the most abundant species and
fragment ions are minimized.
[0261] In preparing the sample for analysis the analyte is mixed
into a matrix of molecules which resonantly absorb at the laser
wavelength. Solid matrix materials for this use include
3-hydroxypicolinic acid (Wu et al., 1993),
alpha-cyano-4-hydroxycinnamic acid (Youngquist et al., 1994),
nicotinic acid (Hillenkamp, 1988), and ice (Nelson et al., 1989),
although a preferred material is 3-hydroxypicolinic acid.
[0262] Examples 4 and 5 include detailed descriptions of MALDI-TOF
mass spectral analyses of modified oligonucleotide compositions
according to the present invention. Example 8, in conjunction with
FIG. 15A and FIG. 15B, illustrates the difference in fragment
information obtained for cleaved primer extension segments
according t the invention (FIG. 15B) versus non-cleaved full
primer-extension segments (FIG. 15A).
[0263] As described in Example 3, a synthetic 17-mer DNA probe
containing a cleavable ribose in the 7-position was selectively
cleaved by ammonium hydroxide treatment. Mass spectra of the intact
mixed base primer prior to (FIG. 3A) and after (FIG. 3B) ammonium
hydroxide treatment reveal the selective cleavage of the ribose
linkage. As shown in FIG. 3A, two sizable peaks were observed for
the intact 17-mer corresponding to the di-protonated molecular ion
[M+2 H].sup.2+ and the protonated molecular ion [M+H].sup.+.
Following ammonium hydroxide treatment, peaks corresponding to the
expected cleavage products, the 7-mer, the 10-mer, and intact
17-mer, were readily observable and identifiable, as illustrated in
FIG. 3B.
[0264] Similarly, mass spectral analysis was carried out on a
biotinylated 18-mer containing a ribose in the 10 position and
captured on streptavidin-coated beads, as described in Example 5.
The immobilized primer was washed after surface binding, followed
by treatment with ammonium hydroxide to effect selective cleavage
of the immobilized primer at the ribose site. FIG. 4 illustrates
the mass spectrum of the 8-mer resulting from selective cleavage of
the ribose site within the immobilized primer.
[0265] 4.4 Utility
[0266] 4.4.1 Genomic Sequencing
[0267] The method of the present invention may be used for both
"shotgun-type" sequence analysis and "directed walk". In the
shotgun approach, a random sequence of DNA is selected and used to
prime on an unknown target. This approach uses large numbers of
primers to increase the possibility of successfully hybridizing
with an unknown target sequence. In one embodiment of this
approach, a multi-well assay format is used where each well has a
different primer and the same substrate (i.e. the target DNA
molecule) is added to each well under hybridization conditions. The
primers in the wells are the modified primers of the present
invention where immobilization to the well surface is through the
primer immobilization site. Primer extension reactions are carried
out. Extension products are only formed in wells where
complementary sequences exist between the primer and the substrate.
Each well is examined for the presence of extension products.
Extension products are then sequenced and sequences assembled for
any given target DNA molecule based on the known sequences of the
primers that yielded extension products and base sequence overlap
from the extension product sequences (i.e. alignment of the
extension product sequences). In using the modified primers of the
present invention, the amount of sequence information for the
extension segments is maximized over that obtained with similar
techniques employing conventional primers, due to cleavage and
removal of a large portion of the primer prior to fragment analysis
(e.g., increased read lengths). Further, the method, when coupled
with analysis by mass spectrometry, is fast and can provide large
amounts of data in a relatively short period of time.
[0268] In a related approach, the present method may be used to
sequence or fingerprint short reads of cDNA inserts for purposes of
gene mapping and identification. These short reads identify each
insert uniquely and are called Expressed Sequence Tags (ESTs) or
Expressed Fingerprint Tags (EFTs). In preparing a cDNA library,
cDNA copies of mRNAs are first inserted into a standard cloning
vector such as pBlueScript. A modified primer according to the
present invention is designed to hybridize to the pBlueScript
vector sequence with its 3' end immediately adjacent to the cDNA
insert. Primer extension and sequencing or fingerprinting reactions
are then carried out which read into the insert and identify its
sequence or fingerprint. In order to identify a unique length of
sequence, a minimum read length for the extension segment is
typically 30 bases, although a preferred read length is at least
about 40 bases.
[0269] In an alternative embodiment, an array of the immobilized,
cleavable primers can be formulated (Fodor et al. 1991; Southern et
al. 1992). In this aspect of the invention, the array consists of
the modified primers of the present invention where the cleavable
linkage is, for example, a photocleavable linkage (e.g., backbone
nitrobenzyl group) and the primer is attached to the support matrix
through the immobilization site of the modified primer. In this
embedment, the target DNA molecule is hybridized to the primers,
primer extension reactions are carried out and the different
sequence primers are sequentially cleaved and the presence or
absence of an extension product is determined. When extension
products are detected their sequences can be determined as
described above.
[0270] In the directed walk approach, a known DNA sequence is used
as the primer sequence, thus providing an initiation point for
sequencing in both directions away from the known region. Each
newly identified sequence is then used to direct the synthesis of
new primers to enable progression of the sequence walk.
[0271] 4.4.2 Diagnostics
[0272] A number of synthetic oligonucleotides are available or may
be readily synthesized which are complementary to target nucleic
acid sequences (e.g., RNA or DNA) and may be used as probes to
detect the presence of certain bacteria, viruses, fungi, parasites
and the like.
[0273] The oligonucleotide compositions of the present invention
may be used, for example, to detect the presence of specific DNA or
RNA sequences or fingerprints corresponding to the following
targets (i) herpes viruses such as cytomegalovirus (CMT), Epstein
Barr, and Simplex Herpesvirus; (ii) hepatitis viruses such as
hepatitis A, B, G, and D; (iii) papillomaviruses such as human
papilloma virus 6, 11, 16, 18, and 33; (iv) retroviruses such as
human immunodeficiency virus 1 (HIV I), HIV II, human T-cell
lymphoblastic virus I (HTLV I), HTLV II; (v) animal viruses such as
pig parvovirus and pig mycoplasma hypneumoniae, parvoviruses such
as parvovirus B 19; (vi) picornaviruses such as rhinovirus
(enterovirus) and rhinovirus HRV 2-14; (vii ) bacteria such as
mycobacterium avium, mycobacterium tuberculosis, chlamydia
trachomatis, Escherichia coli, streptococci and staphylococci; and
(viii) parasites such as trypanosoma, toxoplasma, and
plasmodia.
[0274] Modified primers of the present invention having a primer
sequence, such as a sequence specifically hybridizable to a nucleic
acid from the microorganism of interest, are hybridized to nucleic
acids in a sample. Primer extension reactions and isolation of
extension products is carried out as described above. Presence of
the extension product indicates the presence of a nucleic acid from
the microorganism in the sample. The modified primers and sizing
method of the present invention provide a method for rapid, high
throughput screening for the presence of specific, target sequences
or fingerprints using mass spectrometry.
[0275] In a related embodiment, the modified primers of the
invention can be used to identify pathogens by direct sequencing or
fingerprinting. In one such approach, a particular region of
genomic DNA is identified which has segments that are common among
a large population of pathogens (e.g., conserved regions) located
next to regions that contain unique sequences for each pathogen
(e.g., variable regions). One such exemplary sequence is from DNA
that is transcribed into bacterial 16 S ribosomal RNA, or 16 S rRNA
(Olsen et al. 1992). All 16 S-like rRNAs contain the same core
structure. Nucleotides which are conserved in 90% of the available
bacterial 16 S rRNA sequence have been identified (Schmidt et al.,
1994).
[0276] Pathogen identification using rRNA as described above is
carried out as follows. In accordance with the present invention, a
primer is constructed to hybridize to a select region of the 16 S
rRNA consensus sequence, for example, sequence 1047-1065 in 16 S
rRNA, where (i) the primer has the sequence
5'ACGACANCCATGCANCACC-3' (SEQ ID NO:9) or 5'ACGACATCCATGCATCACC-3'
(SEQ ID NO: 19), and (ii) reads into the hypervariable region,
e.g., sequence 995-1046. Upon analysis of the primer extension
segments by mass spectrometry, a single pathogen, if present, can
be uniquely identified by determining the sequence or fingerprint
along the hypervariable region, with a desirable read length of at
least 20 bases, and preferably, at least 40.
[0277] Alternatively, instead of selecting a conserved region
adjacent a hypervariable region, a series of unique primers can be
created that will hybridize to a hypervariable or unique region of
a selected pathogen. Enzymatic extension of these primers provides
sequence or fingerprint information about an adjacent segment of
hypervariable region. This methodology enables specific
identification of each pathogen present in a mixed population.
Utilizing this approach, one may target different hypervariable
regions for each target pathogen. This approach may be preferred
for identifying viruses for which there is often very little
conservation among other viruses or bacteria.
[0278] In addition to determining the presence of a nucleic acid in
a sample from such microorganisms, the present invention
facilitates the determination of the specific sequences or
fingerprints present in a sample. For example, specific variants of
HIV or trypanosomes can be identified by fingerprinting or
sequencing as well as the presence or absence of genes responsible
for antibiotic resistance.
[0279] The modified primers can likewise be used in diagnostic
methods where mutant sequences are distinguished from wild-type
sequences by sequence variations, such as, deletions, insertions,
point mutations. Numerous potential target sites can be evaluated
by this method including target sites selected from DNA sequences
that vary in length (e.g., BCR/ABL) as well as those that vary in
sequence (e.g., sickle cell anemia). The sizing methodology of the
present invention is particularly suited for the former application
(e.g., target sites of varying length). Hybridizations of the
modified primers to target nucleic acids are carried out according
to standard procedures, with suitable adjustment of the
hybridization conditions to allow modified primer hybridization to
the target region.
[0280] Exemplary genetic disorders for detection using the modified
primers of the present invention include sickle cell anemia and
alpha1-antitrypsin deficiency (Watson et al., 1992). As shown in
FIG. 7A, sickle cell anemia results from a mutation that changes a
glutamic acid residue (coded by the triplet GAG) for a valine
residue (coded by GTG) at position 6 in the beta-globin chain of
hemoglobin. This base change (A to T) destroys the recognition
sequence for a number of restriction enzymes, including MstII. A
modified primer for detecting this disorder would typically contain
a cleavable site as indicated in FIG. 7A, located about 2
nucleotides from the end of the primer and preferably about 10-20
nucleotides upstream from the known mutation site.
[0281] Also detectable using the modified primers of the present
invention is alpha1-antitrypsin deficiency, a disorder
characterized by uninhibited production of elastase, a protease
which destroys the elastic fibers of the lung causing patients to
suffer from pulmonary emphysema. The alpha1-antitrypsin gene has
been cloned and as shown in FIG. 7B, the mutant gene, a fragment of
which is presented by SEQ ID NO:7, corresponds to a single base
change that leads to an amino acid substitution (glutamine to
lysine) at residue 342 as indicated by SEQ ID NO:8. A portion of
the wild-type alpha1-antitrypsin gene, as shown in FIG. 7B (top),
is presented by SEQ ID NO:5. A fragment of the protein produced in
individuals having the wild type alpha1-antitrypsin gene is
presented by SEQ ID NO:6. Other diseases for which the
corresponding gene mutations have been identified and which may be
detected using the modified primers of the present invention
include Duchenne muscular dystrophy, factor X deficiency,
hemophilia and phenylketonuria. Modified primers used for detecting
such disorders typically contain a cleavable site located near the
end of the primer, where the end of the primer is upstream from the
known mutation site (e.g., within about 20 base pairs from a
mutation site detected in a 40-mer).
[0282] Detection of a point mutation using the methods described
herein is provided in Example 7 and further illustrated in FIG. 14A
and FIG. 14B.
[0283] Another diagnostic example is the detection of BCR-ABL
transcripts, which are found in the majority of chronic myelogenous
leukemia (CML) patients and in Ph.sup.+ acute lymphocytic leukemia
patients, and are believed to be necessary for the maintenance of
leukemic phenotype (Szczylik et al., 1991; Gale et al., 1984;
Collins et al., 1984; Daley et al., 1988). The BCR-ABL transcripts
are the result of a translocation of the proto-oncogene ABL
(chromosome 9) to the breakpoint cluster region (bcr) (chromosome
22), resulting in the formation of BCR-ABL hybrid genes. In this
embodiment, the modified primers of the present invention would
have their 3' end before the breakpoint region. Primer extension
reactions would then proceed across the breakpoint region if
present, or continue through the normal transcript region if no
breakpoint was present. The sequence of such primer extension
products are diagnostic of whether a breakpoint fusion exists in
any given sample of nucleic acids.
[0284] The modified primers can also be employed in DNA
amplification reactions (e.g., Mullis, 1987a; Mullis et al., 1987b)
for detecting the presence of specific sequences in samples by
sizing or sequencing or for preparing quantities of DNA for
sequencing reactions. In this embodiment of the invention, modified
primers containing immobilization sites that can be attached to the
solid support following amplification are particularly useful
(e.g., biotin and digoxigenin). Amplified products can be captured,
the modified primers cleaved, and the resulting amplification
products isolated.
[0285] In particular, the present method may be utilized to
identify pathogens by the sizing of PCR.TM. products. Briefly,
primers are first selected to hybridize with a sequence unique to
the target pathogen(s) of interest. The primers are chosen for use
in a multiplex situation (e.g., one in which several different
pathogens may be present) to produce PCR.TM. products of varying
sizes, with each size correlating to a unique PCR.TM. product for a
specific pathogen.
[0286] Such a study for determining the presence of three different
pathogens (e.g., Pseudomonas aeruginosa, Escherichia coli, and
Staphylococcus aureaus) is carried out by adding to a sample
containing, in addition to a DNA analyte, modified primers for each
of the above pathogens designed to produce PCR.TM. amplification
products having sizes of, for example, 65, 70, and 75 base pairs,
respectively.
[0287] The PCR.TM. products are reduced in size by as many as 20-25
nucleotides by cleavage at the cleavable site (in the modified
primer). This results in shifting the corresponding peaks into a
more readily resolvable range of the mass spectrometer and permits
multiplexing of greater numbers of PCR.TM. products.
[0288] The cleaved-amplification products are detected and sized
using mass spectrometry, according to the method of the present
invention.
[0289] The following examples illustrate, but in no way are
intended to limit the scope of the present invention.
[0290] 4.5 Materials and Methods
[0291] Protected nucleotide H-phosphonates such as
Bz-DMT-deoxyadenosine H-phosphonate, iBu-DMT-deoxyguanosine
H-phosphonate, fully protected deoxynucleoside phosphoramidites,
protected deoxynucleoside diesters and triesters, nucleotide
dimers, and solid phase supports may be obtained from Sigma
Chemical Co. (St. Louis, Mo.).
Bis(trifluoromethanesulfonyl)diisopropylsilane may be obtained from
Petrarch System Inc. (Bertram, Pa.). Phosphoramidites may be
obtained from Applied Biosystems (Foster City, Calif.). Standard
chemical reagents and solvents may be obtained from Aldrich
Chemical Company (St. Louis, Mo.).
EXAMPLE 1
Preparation of a Modified Oligonucleotide Containing A
3'-5'-Cleavable Linkage
[0292] Nucleoside dimers containing the following
3'-5'-internucleoside cleavable linkages are prepared as
follows.
A. 3',5'-Dialkoxysilane Internucleoside Linkage
[0293] The 3'-O-functionalized nucleoside intermediate,
3'-O-(diisopropylsilyl)-2'-deoxynucleoside triflate (1) is prepared
by first adding bis(trifluoromethanesulfonyl)diisopropylsilane (1
mmol) to an equimolar amount of the sterically hindered base,
2,6-di-tert-butyl-4-methylpyridine, dissolved in dry acetonitrile,
under an inert atmosphere. The resulting solution is cooled to
-40.degree. C. in a cooling bath, to which is added a solution of a
5'-(O)-protected nucleoside,
5'-(dimethoxytrityl)-2'-deoxynucleoside (0.9 mmol) and
2,6-di-tert-butyl-4-methylpyridine (0.2 mmol) in dimethylformamide
over a 10 min period. The resulting reaction mixture is stored at
40.degree. C. for 1 h and then allowed to warm to room temperature.
The 3'-O-diisopropylsilyl triflate product is isolated by
precipitation from water, with yields typically ranging from
90-100%. Isolation is not required, and preferably, the reactive
intermediate is coupled directly with unprotected nucleoside to
form the desired dimer.
[0294] The above procedure is used to form the 3'-silyl derivatives
of the protected nucleosides 5'-(O)-dimethoxytrityl-thymidine,
N6-benzoyl-2'-deoxy-5'-(O)-DMT-adenosine,
N4-benzoyl-2'-deoxy-5'-(O)-DMT-cytidine, and
N2-isobutyl-2'-deoxy-5'-(O)-dimethoxytrityl-guanosine with minimal
formation of the undesired 3',3' symmetrical dimers.
[0295] Intermediate (1) is reacted with nucleoside, such as
thymidine, by stirring a mixture of (1) and nucleoside for
approximately 1 h room temperature. The coupled dimer is isolated
by adding the reaction mixture dropwise to a vigorously-stirred
ice/water mixture. The mixture is filtered to give a white solid,
which is then dried and purified by column chromatography on silica
gel (eluent: ethyl acetate/hexane gradient). The protected dimer,
5'-(O)-DMT-3'-(O)-(5'-(O)-nucleosidyldiisopropylsilyl)thymidine
(2), is typically isolated in yields ranging from 50-75%.
[0296] The prepared dimers are then functionalized for use in
automated solid phase synthesis to form primers containing a
dialkoxysilane cleavable site.
[0297] The dimer, such as (2) above, is converted to the
corresponding 3'-(2-cyanoethyl-N,N-diisopropylphosphoramidite) by
dissolving the 5'-DMT dimer in tetrahydrofuran and adding the
resulting solution dropwise to a stirred solution containing 4-DMAP
(4-dimethylaminopyridine), diisopropylethylamine and
2-cyanoethyl-N,N-diisopropylphosphoramidochloridite in THF under
nitrogen and maintained at room temperature. The reaction mixture
is stirred for 2 h, added to ethyl acetate, washed with brine, and
dried over magnesium sulfate. The crude product is then purified by
column chromatography on silica using 1:1 ethyl acetate/hexane as
the eluent.
[0298] The phosphoramidite-functionalized timer is then employed
for use in automated solid phase synthesis using a programmable DNA
synthesizer to form an oligonucleotide primer containing a
3'-5'-diisopropylsilyl ether cleavable site.
[0299] Cleavage: After carrying out the desired hybridization and
extension reactions with an immobilized primer containing a
dialkoxysilane internucleotide linkage as described above, the
silyl-ether (Si--O) linkage is selectively cleaved by treatment
with fluoride ion (Green, 1975) to release the extension product,
typically containing no more than about five nucleotides derived
from the modified primer molecule.
B. 3'(S)-Phosphorothioate Internucleoside Linkage
[0300] The functionalized nucleoside,
5'-(0)-monomethoxytrityl-3'-(S)-thiophosphoramidite, is prepared as
follows. The 3'-S-functionalized starting material,
5'-(O)-monomethyoxytrityl-3'-(S)-benzoyl-thymidine (3), is prepared
according to the method of Cosstick et al. (1988). Debenzoylation
is carried out by treating a solution of
5'-(O)-monomethoxytrityl-3'-(S)-benzoylthymidine dissolved in
argon-saturated ethanol and maintained at 5.degree. C. with 10N
sodium hydroxide. The resulting solution stirred for approximately
1 h. The product, 5'-O-MMT-3'-thiothymidine (4), is purified by
column chromatography on silica gel. The
5'-(O)-MMT-3'-(S)-thymidine (4) is then converted to the
corresponding thiophosphoramidite by reaction with
2-cyanoethyl-N,N-diisopropylaminophosphomonochloridite under
standard conditions (McBride et al., 1983). The
3'-(S)-phosphoramidite (5) is suitable for coupling to a second
nucleoside to form a dimer containing a cleavable phosphorothioate
site or for use in automated solid phase synthesis to prepare an
oligonucleotide containing a phosphorothioate internucleoside
linkage.
[0301] Chemical synthesis of the phosphorothioate dimer is carried
out as follows. A solution of 3'-(O)-acetylthymidine in
acetonitrile is added dropwise over a 20 min period to a stirred
solution of the 3'-(S)-phosphoramidite (5) in acetonitrile
saturated with 5'(4-nitrophenyl)tetrazole. Use of the tetrazole
activating agent reduces the probability of the self-condensation
reaction occurring between two thiophosphoramidite molecules. The
resulting thiophosphite dimer (6) is oxidized in situ by quenching
the reaction mixture with 2,6-lutidine, followed by addition of an
oxidant such as TBA periodate in dichloromethane. The fully
protected phosphorothioate dimer (7) is deprotected by treatment
with 5-butylamine, 80% aqueous acetic acid, followed by
concentrated aqueous ammonia to yield the
3-(S)-phosphorothioate-linked thymidine dimer (8).
[0302] Formation of an oligonucleotide probe containing a
3'-(S)-phosphorothioate cleavable linkage is performed by solid
phase synthesis on controlled pore class using a DNA synthesizer.
The protocol and reaction conditions for conducting the solid phase
reaction cycle are adjusted according to the primer products
desired using standard solid phase phosphoramidite procedures. The
functionalized 3'-(S)-phosphoramidite nucleoside (5), prepared as
described above, is utilized to introduce the
3'-(S)-phosphorothioate moiety into the oligonucleotide primer in
the presence of the functionalized tetrazole reagent,
5-(para-nitrophenyl)tetrazole.
[0303] Cleavage: After carrying out hybridization, extension, and
washing of an immobilized modified primer containing a
3'-(S)-phosphorothioate internucleotide bond, selective cleavage of
the phosphorus-sulfur bond is carried out by treatment with aqueous
silver nitrate.
C. 5'(S)-Phosphorothioate Internucleoside Linkage
[0304] Synthesis of an oligonucleotide containing a
5'-phosphorothioate internucleoside linkage is carried out by first
synthesizing a derivatized phosphoramidite,
5'-(S)-trityl-deoxythymidine-3'-(O)-(2-cyanoethyl-N,N-diisopropylamino)ph-
osphite as described below.
[0305] 5'-(O)-p-toluenesulfonyl thymidine (9) is prepared by
reacting thymidine with one equivalent of p-toluenesulfonyl
chloride in pyridine. The reaction mixture is stirred for 3 h at
room temperature, cooled in ice and quenched by addition of water.
Following dissolution in ethyl acetate, and sequential washing with
sodium bicarbonate and brine, the solution is dried over sodium
sulfate and the solvent is removed in vacuo. The desired
5'-tosylate product (9) is readily recrystallized from ethyl
acetate/methanol, thus avoiding the need for a protecting group at
the 3'-OH position.
[0306] The 5'-tosylate is then converted to the corresponding
5'-(S)-trityl-deoxythymidine (10). A solution of the
5'-(O)-tosylthymidine (9) in ethanol is added to a reaction flask
containing a five-fold molar excess of 7.0 M sodium hydroxide and
triphenylmethyl mercaptan in ethanol (which generates the
corresponding reactive thiolate in situ). The reaction mixture is
refluxed for 8 h under an inert atmosphere, filtered to remove
residual solids, dissolved in ethyl acetate, and the solution is
washed, dried, and evaporated in vacuo. The crude product is
purified by chromatography on silica gel using a methanol/methylene
chloride gradient.
[0307] The desired reactive nucleoside phosphoramidate,
5'-(S)-trityl-deoxythymidine-3'-(O)-(2-cyanoethyl-N,N-diisopropylamino)ph-
osphite (11), is prepared by treating a solution of the protected
nucleoside, 5'-(S)-trityl)-deoxythymidine (10), in dry 1:1
acetonitrile/methylene chloride with an equimolar amount of
tetrazole, followed by addition of a 1.5 molar excess of
2-cyanoethyoxy-bis-(N,N-diisopropylamino)phosphine. The reaction
mixture is stirred for about 1 h at room temperature and
subsequently quenched by addition of butanol. The solution is
diluted with ethyl acetate, washed, drived over anhydrous sodium
sulfate, filtered, and evaporated to dryness. The crude product is
purified by flash chromatography.
[0308] Incorporation of the desired 5'(S)-phosphorothioate
cleavable site into an oligonucleotide probe is carried out by
utilizing standard solid phase phosphoramidite chemistry.
D. 5'(N)-Phosphoramidate Internucleoside Linkage
[0309] Oligonucleotide fragments containing a 5'-(N)
phosphoramidate internucleotide bond are prepared as follows.
[0310] Thymidine is transformed to the corresponding 5'-azide
derivative (12) by treatment with sodium azide and
triphenylphosphine in carbon tetrabromide according to the
procedure of Hasa et al. (1976). Reduction of
5'-azido-deoxythymidine (12) is then carried out by hydrogenation
over Pd/C catalyst to form the corresponding 5'-amino derivative
(13).
[0311] Formation of the corresponding 5'-N-protected nucleoside is
carried out by dissolving (13) (25 mmol) in anhydrous pyridine (150
ml), to which is added 4-DMAP (17 mmol), triethylamine (17 mmol),
and 4-methoxytrityl chloride (60 mmol), and the resulting reaction
mixture is stirred for 2 h at room temperature. Methanol is added
to the reaction flask and the resulting mixture is added to a
solution of saturated sodium bicarbonate, extracted with
chloroform, and the organic extracts dried over anhydrous sodium
sulfate. The organic layer is evaporated to dryness and the
resulting crude residue is purified by column chromatography over
silica gel to yield
5'-amino-5'-deoxy-5'-(N)-(4-methoxytrityl)thymidine (13). (Cleavage
of the N-MeOTr protecting group is carried out by treatment with 3%
dichloroacetic acid in 1,2-dichloroethane).
[0312] The desired functionalized nucleoside,
5'-amino-5'deoxy-5'-(N)-(4-methoxytrityl)thymidine-3'-(2-cyanoethyl)-N,N--
diisopropylphosphoramidite (14), containing a reactive
phosphoramidite moiety suitable for incorporation into an
oligonucleotide fragment is synthesized as follows.
[0313] The 5'-amino-protected thymidine (13) (4 mmol) is dissolved
in anhydrous methylene chloride (60 ml), to which is added dry
bis(diisopropylammonium)tetrazolide (3 mmol) and
(2-cyanoethoxy)bis(diisopropylamino)phosphine, (8 mmol). The
mixture is stirred for 1 h at room temperature, poured into a
saturated sodium bicarbonate solution, and extracted several times
with chloroform. The combined organic extracts are rinsed with
brine, dried, and evaporated to dryness. The crude residue is
dissolved in a minimal amount of methylene chloride and
precipitated by addition of pentane to yield a mixture of the
disatereomeric product, 14.
[0314] The functionalized nucleoside 14 is then selectively
introduced into an oligonucleotide fragment to form an
oligonucleotide containing a 5'-(N)-phosphoramidate cleavable site.
The 5'-amino nitrogen of thymidine derivative 14 is the reactive
center at which phosphoramidate bond formation takes place. The
modified nucleoside 14 is introduced in the course of a standard
cycle into a growing DNA fragment synthesized on a solid support as
follows. Insertion of the phosphoroamidate group is performed at a
specific site to form the desired nucleotide fragment containing a
selectively cleavable site.
[0315] Formation of the following modified exemplary sequence:
d(T-T-C-A-T-G-C-A-A-(phosphoramidate)-T-C-C-G-A-T-G) (SEQ ID NO:1)
is performed as follows. The DNA fragment is synthesized in a
stepwise fashion beginning from the hexamer sequence
(d(C-C-G-A-T-G) (Gromova, 1987). The hexamer is synthesized on
controlled pore glass as solid support using standard procedures
(Bannwarth et al., 1986; Bannwarth, 1987). The introduction of key
intermediate 14 is performed during a standard cycle utilizing
slightly longer times for the coupling of 14 and for deblocking of
the 4-methoxytrityl protecting group. Following cleavage of the
5'-MeOTr group of 14, the synthesis is continued using standard
phosphoramidites as the building units to form the desired 16-mer
sequence.
[0316] The support material is treated with concentrated ammonia at
56.degree. C. overnight to cleave the 16-mer product from the solid
support. Following removal of the support material, the ammonia is
removed by vacuum evaporation, and the remaining residue is
dissolved in water/dioxane and subsequently precipitated by
addition of THF. The resulting 1 6-mer is then purified by either
gel electrophoresis or HPLC.
[0317] Selective chemical cleavage of the phosphoramidate
internucleotide bond is carried out under mild acidic conditions to
form the corresponding phosphate and amino-functionalized
fragments. Treatment with 80% acetic acid at room temperature for
between 2-6 h results in selective cleavage of the internucleotide
phosphoramidate bond while leaving the unmodified portion of the
DNA fragment intact.
EXAMPLE 2
Attachment to the Solid Support
A. Streptavidin Affinity Immobilization
[0318] A modified primer from Example 1 above containing a
cleavable site is immobilized by attachment to a functionalized
solid support material. In some cases the cleavable site-containing
primer is modified as described below.
[0319] For attaching an oligonucleotide primer to a
streptavidin-coated support a biotinylated primer is typically
used. Biotinylation is carried out as follows.
[0320] A primer containing a cleavable site is prepared as in
Example 1, with a minor modification: the primer is synthesized to
contain a reactive amino site for biotinylation. An amino group is
introduced during solid phase synthesis using a standard DNA
synthesizer, such as Applied Biosystems 393 DNA/RNA
Synthesizer.
[0321] To selectively introduce the internal amino function, the
modified nucleoside phosphoramidite Amino-Modifier dT, containing a
base labile trifluoroacetyl group protecting a primary amine
attached to thymine with a 10 atom spacer arm, is added at an
appropriate phase of the DNA synthesis cycle. Upon completion of
the oligonucleotide synthesis, the primer is cleaved from the
support by standard methods. The remaining base-protecting groups
as well as the trifluoroacetyl amino protecting group are removed
by treatment with fresh, concentrated ammonium hydroxide at
40.degree. for 15-17 h. The solution is dried by rotary
evaporation, and the residue is redissolved in 200 microliters of
water.
[0322] The amino-modified primer (approximately 0.25 micromoles) is
reacted with NHS-LC-Biotin (Pierce, Rockford, Ill.) which has an 11
carbon spacer between the biotin group and the
N-hydroxylsuccinimide activated carboxyl group. Aliquots of a 50 mM
NHS-LC-biotin solution in DMF are added to the primer solution
containing 0.1 M sodium bicarbonate/sodium carbonate buffer at pH 9
over a 1 h period. The solution is maintained at room temperature
overnight and the biotinylated primer is then purified by reverse
phase HPLC.
[0323] The biotinylated primer is then immobilized by attachment to
streptavidin-coupled magnetic beads (Dynabeads M-280, Dynal, Inc.,
Great Neck, N.Y.) as described in Dynabeads M-280 Technical
Handbook: Magnetic DNA Technology 6, Dynal, Inc. A
neodymium-iron-boron magnet is used to immobilize the beads during
supernatant removal and washing steps.
B. Immobilization via a Thiourea Linkage
[0324] 5'-Amino-modified oligonucleotide primers containing a
cleavable linkage are prepared as described in Examples 1 and 2A
above.
[0325] Glass slides are activated in a two-stage process for
coupling to amino-functionalized oligonucleotides. The glass
surface is first functionalized by reaction with
aminopropyltrimethoxysilane to form an amino-derivatized surface.
To carry out the amino-funtionalization, clean microscope slides
are immersed for 2 min in a 1% solution of
3-aminopropyltrimethoxysilane solution in 95% acetone/water. The
slides are then washed several times with acetone (5 min per wash),
and dried for 45 min at 110.degree. C.
[0326] Following amino-derivatization, the glass slides are treated
with excess p-phenylenediisothiocynate to convert the amino groups
to amino-reactive phenylisothiocyanate groups suitable for coupling
to amino-functionalized oligonucleotides. The amino-derivatized
glass plates are treated for 2 h with solution of 0.2%
1,4-phenylene diisothiocynate solution in 10% pyridine/DMF,
followed washing with methanol and acetone.
[0327] A 2 mM solution of the amino-modified primer in sodium
carbonate/bicarbonate buffer (2 microliters) is applied directly to
the activated glass plate surface and the resulting slides are then
incubated 37.degree. C. in a covered Petri dish containing a
minimal amount of water for about 2 h. The plates containing
thirourea-linked primer are then washed sequentially with 1%
ammonium hydroxide, and water, followed by air drying at room
temperature.
C. Immobilization via Hg-S Affinity Binding
[0328] An amino-modified oligonucleotide primer is prepared as
described above. Conversion of the 5'-amino group to a thiol is
carried out by reacting 5.0 A.sub.260 units of the amine-containing
primer dissolved in 1.0 ml of 0.2 molar
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.7) with
1.6 ml of 10 mM dithiobis(succinimidylpropionate) in dry
acetonitrile for 1 h at 20.degree. C. An additional 1.0 ml of 10 mM
dithiobis(succinimidylpropionate) in acetonitrile is then added to
the reaction vessel and the resulting mixture is stirred for an
additional hour. Addition of dithioerythritol (3.5 ml of a 20 mM
solution in 0.2 M Tris buffer) is followed by stirring for 1 h at
37.degree. C. The thiol-derivatized primer solution is concentrated
under vacuum to form a concentrate which is further purified using
reverse phase HPLC followed by lyophilization.
[0329] In an alternate approach for synthesizing thiol-modified
oligonucleotide primers, the 5'-phosphate of an oligonucleotide
primer is esterified with 6-mercapto-hexanol using a
beta-cyanoethyl-phosphoramidite C6-Thiol-Midifier (Clontech
Laboratories, Inc., Palo Alto, Calif.).
[0330] The thiol-derivatized primer is then immobilized on
p-chloromercuribenzoate derivatized agarose by mixing a solution of
the thiol-derivatized primer (0.25 A.sub.260 units) dissolved in
140 microliters of 1.0 M sodium chloride, 1.0 mM
ethylenediaminetetraacetic acid disodium salt (EDTA), and 50 mM
Tris HCl (pH 8) with 50 il of p-chloromercuribenzoate derivatized
agarose for 3 min at 20.degree. C.
EXAMPLE 3
Selective Cleavage of a Synthetic DNA Probe Containing: Cleavable
Ribose
[0331] A synthetic DNA probe containing a cleavable ribose site was
selectively cleaved by ammonium hydroxide treatment. The 17-mer,
having the sequence: 5'-AAA TAC ATC riboGCT TGA AC-3' (SEQ ID
NO:10) was prepared to contain a cleavable ribose in the
7-position. The modified probe was treated with aqueous 3% ammonium
hydroxide for 15 min at room temperature (pH 10) to effect
selective cleavage of the ribose moiety.
EXAMPLE 4
Mass Spectral Analysis of the Selective Cleavage Products of a
Ribose-Containing DNA Probe
[0332] The cleavage products from Example 3 were analyzed using
matrix assisted laser desorption with concomitant ionization
(MALDI) in conjunction with time-of-flight (TOF) mass
spectrometry.
[0333] The experimental apparatus used for analyzing the sample
fragments was composed of an excitation source, a sample
manipulator and TOF mass spectrometer. The excitation sources used
for desorption were a Nd:YAG laser (model DCR-1, Spectra-Physics,
Mountain View, Calif.) with spatially filtered 5 ns pulses
frequently tripled to 355 nm or quadrupled to 266 nm and a 35-ps
pulse Nd:YAG laser (model PY610C-10, Continuum, Santa Clara,
Calif.) operating at 355 nm. Both of the lasers were operated at a
10 Hz repetition rate, with a 5 nm pulse width. The desorption
laser beam maintained at an incident angle of 45.degree. was
focused onto the sample with a 250 mm focal length quart lens to an
elliptical spot size of approximately 100 to 150 micrometers. A
Glan-laser polarizer (Newport Corporation, Fountain Valley, Calif.)
was placed in a rotation stage in the beam path for continuously
variable attenuation, allowing adjustment of the polarized Nd:YAG
laser energy density from below 1 mJ/cm.sup.2. The optimum energy
density for desorption was found to be in the range of 2 to 20
mJ/cm.sup.2.
[0334] Sample preparation was carried out as follows. The
oligonucleotide fragments were dissolved in deionized water at room
temperature to concentrations of about 50 micromoles/liter. A
separate saturated solution of 3-hydroxypicolinic acid (3-HPA) in
50% water/acetonitrile was freshly prepared, and the two solutions
were mixed to provide a sample solution containing 3-HPA and
analyte in a molar ration of about 10,000:1. A 2 microliters
aliquot of the sample solution was pipetted onto the sample stage
and spread to an area of 2 mm diameter. The sample was dried under
a gentle flow of nitrogen prior to insertion into the vacuum
system.
[0335] The sample stage, consisting of either a smooth silver foil
or a polished silicon wafer, was mounted on a manipulator which
alloy three translational and one rotational degrees of freedom.
The studies were carried out at room temperature. The sample region
was evacuated by a 300 liter per second turbomolecular pump. The
drift and detection regions were evacuated using a cryopump with
nominal 1500 liter per second pumping speed. The base pressure of
the chamber was 3.times.10.sup.-9 Torr, and the normal working
pressure, within about five minutes of sample introduction, was
5.times.10.sup.-8 Torr.
[0336] The ions produced during desorption were extracted
perpendicular to the sample surface into the time-of-flight mass
spectrometer by biasing the sample with a voltage of 28 kV, with
the drift and extraction potentials at ground. The sample-extractor
distance was 5 mm, and an einzel lens about 5 cm from the sample
was used to focus the ions. Both linear and reflecting TOF mass
spectrometric geometries were examined. For reflecting TOF-MS, a
two stage electrostatic reflector was used and the effective drift
path was 2.0 m. A dualmicrochannel plate detector was used. The
detector was placed beside the electrostatic deflector due to space
constraints of the vacuum chamber. Deflecting voltage was applied
to horizontal deflecting plates and the beam path was bent in order
to direct the ions to the detector for the linear geometry. The
total flight distance was 1 m for the linear geometry. The four
degree bend was sufficient to block the line-of-sight between the
ion creation region and the detector to prevent any energetic
neutral flux created in the ionization region from reaching the
detector. For reflecting TOF measurements, the beam path was bent
in the opposite direction. To avoid detector saturation caused by
the high abundance of ionized matrix molecules in studies performed
at higher laser powers, the low mass matrix ions were deflected
away from the detector by a pulsed electric field of 200 V/cm.
[0337] The signal output of the microchannel plates was amplified
and then digitized with a time resolution of 10 to 50 ns/channel
and typically summed over 100 laser pulses. Mass calibration was
performed by analyzing a variety of known masses, such as alkalis
at low mass, isotopically resolved fullerenes, mixtures of
gramicidin S, bovine insulin, horse heart cytochrome C, and horse
heart myoglobin.
[0338] The resulting time-of-flight mass spectra are illustrated in
FIG. 3A and FIG. 3B. FIG. 3A is a mass spectrum of the 17-mer
synthetic mixed base primer containing a cleavable ribose linkage
prior to ammonium hydroxide treatment. Two sizable peaks were
observed for the intact 17-mer corresponding to the di-protonated
molecular ion [M+2H].sup.2+ and the protonated molecular ion,
[M+H].sup.+.
[0339] The resulting oligomer fragments obtained following ammonium
hydroxide treatment were then analyzed, as shown in FIG. 3. As
indicated in the mass spectrum, peaks corresponding to the expected
cleavage products, the 7-mer, the 10-mer, and intact 17-mer, were
readily observable (and identifiable).
EXAMPLE 5
Capture and Selective Cleavage of a Biotinylated Primer Having a
Cleavable Ribose in the 10 Position
[0340] A biotinylated 18-mer containing a ribose in the 10
position, 5'-biotin-ATCTTCCTG-ribo-GCAAACTCA-3', SEQ ID NO:11,
(Keystone Laboratories, Inc., Menlo Park, Calif.), was captured on
streptavidin-coated beads (DynaBeads M-280, Dynal, Inc., Great
Neck, N.Y.). The immobilized primer was then washed after surface
binding, followed by treatment with ammonium hydroxide as described
in Example 3 above to effect selective cleavage of the immobilized
primer at the ribose site.
[0341] The modified primer, containing the biotin immobilization
attachment site and the cleavable ribose site, was analyzed both
prior to capture and subsequent to selective cleavage. The samples
were analyzed using MALDI in conjunction with TOF mass
spectrometry, as described in Example 4 above. FIG. 4 illustrates
the mass spectrum of the 8-mer resulting from selective cleavage of
the ribose site within the immobilized primer.
EXAMPLE 6
Immobilization of a Cleavable Extended Primer by Hybridization to
an Intermediary Solid-Phase 1 Bound Oligonucleotide
[0342] A modified Ml 3 reverse primer containing a 5'-(S)-thymidine
located 5 nucleotides from 35 the 3' end, having the sequence
presented herein as SEQ ID NO: 12, was hybridized to a single
stranded target molecule (SEQ ID NO:14) in hybridization medium
containing annealing buffer, 10.times. "THERMOSEQUNASE" DNA
Polymerase) in the presence of a mixture of deoxynucleotides and
dideoxynucleosides to produce a set of oligonucleotide fragments
corresponding to the locations of adenine within the target (i.e.
thymidine within the reaction product). The reaction was performed
using standard cycle sequencing protocols and an 8:1 ratio of
primer to template. Due to the primer-to-template ratio employed,
the resulting set of primer extension products were primarily (89%)
in single stranded form.
[0343] Following primer extension, an intermediary oligonucleotide
complementary to the M13 reverse primer and biotinylated at the 3'
end, having SEQ ID NO:13, was added to the mixture and annealed to
the primer using a standard heat/cool annealing process: 95.degree.
C. for 2 min 30 sec; 25 cycles at 95.degree. C. for 15 sec,
45.degree. C. for 20 sec, 55.degree. C. for 10 sec, 70.degree. C.
for 20 sec), 5 cylces at 95.degree. C. for 30 sec, and 70.degree.
C. for 20 sec, followed by cooling from 95.degree. C. to 70.degree.
C. for 1 min at the rate of0.1.degree. C. per second, and
subsequent maintenance of the sample at 4.degree. C. Streptavidin
coated magnetic beads (MPG-Streptavidin, CPG, Inc., Lincoln Park,
N.J.) were then added to the mixture to capture the biotinylated
intermediary oligonucleotide/extended primer hybrid. The
immobilized product was washed to remove enzymes, triphosphates and
salts in a multistep wash process. The sample was then treated with
silver nitrate (5 microliters, 0.01 mM, Aldrich, Milwaukee, Wis.)
and DTT to release the extension segments into solution. This
solution was (i) separated from the solid phase bound intermediary
oligonucleotide-first primer region complex, (ii) mixed with
3-hydroxypicolonic acid, (iii) dried onto a silicon plate and (iv)
analyzed by MALDI TOF mass spectrometry as described in Example 4
above. The released extension segments are shown in FIG. 13.
[0344] As seen in FIG. 13, the method allows detection of
oligonucleotide extension segments with read lengths up to at least
about 33 base pairs with good resolution.
EXAMPLE 7
Detection of Point Mutation(S) Using a Single Base Fingerprint
[0345] Two DNA templates, one a synthetic 73-mer (presented herein
as SEQ ID NO:14) with a sequence identical to wild type M13
plasmid, corresponding to template "16-C/19-G", and the other a
mutant plasmid, (having a partial sequence included herein as SEQ
ID NO:15), referred to as template "16-A/19-T", were used in primer
extension reactions. The sequences of the templates differed only
at base positions 16 and 19, relative to the 3' end (counting
upstream from the end of the priming region), with the first
template possessing a cytosine at position 16 and a guanine at
position 19, as presented in SEQ ID NO:14, while the second
template, corresponding to SEQ ID NO:15, contained an adenine and
thymine substituted at positions 16 and 19, respectively.
[0346] Each of the templates was subjected to enzymatic extension
in the presence of ddC using a primer with the sequence presented
herein as SEQ ID NO:16. The resulting product mixtures, containing
ddC-terminated oligonucleotide fragments derived from the parent
templates, were then analyzed by MALDI TOF mass spectrometry as
described above and illustrated in FIG. 14A (corresponding to the
reaction product(s) derived from template 16-C/19-G, SEQ ID NO:17)
and FIG. 14B (corresponding to the reaction product(s) derived from
template 16-A/19-T, SEQ ID NO:18).
[0347] As can be seen from the resulting spectra of the reaction
products, the exact mass values corresponding to the differences
between select peaks in each of the spectra were calculated,
confirming the presence of two single nucleotide substitutions at
positions 16 and 19 from the 5' end. As demonstrated in FIG.
14A-14B, the measured mass values for the peak-to-peak differences
are shown in large type, while the actual/theoretical mass values
are shown in small type. The G-to-T base substitution is indicated
by the difference in the Am values for template 16-C/19-G (mass
peak b-mass peak a-618.9) versus template 16-A/19-T (mass peak
b-mass peak a=593). The observed mass difference of 25 (618.9 minus
593.4) corresponds to the difference in mass between guanine
(MW=51) and thymine (MW=126). Confirmation of a single base
substitution occurring at position 19 as a result of a C to A
mutation was similarly determined (e.g., Am.sub.d-b versus
Am.sub.g-f). The single base substitution at position 19 was
further confirmed by the absence of a peak at position 19 in the
spectrum corresponding to template 16-A/19-T (FIG. 14B).
EXAMPLE 8
Comparison of Primer Extension Reactions Using Cleavable Versus
Full Primer
[0348] A modified M13 reverse primer containing a 5'-biotin group
and a thiol-thymidine located 5 nucleotides from the 3' end of the
primer (SEQ ID NO:16) was hybridized to a single stranded target
molecule (SEQ ID NO:14), followed by enzymatic extension in the
presence of a mixture of deoxynucleotides and dideoxy-T to produce
a set of oligonucleotide fragments corresponding to the locations
of adenine within the target, following the procedure described in
Example 6. Following the ddT extension reaction, the biotinylated
primer/extension product was captured (immobilized) on
streptavidin-coated magnetic beads. The bead-immobilized
primer/extension products were then subjected to a series of wash
steps to remove template, enzyme, triphosphates and additional
salts. The streptavidin-coated magnetic beads, now containing
immobilized primer/extension products, were then divided into two
tubes, the contents of the first tube was treated with silver
nitrate and DTT, to cleave the primer at the thiolthymidine located
5 nucleotides from the 5'-end of the primer and to release the
extension segments into solution. The contents of the second tube
were boiled to effect disruption of the biotin/streptavidin bond
and release the full primer/extension products and into solution.
The two samples were then separately mixed with 3-hydroxypicolinic
acid, dried, and analyzed by MALDI TOF mass spectrometry as
described above.
[0349] The resulting mass spectra are shown in FIG. 15A (full
length primer-extension segments) and FIG. 15B (cleaved
primer-extension segments having increased read length). As can be
seen, the resolution quality and read length of spectra of cleaved
primer-extension segment samples (FIG. 15B) according to the
present method are superior to those of the full
primer-boil/release sample (FIG. 15A). The broad peak centered
around base No. 15 in the uncleaved primer sample (FIG. 15A) is due
to primer dimerization, and is an artifact that occasionally occurs
when the sample includes a large amount of primer. Cleavage of the
primer removes this artifact, as can be seen in FIG. 15B.
[0350] While the invention has been described with reference to
specific methods and embodiments, it will be appreciated that
various modifications and changes may be made without departing
from the invention.
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Sequence CWU 1
1
* * * * *