U.S. patent application number 10/145970 was filed with the patent office on 2003-06-19 for methods of screening nucleic acids using mass spectrometry.
Invention is credited to Becker, Christopher H., Monforte, Joseph A., Shaler, Thomas A., Tan, Yuping.
Application Number | 20030113745 10/145970 |
Document ID | / |
Family ID | 21756520 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113745 |
Kind Code |
A1 |
Monforte, Joseph A. ; et
al. |
June 19, 2003 |
Methods of screening nucleic acids using mass spectrometry
Abstract
Methods for screening nucleic acids for mutations by analyzing
nonrandomly fragmented nucleic acids using mass spectrometric
techniques and to procedures for improving mass resolution and mass
accuracy of these methods of detecting mutations. Kits for
performing the methods are provided.
Inventors: |
Monforte, Joseph A.;
(Berkeley, CA) ; Shaler, Thomas A.; (Fremont,
CA) ; Tan, Yuping; (Fremont, CA) ; Becker,
Christopher H.; (Palo Alto, CA) |
Correspondence
Address: |
STEPHANIE L. SEIDMAN
HELLER EHRMAN WHITE & MCAULIFFE LLP
7TH FL.
4350 LA JOLLA VILLAGE DRIVE
SAN DIEGO
CA
92122-1246
US
|
Family ID: |
21756520 |
Appl. No.: |
10/145970 |
Filed: |
May 13, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10145970 |
May 13, 2002 |
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09515277 |
Feb 29, 2000 |
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6468748 |
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10145970 |
May 13, 2002 |
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08811505 |
Mar 4, 1997 |
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6051378 |
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60012752 |
Mar 4, 1996 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 1/6827 20130101; C12Q 1/6827 20130101; C12Q 1/683 20130101;
C12Q 1/683 20130101; C12Q 1/6827 20130101; C12Q 2565/519 20130101;
C12Q 2565/518 20130101; C12Q 2565/607 20130101; C12Q 2521/307
20130101; C12Q 2521/307 20130101; C12Q 2563/167 20130101; C12Q
2563/167 20130101; C12Q 2565/607 20130101; C12Q 2523/107
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Goverment Interests
[0002] This invention was supported in part by a Financial
Assistance Award from the United States Department of Commerce,
Advanced Technology Program, Cooperative Agreement #70NANB5H 1029.
The U.S. Government may have rights in this invention.
Claims
We claim:
1. A method of detecting mutations in a target nucleic acid
comprising: obtaining a target nucleic acid in single-stranded
form; hybridizing the single-stranded target nucleic acid to one or
more sets of fragmenting probes to form hybrid target nucleic
acid/fragmenting probe complexes comprising at least one
double-stranded region and at least one single-stranded region;
nonrandomly fragmenting the target nucleic acid by cleaving the
hybrid target nucleic acid/fragmenting probe complexes at every
single-stranded region with at least one single-strand-specific
cleaving reagent to form a set of nonrandom length fragments
(NLFs); and determining masses of the members of the set using mass
spectrometry.
2. The method of claim 1, wherein at least one member of the set of
single-stranded NLFs optionally has one or more nucleotides
replaced with mass-modified nucleotides.
3. The method of claim 2, wherein the determining step optionally
further comprises utilizing internal self-calibrants to provide
improved mass accuracy.
4. The method of claim 1, wherein the set of fragmenting probes
leaves single-strand gaps between double-stranded regions formed by
hybridization of the set of fragmenting probes to the target
nucleic acid.
5. The method of claim 4, wherein the hybridizing step further
comprises: providing two sets of single-stranded target nucleic
acid and separately hybridizing a first set of fragmenting probes
to a first set of single-stranded target nucleic acid and a second
set of fragmenting probes to a second set of single-stranded target
nucleic acid, wherein the members of the second set of fragmenting
probes comprise at least one single-stranded nucleotide sequence
complementary to regions of the target nucleic acid that are not
complementary to any nucleotide sequences in any members of the
first set of fragmenting probes.
6. The method of claim 5, wherein the members of the first set of
fragmenting probes comprise nucleotide sequences that overlap with
nucleotide sequences of the members of the second set of
fragmenting probes.
7. The method of claim 1, wherein the single-strand-specific
cleaving reagent is a single-strand-specific endonuclease.
8. The method of claim 1, wherein the single-strand-specific
cleaving reagents are single-strand specific chemical cleaving
reagents.
9. The method of claim 8, wherein the single-strand specific
chemical cleaving reagents are selected from the group consisting
of hydroxylamine, hydrogen peroxide, osmium tetroxide and potassium
permanganate.
10. The method of claim 1, further comprising after the nonrandomly
fragmenting step: hybridizing one or more of the NLFs to one or
more capture probes, wherein the capture probes comprise a
single-stranded region complementary to at least one of the NLFs
and a first binding moiety, binding the first binding moiety to a
second binding moiety attached to a solid support, wherein the
binding occurs either before or after the hybridizing of the NLFs
to one or more capture probes, isolating a set of single-stranded
NLFs.
11. The method of claim 1, wherein the fragmenting probes comprise
a single-stranded nucleotide sequence and a first binding moiety,
further comprising: after the nonrandomly fragmenting step, binding
the first binding moiety to a second binding moiety attached to a
solid support; and isolating the set of single-stranded NLFs.
12. A method of detecting mutations in a target nucleic acid
comprising: obtaining a target nucleic acid in single-stranded
form; nonrandomly fragmenting the target nucleic acid with one or
more restriction endonucleases to form a set of nonrandom length
fragments (NLFs); hybridizing one or more of the set of NLFs or a
subset thereof to one or more oligonucleotide probes, wherein each
of the oligonucleotide probes comprises a nucleic acid comprising a
single-stranded region and a first binding moiety, binding the
first binding moiety to a second binding moiety attached to a solid
support either before or after the hybridizing step; isolating the
set or subset of single-stranded NLFs; and determining masses of
the members of the set using mass spectrometry.
13. The method of claim 12, wherein all of the oligonucleotide
probes consist of one of either full-length positive or full-length
negative single strands of the target nucleic acid and a first
binding moiety.
14. The method of claim 12, wherein the binding between the first
binding moiety and the second binding moiety is a covalent
attachment.
15. The method of claim 12, wherein one binding moiety is a member
selected from the group consisting of an antibody, a hormone, an
inhibitor, a co-factor portion, a binding ligand and a
polynucleotide sequence, and the other binding moiety is a
corresponding member selected from the group consisting of an
antigen capable of recognizing the antibody, a receptor capable of
recognizing the hormone, an enzyme capable of recognizing the
inhibitor, a cofactor enzyme binding site capable of recognizing
the co-factor portion, a substrate capable of recognizing the
binding ligand and a complementary polynucleotide sequence.
16. The method of claim 12, wherein the isolating further
comprises: washing the set of NLFs bound to the solid support with
a solution comprising volatile salts selected from the group
consisting of ammonium bicarbonate dimethyl ammonium bicarbonate
and trimethyl ammonium bicarbonate.
17. A method of detecting mutations in a target nucleic acid
comprising: obtaining a target nucleic acid in single-stranded
form; hybridizing the single-stranded target nucleic acid to one or
more restriction site probes to form hybridized target nucleic
acids having double-stranded regions, wherein the restriction site
probes have hybridized to the single-stranded target nucleic acid
and at least one single-stranded region; nonrandomly fragmenting
the hybridized target nucleic acids by contacting it with one or
more restriction endonucleases that cleave at restriction sites
within the double-stranded regions to produce a set of
single-straneded nonrandom length fragments (NLFs); and determining
masses of the members of the set using mass spectrometry.
18. The method of claim 17, further comprising after the
nonrandomly fragmenting step, hybridizing the NLFs to one or more
capture probes, wherein the capture probes comprise a
single-stranded region complementary to at least one or the NLFs
and a first binding moiety, binding the first binding moiety to a
second binding moiety attached to a solid support, wherein the
binding occurs either before or after the hybridizing of the NLFs
to one or more capture probes, isolating a set of single-stranded
NLFs.
19. The method of claim 18, wherein the cleaved restriction site
probes comprise a single-stranded region complementary to half of a
restriction endonuclease site and a first binding moiety; and
further comprising, after the nonrandomly fragmenting step, binding
the first binding moiety to a second binding moiety attached to a
solid support, and isolating a set of single-stranded NLFs.
20. A method of detecting mutations in a target nucleic acid
comprising: obtaining a target nucleic acid in single-stranded
form; exposing the target nucleic acid to conditions permitting
folding of the single-stranded target nucleic acid to form a
three-dimensional structure having intramolecular secondary and
tertiary interactions; nonrandomly fragmenting the folded target
nucleic acid with at least one structure-specific endonuclease to
form a set of single-stranded nonrandom length fragments (NLFs);
modifying either the target nucleic acid or the set of
single-stranded NLFs such that members of the set of
single-stranded NLFs comprise a single-stranded nucleotide sequence
and at least one first binding moiety; binding the first binding
moiety to a second binding moiety attached to a solid support;
isolating the set of single-stranded NLFs; and determining masses
of the members of the set using mass spectrometry.
21. The method of claim 20, wherein the isolated set of
single-stranded NLFs comprise any NLFs having a 5' end of the
target nucleic acid.
22. A method of detecting mutations in a target nucleic acid
comprising: obtaining a target nucleic acid in single-stranded
form; exposing the nucleic acid to conditions permitting folding of
the single-stranded target nucleic acid to form a three-dimensional
structure having intramolecular secondary and tertiary
interactions; nonrandomly fragmenting the folded target nucleic
acid with at least one structure-specific endonuclease to form a
set of single-stranded nonrandom length fragments (NLFs);
hybridizing one or more of the set of NLFs to one or more capture
probes, wherein the capture probes comprise a single-stranded
nucleotide sequence and a first binding moiety; binding the first
binding moiety to a second binding moiety attached to a solid
support either before or after the hybridizing step; isolating a
set of single-stranded NLFs; and determining masses of the members
of the set using mass spectrometry.
23. The method of claim 22 wherein the isolated set of
single-stranded NLFs comprise any NLFs having a 5' end of the
target nucleic acid.
24. The method of claim 22, wherein the structure-specific
endonuclease is selected from the group consisting of: T4
endonuclease VII, RuvC, MutY and the endonucleolytic activity from
the 5'-3' exonuclease subunit of thermo-stable polymerases.
25. A method of detecting mutations in a target nucleic acid
comprising: obtaining a target nucleic acid in single-stranded
form; hybridizing the single-stranded target nucleic acid to one or
more wild type probes; nonrandomly fragmenting the target nucleic
acid with one or more mutation-specific cleaving reagents that
specifically cleave at any regions of nucleotide mismatch that form
between the target nucleic acid and any of the wild type probes to
form a set of single-stranded nonrandom length fragments (NLFs);
and determining masses of the members of the set using mass
spectrometry
26. The method of claim 25, wherein the nonrandomly fragmenting
step further comprises: digesting the first set of nonrandom length
fragments with one or more restriction endonucleases or cleaving
the first set of nonrandom length fragments with one or more
single-strand-specific cleaving reagents.
27. The method of claim 25, wherein members of the set of
single-stranded NLFs comprise a single-stranded region and at least
one first binding moiety, further comprising, after the nonrandomly
fragmenting step, binding the first binding moiety to a second
binding moiety attached to a solid support; and isolating a set of
single-stranded NLFs.
28. The method of claim 25, wherein the obtaining step further
comprises: hybridizing members of the set of NLFs to one or more
capture probes, wherein the capture probes comprise a
single-stranded nucleotide sequence and at least one first binding
moiety, binding the first binding moiety to a second binding moiety
attached to a solid support; and isolating a set of single-stranded
NLFs.
29. The method of claim 25, wherein the obtaining step further
comprises: isolating a set of single-stranded NLFs comprising any
NLFs having a 5' end of the target nucleic acid.
30. A method of detecting mutations in a target nucleic acid
comprising: nonrandomly fragmenting the target nucleic acid with
one or more restriction endonucleases to form a set of
double-stranded nonrandom length fragments (NLFs), wherein the
nonrandomly fragmenting further comprises including volatile salts
in the restriction buffer; and determining masses of the members of
the set of double-stranded NLFs, wherein the determining does not
involve sequencing of the target nucleic acid.
31. A method of detecting mutations in a double-stranded target
nucleic acid comprising: nonrandomly fragmenting the target nucleic
acid using one or more restriction endonucleases to form a first
set of nonrandom length fragments (NLFs); hybridizing members of
the first set of NLFs to a set of wild type probes; nonrandomly
fragmenting one or more members of the set of NLFs with one or more
mutation-specific cleaving reagents that specifically cleave at any
regions of nucleotide mismatch that form between members of the
first set of NLFs and complementary members of the set of wild type
probes, wherein the nonrandomly fragmenting step forms a second set
of NLFs; and determining masses of members of the second set of
NLFs using mass spectrometry, wherein the determining does not
require sequencing of the target nucleic acid.
32. The method of claim 31 further comprising: obtaining the set of
wild type probes by nonrandomly fragmenting a wild type target
nucleic acid using the same restriction endonucleases used to form
the first set of NLFs.
33. The method of claim 32, wherein the steps of nonrandomly
fragmenting of the target nucleic acid and obtaining the set of
wild type fragmenting probes are performed simultaneously in a
single solution.
34. The method of claim 32 further comprising, before the
determining step, isolating the second set of NLFs wherein the
members of the second set comprise double-stranded nucleotide
sequences and a first binding moiety; and binding the first binding
moiety to a second binding moiety attached to a solid support.
35. The method of claim 32 further comprising before the
determining step, isolating the second set of NLFs wherein the
isolating comprises hybridizing members of the second set of NLFs
to one or more capture probes, wherein the capture probes comprise
a single-stranded nucleotide sequence and a first binding moiety,
binding the first binding moiety to a second binding moiety
attached to a solid support.
36. A method of detecting mutations in a target nucleic acid
comprising: nonrandomly fragmenting the target nucleic acid, using
a solution comprising one or more volatile salts to form a set of
nonrandom length fragments (NLFs); and determining masses of
members of the set of NLFs using mass spectrometry, wherein the
determining does not involve sequencing of the target nucleic
acid.
37. A kit for detecting mutations in one or more target nucleic
acids in a sample comprising: (a) one or more sets of fragmenting
probes, wherein the fragmenting probes are complementary to a
sequence of one or more of the target nucleic acids; (b) a
single-strand specific cleaving regent; (c) a solid support for
isolating single-stranded target nucleic acids that have been
nonrandomly fragmented into single-stranded nonrandom length
fragments; and (d) matrix for performing mass spectrometry
analyses.
38. The kit of claim 37, wherein the single-strand specific
cleaving reagent is a single-strand-specific chemical cleaving
reagent selected from the group consisting of hydroxylamine,
hydrogen peroxide, osmium tetroxide and potassium permanganate.
39. The kit of claim 37, wherein the single-strand specific
cleaving reagent is a nuclease selected from the group consisting
of Mung bean nuclease, Nuclease S1 and RNase A.
40. A kit for detecting mutations in one or more target nucleic
acids in a sample comprising: (a) one or more sets of restriction
site probes, wherein the probes comprise a single-stranded sequence
capable of hybridizing to a sequence of the one or more target
nucleic acids; (b) one or more restriction endonucleases that
cleave at restriction sites within the restriction site probes; and
(c) a solid support for isolating single-stranded target nucleic
acids that have been nonrandomly fragmented into single-stranded
nonrandom length fragments.
41. The kit of claim 40, wherein the restriction endonuclease is a
Class IIS restriction endonuclease.
42. The kit of claim 40, wherein the restriction site probe
comprises two regions, a first region that is single-stranded and
complementary to a specific sequence within the target nucleic
acid, and a second region that is double-stranded and contains a
restriction recognition site for a Class IIS restriction
endonuclease.
43. The kit of claim 40, further comprising matrix for performing
mass spectrometry analyses.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of co-pending allowed
U.S. application Ser. No. 09/515,277, filed Feb. 29, 2000. This
application is also a continuation of U.S. application Ser. No.
08/811,505, filed Mar. 4, 1997, now U.S. Pat. No. 6,051,378.
Benefit of priority under 35 U.S.C. .sctn.119(e) is claimed to U.S.
provisional application Serial No. 60/012,752, filed Mar. 4,
1996.
TECHNICAL FIELD
[0003] This invention relates generally to methods for screening
nucleic acids for mutations by analyzing fragmented nucleic acids
using mass spectrometry.
INTRODUCTION
[0004] 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 associated with each disorder. The definitive diagnostic
test for any specific genetic disease (or predisposition to
disease) will be the identification of mutations in affected cells
that result in alterations of gene function. Furthermore, response
to specific medications may depend on the presence of mutations.
Developing DNA (or RNA) screening as a practical tool for medical
diagnostics requires a method that s inexpensive, accurate,
expeditious, and robust.
[0005] Genetic mutations can manifest themselves in several forms,
such as point mutations where a single base is changed to one of
the three other bases, deletions where one or more bases are remove
from a nucleic acid sequence and the bases flanking the deleted
sequence are directly linked to each other, and insertions where
new bases are inserted at a particular point in a nucleic acid
sequence adding additional length to the overall sequence. Large
insertions and deletions, often the result of chromosomal
recombination and rearrangement events, can lead to partial or
complete loss of a gene. Of these forms of mutation, in general the
most difficult type of mutation to screen for and detect is the
point mutation because it represents the smallest degree of
molecular change. The term mutation encompasses all the
above-listed types of differences from wild type nucleic acid
sequence. Wild type is a standard or reference nucleotide sequence
to which variations are compared. As defined, any variation from
wild type is considered a mutation including naturally occurring
sequence polymorphisms.
[0006] Although a number of genetic defects can be linked to a
specific single point mutation within a gene, e.g. sickle cell
anemia, many are caused by a wide spectrum of different mutations
throughout the gene. A typical gene that might be screened using
the methods described here could be anywhere from 1,000 to 100,000
bases in length, though smaller and larger genes do exist. Of that
amount of DNA, only a fraction of the base pairs actually encode
the protein. These discontinuous protein coding regions are called
exons and the remainder of the gene is referred to as introns. Of
these two types of regions, exons often contain the most important
sequences to be screened. Several complex procedures have been
developed for scanning genes in order to detect mutations, which
are applicable to both exons and introns.
[0007] Gel Electrophoresis: Several of the procedures described
below use some form of gel electrophoresis. Therefore it is
worthwhile to briefly consider this separation technology before
proceeding to the specific methods. In terms of current use, most
of the methods to scan or screen genes employ slab or capillary gel
electrophoresis for the separation and detection step in the
assays. Gel electrophoresis of nucleic acids primarily provides
relative size information based on mobility through the gel matrix.
If calibration standards are employed, gel electrophoresis can be
used to measure absolute and relative molecular weights of large
biomolecules with some moderate degree of accuracy; even then
typically the accuracy is only 5% to 10%. Also the molecular weight
resolution is limited. In cases where two DNA fragments with
identical number of base pairs can be separated, using high
concentration polyacrylamide gels, it is still not possible,to
identify which band on a gel corresponds to which DNA fragment
without performing secondary labeling experiments. Gel
electrophoresis techniques can only determine size and cannot
provide any information about changes in base composition or
sequence without performing more complex sequencing reactions.
Gel-based techniques, for the most part, are dependent on labeling
methods to visualize and discriminate between different nucleic
acid fragments.
[0008] DNA Sequencing: The principal approach currently used to
screen for genetic mutations is DNA sequencing. Sequencing
reactions can be performed to screen the full genetic target base
by base. This process, which can pinpoint the exact location and
nature of mutation, requires labeling DNA, use of polyacrylamide
gels, and a multiplicity of reactions to assess all bases over the
length of a gene, all of which are slow and labor intensive
procedures. [J. Bergh et al. "Complete Sequencing of the p53 Gene
Provides Prognostic Information in Breast Cancer Patients,
Particularly in Relation to Adjuvant Systemic Therapy and
Radiotherapy," Nature Medicine 1, 1029 (1995)]
[0009] For DNA sequencing, nucleic acids comprising different exons
or small clusters of exons are individually amplified, often using
polymerase chain reaction (PCR). The amplifications are normally
performed separately although some multiplexing of reactions is
possible. The amplified nucleic acids typically range from one
hundred to several thousand bases in length. Following
amplification, the PCR products can serve as templates for standard
dideoxy-based Sanger sequencing reactions. The four different
sequencing reactions are run (or for fluorescence detection, one
reaction with four different dye terminators) and then analyzed by
polyacrylamide gel electrophoresis. Each sequencing run yields
about 300 to 600 bases of sequence which typically must be read
with at least a two to three-fold redundancy in order to assure
accuracy. Using slab gel, the analysis process typically takes
several hours.
[0010] SSCP: The single strand conformational polymorphism assay
takes advantage of structural variation within DNA that results
from mutation. The method involves folding the single-stranded form
of a given nucleic acid sequence into a thermodynamically directed
secondary and tertiary structure. In most cases, mutated sequences
form different structures than the wild type sequence, thus
permitting separation of mutated and wild type sequences by gel
electrophoresis. Like sequencing, this assay is complicated by the
need to label molecules and run polyacrylamide gels. In a typical
case, mutations can be located within a general range of 50 to 200
base pairs, but the exact nature of the mutation cannot be
identified. [M. Orita et al., "Detection of Polymorphisms of Human
DNA by Gel Electrophoresis as Single-Stranded Conformation
Polymorphisms," Proc. Natl. Acad. Sci. USA 86, 2766 (1989)]
[0011] DGGE: Like SSCP, denaturing gradient gel electrophoresis
assays also differentiate based on structural variation, but
require the use of gradient gels, which are difficult to prepare.
The different thermodynamic stability of structures formed by the
mutant sequence, as opposed to wild type, lead to differences in
the temperature and/or pH at which the molecule will denature. DGGE
mutation identification and localization properties are similar to
those for SSCP though sensitivity is higher for DGGE because not
all mutations cause the structural changes that the SSCP method
depends upon for detection. [E. S. Abrams, S. E. Murdaugh & L.
S. Lerman, "Comprehensive Detection of Single Base Changes in Human
Genomic DNA Using Denaturing Gradient Gel Electrophoresis and a GC
Clamp," Genomics 7, 463 (1990)]
[0012] EMC: Enzyme mismatch cleavage utilizes one or more enzymes
that are capable of recognizing interruptions in base pairing
within a double-stranded nucleic acid molecule, e.g. base-base
mismatches, bulges, or internal loops. A given length of DNA or RNA
is prepared in heterozygous form, with one strand composed of wild
type nucleic acid and the other stand containing a potential
mutation. At the specific site where the mutation forms a mismatch
with the wild type sequence, a structural perturbation occurs. An
enzyme such as T4 endonuclease VII, RuvC, RNase A, or MutY, can
recognize such a structural perturbation and can site-specifically
cut the double-stranded nucleic acid, creating smaller molecules
whose sizes indicate the presence and location of the mutation. As
with the previously discussed methods, this approach as currently
used, also requires labeling and gel electrophoresis. With this
method, the site of mutation can be localized to within a few base
pairs but the exact nature of the mutation cannot be determined.
[R. Youil, B. W. Kemper & R. G. H. Cotton, "Screening for
Mutations by Enzyme Mismatch Cleavage with T4 Endonuclease VII,"
Proc. Natl. Acad. Sci. USA 92, 87 (1995)]
[0013] CCM: A variation of EMC is to replace the enzymatic cleavage
step with chemical cleavage. Chemical cleavage mismatch analysis
involves the use of reagents such as osmium tetroxide to react with
mismatched thymine residues or hydroxylamine to react with
mismatched cytosine residues. Cleavage of the modified mismatched
residues occurs when the modified bases are subsequently treated
with piperidine or another oxidizing agent. The effectiveness of
the method is similar to EMC. [J. A. Saleeba & R. G. H. Cotton,
"Chemical Cleavage of Mismatch to Detect Mutations," Methods in
Enzymology 217, 286 (1993)]
[0014] Hybridization Arrays: Several approaches to screening for
mutations involve the probing of a target nucleic acid by an array
of oligonucleotides that can differentiate between normal wild type
nucleic acids and mutant nucleic acids. These arrays involve the
performance of hundreds or thousands of hybridization reactions in
parallel with different site-directed oligonucleotides and requires
sophisticated and costly probe arrays. Hybridization arrays can
identify the location and type of mutation in many, but not all
cases. For example, semihomologous sequential insertions or targets
with repeating sequences and/or repeating sequential motifs cannot
be analyzed by hybridization. [A. C. Pease et al., "Light-Generated
Oligonucleotide Arrays for Rapid DNA Sequence Analysis," Proc.
Natl. Acad. Sci. USA 91, 5022 (1994)]
[0015] Simple screens: For mutations localized within a given gene,
such as the cystic fibrosis .DELTA.F508 deletion, it is also
possible to perform a single PCR or ligase chain reaction (LCR)
assay or simple hybridization assays tailored to these specific
sites. PCR and LCR results are presently determined by the use of
labeled molecules, where radioactive emissions, fluorescence,
chemiluminescence or color changes are detected directly. These
simple screens amount to a yes/no answer and do not directly
identify the nature of the mutation, only whether or not a reaction
took place. [P. Fang et al., "Simultaneous Analysis of Mutant and
Normal Alleles for Multiple Cystic Fibrosis Mutations by the Ligase
Chain Reaction," Human Mutation 6, 144 (1995)]
[0016] All of the methods in use today capable of screening broadly
for genetic mutations suffer from technical complication and are
labor and time intensive. There is a need for new methods that can
provide cost effective and expeditious means for screening genetic
material in an effort to reduce medical expenses. The inventions
described here address these issues by developing novel,
tailor-made processes that focus on the use of mass spectrometry as
a genetic analysis tool. Mass spectrometry requires minute samples,
provides extremely detailed information about the molecules being
analyzed including high mass accuracy, and is easily automated.
[0017] The late 1980's saw the rise of two new mass spectrometric
techniques for successfully measuring the masses of intact very
large biomolecules, namely, matrix-assisted laser
desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF
MS) [K. Tanaka et al., "Protein and Polymer Analyses up to m/z
100,000 by Laser Ionization Time-of-flight Mass Spectrometry,"
Rapid Commun. Mass Spectrom. 2, 151-153 (1988); B. Spengler et al.,
"Laser Mass Analysis in Biology," Ber. Bunsenges. Phys. Chem. 93,
396-402 (1989)] and electrospray ionization (ESI) combined with a
variety of mass analyzers [J. B. Fenn et al., Science 246, 64-71
(1989)]. Both of these two methods are suitable for genetic
screening tests. The MALDI mass spectrometric technique can also be
used with methods other than time-of-flight, for example, magnetic
sector, Fourier-Transform, ion cyclotron resonance, quadropole, and
quadropole trap. One of the advances in MALDI analysis of
polynucleotides was the discovery of 3-hydroxypicolinic acid as an
ideal matrix for mixed-base oligonucleotides. Wu, et al., Rapid
Comm'ns in Mass Spectrometry, 7:142-146 (1993).
[0018] MALDI-TOF MS involves laser pulses focused on a small sample
plate comprising analyte molecules (nucleic acids) embedded in
either a solid or liquid matrix comprising a small, highly
absorbing compound. The laser pulses transfer energy to the matrix
causing a microscopic ablation and concomitant ionization of the
analyte molecules, producing a gaseous plume of intact, charged
nucleic acids in single-stranded form. If double-stranded nucleic
acids are analyzed, the MALDI-TOF MS typically results in mostly
denatured single-strand detection. The ions generated by the laser
pulses are accelerated to a fixed kinetic energy by a strong
electric field and then pass through an electric field-free region
in vacuum in which the ions travel with a velocity corresponding to
their respective mass-to-charge ratios (m/z). The smaller m/z ions
will travel through the vacuum region faster than the larger m/z
ions thereby causing a separation. At the end of the electric
field-free region, the ions collide with a detector that generates
a signal as each set of ions of a particular mass-to-charge ratio
strikes the detector. Usually for a given assay, 10 to 100 mass
spectra resulting from individual laser pulses are summed together
to make a single composite mass spectrum with an improved
signal-to-noise ratio.
[0019] The mass of an ion (such as a charged nucleic acid) is
measured by using its velocity to determine the mass-to-charge
ratio by time-of-flight analysis. In other words, the mass of the
molecule directly correlates with the time it takes to travel from
the sample plate to the detector. The entire process takes only
microseconds. In an automated apparatus, tens to hundreds of
samples can be analyzed per minute. In addition to speed, MALDI-TOF
MS has one of the largest mass ranges for mass spectrometric
devices. The current mass range for MALDI-TOF MS is from 1 to
1,000,000 Daltons (Da) (measured recently for a protein). [R. W.
Nelson et al., "Detection of Human IgM at m/z.about.1 MDa," Rapid
Commun. Mass Spectrom. 9, 625 (1995)].
[0020] The performance of a mass spectrometer is measured by its
sensitivity, mass resolution and mass accuracy. Sensitivity is
measured by the amount of material needed; it is generally
desirable and possible with mass spectrometry to work with sample
amounts in the femtomole and low picomole range. Mass resolution,
m/.DELTA.m, is the measure of an instrument's ability to produce
separate signals from ions of similar mass. Mass resolution is
defined as the mass, m, of a ion signal divided by the full width
of the signal, .DELTA.m, usually measured between points of
half-maximum intensity. Mass accuracy is the measure of error in
designating a mass to an ion signal. The mass accuracy is defined
as the ratio of the mass assignment error divided by the mass of
the ion and can be represented as a percentage.
[0021] To be able to detect any point mutation directly by
MALDI-TOF mass spectrometry, one would need to resolve and
accurately measure the masses of nucleic acids in which a single
base change has occurred (in comparison to the wild type nucleic
acid). A single base change can be a mass difference of as little
as 9 Da. This value represents the difference between the two bases
with the closest mass values, A and T
(A=2'-deoxyadenosine-5'-phosphate=313.19 Da;
T=2'-deoxythymidine-5'-phosp- hate=304.20 Da;
G=2'-deoxyguanosine-5'-phosphate=329.21 Da; and
C=2'-deoxycytidine-5'-phosphate=289.19 Da). If during the mutation
process, a single A changes to T or a single T to A, the mutant
nucleic acid containing the base transversion will either decrease
or increase by 9 in total mass as compared to the wild type nucleic
acid. For mass spectrometry to directly detect these transversions,
it must therefore be able to detect a minimum mass change,
.DELTA.m, of approximately 9 Da.
[0022] For example, in order to fully resolve (which may not be
necessary) a point-mutated (A to T or T to A) heterozygote 50-base
single-stranded DNA fragment having a mass, m, of .about.15.000 Da
from its corresponding wild type nucleic acid, the required mass
resolution is m/.DELTA.m=15,000/9.apprxeq.1,700. However, the mass
accuracy needs to be significantly better than 9 Da to increase
quality assurance and to prevent ambiguities where the measured
mass value is near the half-way point between the two theoretical
masses. For an analyte of 15,000 Da, in practice the mass accuracy
needs to be .DELTA.m.about..+-.3 Da=6 Da. In this case, the
absolute mass accuracy required is (6/15,000)*100=0.04%. Often a
distinguishing level of mass accuracy relative to another known
peak in the spectrum is sufficient to resolve ambiguities. For
example, if there is a known mass peak 1000 Da from the mass peak
in question, the relative position of the unknown to the known peak
may be known with greater accuracy than that provided by an
absolute, previous calibration of the mass spectrometer.
[0023] In order for mass spectrometry to be a useful tool for
screening for mutations in nucleic acids, several basic
requirements need to be met. First, any nucleic acids to be
analyzed must be purified to the extent that minimizes salt ions
and other molecular contaminants that reduce the intensity and
quality of the mass spectrometric signal to a point where either
the signal is undetectable or unreliable, or the mass accuracy
and/or resolution is below the value necessary to detect single
base change mutations. Second, the size of the nucleic acids to be
analyzed must be within the range of the mass spectrometry-where
there is the necessary mass resolution and accuracy. Mass accuracy
and resolution do significantly degrade as the mass of the analyte
increases; currently this is especially significant above
approximately 30,000 Da for oligonucleotides (.about.100 bases).
Third, because all molecules within a sample are visualized during
mass spectrometric analysis (i.e. it is not possible to selectively
label and visualize certain molecules and not others as one can
with gel electrophoresis methods) it is necessary to partition
nucleic acid samples prior to analysis in order to remove unwanted
nucleic acid products from the spectrum. Fourth, the mass
spectrometric methods for generalized nucleic acid screening must
be efficient and cost effective in order to screen a large number
of nucleic acid bases in as few steps as possible.
[0024] The methods for detecting nucleic acid mutations known in
the art do not satisfy these four requirements. For example, prior
art methods for mass spectrometric analysis of DNA fragments have
focussed on double-stranded DNA fragments which result in
complicated mass spectra, making it difficult to resolve mass
differences between two complementary strands. See, e.g., Tang et
al., Rapid Comm'n. in Mass Spectrometry, 8:183-186 (1994).
[0025] Thus, there is a need for cost and time effective methods of
detecting genetic mutations using mass spectrometry, preferably
MALDI or ES, without having to sequence the genetic material and
with mass accuracy of a few parts in 10,000 or better.
SUMMARY OF THE INVENTION
[0026] The present invention provides methods of and kits for
detecting mutations in a target nucleic acid comprising nonrandomly
fragmenting the target nucleic acid to form a set of nonrandom
length fragments (NLFs), determining masses of members of the set
of NLFs using mass spectrometry, wherein the determining does not
involve sequencing of the target nucleic acid.
[0027] In a preferred embodiment, the method of detecting mutations
comprises obtaining a set of nonrandom length fragments in
single-stranded form. The masses of the members of the set of NLFs
can be compared with the known or predicted masses of a set of NLFs
derived from a wild type target nucleic acid that is the wild type
version of the target nucleic acid that is being screened for
mutations. The members of the set of single-stranded NLFs can
optionally have one or more nucleotides replaced with mass-modified
nucleotides, including mass-modified nucleotide analogs. Another
optional aspect of the invention is the inclusion of internal
calibrants or internal self-calibrants in the set of nonrandom
length fragments to be analyzed by mass spectrometry to provide
improved mass accuracy.
[0028] The present invention includes a number of nonrandom
fragmentation techniques for nonrandomly fragmenting a target
nucleic acid.
[0029] In one embodiment, the nonrandom fragmentation technique
comprises hybridizing a single-stranded target nucleic acid to one
or more sets of fragmenting probes to form hybrid target nucleic
acid/fragmenting probe complexes comprising at least one
double-stranded region and at least one single-stranded region,
nonrandomly fragmenting the target nucleic acid by cleaving the
hybrid target nucleic acid/fragmenting probe complexes at every
single-stranded region with at least one single-strand-specific
cleaving reagent to form a set of NLFs. The set of fragmenting
probes can leave single-stranded regions between double-stranded
regions formed by hybridization of the set of fragmenting probes to
the target nucleic acid. A single-stranded region comprises a
portion of a polynucleotide sequence as small as a single
phosphodiester bridge, i.e. the phosphodiester bond across from a
nick, to 450 nucleotides in length.
[0030] The fragmenting probes are oligonucleotides that are
complementary to a nucleotide sequence of the target nucleic acid.
A set of fragmenting probes can be created such that the nucleotide
sequences of the members of the set of fragmenting probes
represents the entire complement to the nucleotide sequence of the
target nucleic acid. For example, a set of fragmenting probes can
provide complete complementary sequence to the target nucleic acid.
Alternatively, a set of fragmenting probes, when hybridized to the
target nucleic acid, can leave single-stranded regions. Also, one
or more sets of fragmenting probes can be used such that the
members of one set of fragmenting probes contain nucleotide
sequences that overlap with nucleotide sequences of members of a
second set of fragmenting probes. In yet another aspect, there are
provided two sets of fragmenting probes, where members of the
second set of fragmenting probes comprise at least one
single-stranded nucleotide sequence complementary to regions of the
target nucleic acid that are not complementary to any nucleotide
sequences in any members of the first set of fragmenting
probes.
[0031] Once the set(s) of fragmenting probes are hybridized to the
target nucleic acid, the single-stranded regions are cleaved using
single-strand-specific cleaving reagents, including enzymatic
reagents as well as chemical reagents. Single-strand-specific
chemical cleaving reagents include hydroxylamine, hydrogen
peroxide, osmium tetroxide, and potassium permanganate.
[0032] Yet another nonrandom fragmentation technique comprises
providing a single-stranded target nucleic acid, hybridizing the
single-stranded target nucleic acid to one or more restriction site
probes to form hybridized target nucleic acids comprising
double-stranded regions where the restriction site probes have
hybridized to the single-stranded target nucleic acid and at least
one single-stranded region, nonrandomly fragmenting the hybridized
target nucleic acids using one or more restriction endonucleases
that cleave at restriction sites within the double-stranded
regions. Another variation on this technique involves use of
universal restriction probes comprising two regions, the first
region being single-stranded and complementary to a specific site
within the target nucleic acid, and the second region being
double-stranded and containing the restriction recognition site for
a particular class IIS restriction endonuclease. Class IIS
restriction endonucleases cleave double-stranded DNA at a specific
distance from their recognition site sequence.
[0033] Another technique for nonrandom fragmentation comprises
fragmenting the target nucleic acid with one or more restriction
endonucleases to form a set of NLFs. This and the other forms of
nonrandom fragmentation can be combined with direct and indirect
capture to a solid support to isolate single-stranded NLFs for mass
spectrometric analysis.
[0034] Another nonrandom fragmentation technique comprises
providing conditions permitting folding of the single-stranded
target nucleic acid to form a three-dimensional structure having
intramolecular secondary and tertiary interactions, and nonrandomly
fragmenting the folded target nucleic acid with at least one
structure-specific endonuclease to form a set of single-stranded
NLFs. A set of nonrandom length fragments can comprise a nested set
of NLFs, wherein each member of the set has a 5' end of the target
nucleic acid. The structure-specific endonucleases useful for
nonrandom fragmentation comprise any nucleases that cleave at
structural transitions within nucleic acids, including: Holliday
junctions, single-strand to double-strand transitions, or at the
ends of hairpin structures.
[0035] Another nonrandom fragmentation method comprises
mutation-specific cleavage by hybridizing a target nucleic acid to
a set of one or more wild type probes and specifically cleaving at
any regions of nucleotide mismatch or base mismatch that form
between the target nucleic acid and a wild type probe. The
mutation-specific cleavage can be accomplished using a
mutation-specific cleaving reagent comprising structure-specific
endonuclease or chemical reagents.
[0036] The nonrandom fragmentation methods described herein can be
combined to form different sets or subsets of nonrandom length
fragments. For example, the base mismatch nonrandom fragmentation
method using wild type probes can be used in concert with a set of
nonrandom length fragments that have already been creating using
any one of the other nonrandom fragmentation methods. These
nonrandom fragmentation methods can also be combined with isolation
methods designed to isolate specific sets of single-stranded
nonrandom length fragments, for example, only those NLFs derived
from the +stand of the target nucleic acid. The isolation methods
include direct capture of the set of NLFs to a solid support or
indirect capture of a set of NLFs to a solid support via a capture
probe capable of binding to a solid support via covalent or
noncovalent binding. The fragmenting, wild type, restriction site,
and universal restriction probes described herein can be also be
used as capture probes for isolating a particular set of NLFs.
[0037] The isolation methods also comprise the use of a solution of
volatile salts to wash away undesired contaminants from the set of
NLFs intended for mass determination in the mass spectrometer. The
volatile salts are useful for removing background noise and can be
easily removed by evaporation of the volatile salts prior to mass
spectrometric analysis. Volatile salt solutions can be used in a
variety of different methods to prepare organic molecules such as
nucleic acids and polypeptides for mass spectrometric analysis.
Thus, a method is described herein of decreasing background noise,
wherein the method comprises obtaining a sample to be analyzed by a
mass spectrometer, washing the sample with a solution of volatile
salts, and evaporating the solution of volatile salts from the
sample.
[0038] The fragmentation and isolation methods separately or
together can also be combined with the use of internal
self-calibrants to improve the mass accuracy of the mass
spectrometric analysis.
[0039] The above methods, separately or in combination, can also be
combined with the use of mass-modified nucleotides and
mass-modified nucleotide analogs incorporated in the target nucleic
acid or a set of NLFs to improve mass resolution between mass
peaks.
[0040] Kits for detecting mutations in one or more target nucleic
acids in a sample are also provided. In preferred embodiments, such
kits comprise one or more single-stranded target nucleic acids, one
or more sets of oligonucleotide probes, wherein each of the probes
is complementary to a portion of the single-stranded target nucleic
acids, and various cleaving reagents, including single-strand
specific cleaving reagents, restriction endonucleases (both Class
II and Class IIS), and mutation-specific cleaving reagents. The
oligonucleotide probes include fragmenting probes, restriction site
probes, and wild type probes. Such kits can also contain a matrix,
preferably 3-hydroxypicolinic acid. The kits may also contain
volatile salt buffers, and buffers providing conditions suitable
for the enzymatic or chemical reactions described above for
nonrandomly fragmenting target nucleic acids and isolating
nonrandom length fragments in preparation for mass spectrometric
analysis. Additionally, the kits may contain solid supports for
purposes of isolating nonrandom length fragments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIGS. 1A and 1B display examples of resolved nucleic acid
fragments (DNA) in the 20,000 to 30,000 Da range using MALDI-TOF
mass spectrometry. Both FIGS. 1A and 1B are positive ion mass
spectra obtained from 200 fmoles of DNA in 3-HPA
(3-hydroxypicolinic acid). Each spectrum is a sum of 100 laser
pulses at 266 nm. FIG. 1A: a single-stranded 72-mer which also
shows a 71-mer. The FWHM resolution is 240, clearly resolving
matrix adducts (labelled M). FIG. 1B: 88-mer parent peak has a
resolution of 330.
[0042] FIG. 2 is a diagram illustrating the basic steps for mass
spectrometric analysis of a nonrandomly-fragmented, double-stranded
target nucleic acid.
[0043] FIG. 3 is a diagram illustrating the expected mass spectrum
for a nonrandomly-fragmented, double-stranded target nucleic acid
that is a heterozygous mix of wild type and mutant nucleic acid
where the mutation is an A to T transversion.
[0044] FIGS. 4A and 4B illustrate the effect on mass resolution of
a mass-substituted base where a T has been replaced by
heptynyldeoxyuridine during amplification of the mutant region.
FIG. 4A depicts a mass spectra of a heterozygous mix of wild type
and mutant where A has mutated to T. Spectral peaks are separated
by 9 mass units. FIG. 4B depicts a mass spectra of a heterozygous
mix of wild type and mutant where A has mutated to T. T has been
replaced by heptynyideoxyuridine during amplification of the mutant
region. Spectral peaks are now separated by 65 mass units.
[0045] FIG. 5 is a diagram illustrating the affect of analyzing
only positive strand fragment from a heterozygous sample in
reducing the number of total fragments and simplifying the mass
spectrum.
[0046] FIG. 6 is a diagram illustrating the use of restriction site
probes to produce nonrandom fragments from single-stranded target
nucleic acid. Note that in the step of purifying nonrandom length
fragments, the small cleaved probes will likely be removed during
purification.
[0047] FIGS. 7A and B illustrate the use of fragmenting probes in
conjunction with single-strand-specific endonuclease to produce
nonrandom fragments from single-stranded target nucleic acid.
[0048] FIG. 8 is a diagram illustrating the use of fragmenting
probes in conjunction with single-strand-specific, base-specific
chemical cleavage to produce nonrandom fragments from
single-stranded target nucleic acid.
[0049] FIGS. 9A and B illustrate the use of fragmenting probes to
produce nonrandom fragments from heterozygous, single-stranded
target nucleic acid in combination with the use of a
mismatch-specific cleaving reagent to further fragment the target
nucleic acid at the site of a mutation.
[0050] FIG. 10 is a diagram illustrating a method of detecting a
mutation using mass spectrometric analysis of nonrandomly
fragmented mutant and wild-type double-stranded nucleic acids that
have been denatured and reannealed and then cleaved at any mismatch
regions.
[0051] FIG. 11 is a diagram illustrating the effect of analyzing
only positive strand fragments from a heterozygous sample in
reducing the number of total fragments and simplifying the mass
spectrum. In this case the positive strand has been nonrandomly
fragmented using both restriction endonuclease treatment and
mismatch-specific cleavage.
[0052] FIG. 12 is a diagram illustrating the use of
structures-specific endonucleases to nonrandomly fragment a folded,
single-stranded target nucleic acid.
[0053] FIGS. 13A and B illustrate the use of a full length capture
probe to isolate and purify a set of single-stranded nonrandom
length fragments. Shown in FIG. 13B as an option is a second step
involving cleavage at mutation-specific mismatch. This mismatch
cleavage is particularly useful for cases where mutant DNA is
hybridized to wild type.
[0054] FIG. 14 is a mass spectrum of a set of nonrandom length
fragments from a target nucleic acid containing a mutation, wherein
the target nucleic acid is nonrandomly fragmented with
hydroxylamine followed by piperidine, resulting in
mutation-specific cleavage at a mismatch. This mass spectrum
illustrates the presence of a nonrandom length fragment of 75 bases
in size, that results from mutation-specific cleavage.
[0055] FIG. 15 is a mass spectrum illustrating hydroxylamine
fragmentation of a wild type control of the mutation-containing
target nucleic acid of FIG. 14. This mass spectrum lacks a fragment
of 75 bases in size due to the lack of a mutation in the wild type
target nucleic acid.
[0056] FIG. 16 is a mass spectrum of a mutation-containing target
nucleic acid that is specifically cleaved with potassium
permanganate at the site of a base mismatch.
[0057] FIG. 17 is a mass spectrum of a set of 5 single-stranded
nonrandom length fragments from an MNL I digest of a wild type
target nucleic acid of 184 nucleotides in length.
[0058] FIG. 18 is a magnified mass spectrum of two fragments, both
26 bases in length, identical in nucleotide sequence except for a
single G to A point mutation, illustrating clear resolution of the
two mass peaks.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0059] The present invention, directed to methods of screening
target nucleic acids to detect mutations using mass spectrometric
techniques to analyze post-amplification nucleic acids, provides
the advantages of technical ease, speed, and high sensitivity
(minute samples are required). The methods described herein yield a
minimal set of products with improved mass resolution and accuracy
and detailed information about the nature and location of the
mutation in the target nucleic acid.
[0060] The present invention involves obtaining from a target
nucleic acid, using a variety of nonrandom fragmentation
techniques, a set of nonrandom length fragments (NLFs) and
determining the mass of the members of the set of NLFs.
[0061] The target nucleic acid can be single-stranded or
double-stranded DNA, RNA or hybrids thereof, from any source,
preferably from a human source, although any source which one is
interested in screening for mutations can be used in the methods
described herein. When the target nucleic acid is RNA, the RNA
strand is the +strand. If desired, the target nucleic acid can be
an RNA/DNA hybrid, wherein either strand can be designated the
+strand and the other, the -strand. The target nucleic acid is
generally a nucleic acid which must be screened to determine
whether it contains a mutation. The corresponding target nucleic
acid derived from a wild type source is referred to as a wild type
target nucleic acid. The target nucleic acids can be obtained from
a source sample containing nucleic acids and can be produced from
the nucleic acid by PCR amplification or other amplification
technique. The target nucleic acids are typically too large to
analyze directly because current mass spectrometric methods do not
have the mass accuracy and resolution necessary to identify a
single base change in molecules larger than 100 base pairs.
Accordingly, the target nucleic acids must be fragmented.
[0062] Nonrandom length fragments are nucleic acids derived by
nonrandom fragmentation of a target nucleic acid, and can comprise
regions or nucleotide sequences that are single-stranded or
double-stranded. Due to the simpler mass spectrum that results from
mass analysis of single-stranded nonrandom length fragments, it is
preferred to determine the masses of sets of single-stranded
nonrandom length fragments. The nonrandom length fragments can also
contain mass-modified nucleotides, which can enhance ease of
analysis, especially when a point mutation has resulted in a very
small mass change (on the order of 9 Da) in a nonrandom length
fragment as compared to the corresponding wild type nonrandom
length fragment. The methods described herein use mass spectrometry
to determine the masses of the set or sets of nonrandom length
fragments to detect mutations in a target nucleic acid.
[0063] The nonrandom fragmentation techniques of the invention are
any methods of fragmenting nucleic acids that provide a defined set
of nonrandom length fragments, where that set of nonrandom length
fragments may be reproducibly obtained by using the same nonrandom
fragmentation method on the same target nucleic acid or its wild
type version. The methods used for nonrandom fragmentation are
designed to optimize the ease of analyzing the resulting mass
spectral data by obtaining a range of fragment sizes that avoids
significant overlap of mass peaks. The nonrandom fragmentation
techniques of the invention include digestion with restriction
endonucleases, structure-specific endonucleases, and specific
chemical cleavage. The enzymatic nonrandom fragmentation techniques
include partial digestion with restriction endonucleases or
structure-specific endonucleases. Partial cleavage occurs when not
every possible cleavage site is cleaved by the cleaving reagents
used, whether enzymatic or chemical.
[0064] Fragmenting probes used in the invention are nucleic acids
comprising a single-stranded nucleotide sequence or region that is
complementary to a nucleotide sequence of a target nucleic acid.
When fragmenting probes are also used as capture probes (i.e. to
bind the fragmenting probe and any complementary nucleic acids
hybridized thereto to a solid support), the fragmenting probes
comprise a first binding moiety that is capable of binding to a
second binding moiety attached to a solid support. Upon
hybridization of a set of fragmenting probes and a target nucleic
acid, the hybrid can be nonrandomly fragmented using one or more
cleaving reagents that specifically cleave single-stranded
regions.
[0065] Restriction site probes are oligonucleotides that when
hybridized to single-stranded target nucleic acid at specific
complementary sequences form complete double-stranded restriction
endonuclease recognition sites cleavable using the restriction
endonuclease capable of cleaving at or near the recognition sites
formed.
[0066] Universal restriction probes comprise two regions, the first
region being single-stranded and complementary to a specific
sequence within the target nucleic acid, and the second region
being double-stranded and containing the restriction recognition
site for a particular class IIS restriction endonuclease.
[0067] Capture probes used in the methods described herein comprise
fragmenting probes, restriction site probes, universal restriction
probes, and any nucleic acids that are bound to a solid support to
isolate sets or subsets of nucleic acids or NLFs. Capture probes
can comprise a cleavable linkage or cleavable moiety that can be
selectively cleaved to release nucleic acids from a solid support
prior to mass spectrometric analysis.
[0068] Wild type probes are nucleic acids derived from a wild type
nucleic acid sequence comprising at least one nucleotide sequence
complementary to a nucleotide sequence of a target nucleic acid or
a member of a set of NLFs. Wild type probes can be restriction site
probes, fragmenting probes, or capture probes comprising a wild
type nucleotide sequence that when hybridized to a complementary
mutation-containing region of a target nucleic acid results in a
base mismatch bulge or loop structure. Wild type refers to a
standard or reference nucleotide sequence to which variations are
compared. As defined, any variation from wild type is considered a
mutation, including naturally occurring sequence polymorphisms.
[0069] The term complementary refers to the formation of sufficient
hydrogen bonding between two nucleic acids to stabilize a
double-stranded nucleotide sequence formed by hybridization of the
two nucleic acids.
[0070] A single-stranded region comprises a portion of a nucleotide
sequence that is capable of being selectively cleaved by
single-strand-specific cleaving reagents or structure-specific
endonucleases, wherein the portion of a nucleotide sequence can
range in size from a single phosphodiester bridge, i.e. the
phosphodiester bond across from a nick, to a nucleotide sequence
ranging from one to 450 nucleotides in length which are not
hybridized to a complementary nucleotide sequence or region.
[0071] The types of mass spectrometry used in the invention include
ESI or MALDI, wherein the MALDI method may optionally include
time-of-flight. The significant multiple charging of molecules in
ESI and the fact that complex mixture analysis is generally
required mean that the ESI mass spectra will consist of a great
many spectral peaks, possibly overlapping and causing confusion.
Because the MALDI MS approach produces mass spectra with many fewer
major peaks, this method is preferred.
[0072] The methods described herein do not require sequencing of
the target nucleic acid (using the sequencing methods that require
four different base-specific chain termination reactions to
determine the complete nucleotide sequence of a nucleic acid) in
order to determine the nature and presence of a mutation within the
target nucleic acid.
[0073] For an initial mutation screen, a useful range of fragment
sizes that will allow detection of a point mutation is around 10 to
100 bases. This size range is where mass spectrometry presently has
the necessary level of mass resolution and accuracy. Thus, the
fragmentation methods used in this invention are designed to
produce from the target nucleic acid, a set of nonrandom length
fragments ranging up to 100 bases in size. For purposes of this
invention, fragmentation methods that produce a set of random
length fragments are not desirable due to the limited
reproducibility of such fragments, the limited information
available from mass spectrometry analysis of such fragments, and
the likelihood of spectral overlap from randomly generated
fragments. For example, nonrandom fragmentation permits
determination of the mass, base composition, and location of the
set of NLFs relative to the target nucleic acid, whereas random
fragmentation methods do not.
[0074] Existing mass spectrometric instrumentation in the case of
MALDI-TOF MS optimally has a mass accuracy of about 1 part in
10,000 (0.01%), four times what is necessary for detecting a single
base change in a 50-base long single-stranded DNA fragment.
Utilization of mass-modified nucleotides (to be described later)
and nearby masses as internal calibrants, provides optimal
resolution and mass accuracy of larger nucleic acids, and can
extend the usable mutation detection range up to 100 bases, if not
higher. Continued advances in mass spectrometric instrumentation
will also push this range higher.
[0075] Examples of the resolving capabilities of MALDI-TOF MS are
displayed in FIGS. 1A and 1B. FIG. 1 shows the positive ion TOF
mass spectra obtained from 200 fmoles of DNA in the matrix 3-HPA.
FIG. 1A (top figure) shows two single-stranded PCR products of
lengths 71 and 72 (mass difference=305 Da=Adenosine) as well as the
72 mer and 72 mer+a single matrix adduct (M) (mass difference=139
Da) to be well resolved (FWHM resolution=240). FIG. 1B (bottom
figure) shows an 88 base length single-stranded product having a
resolution of 330. Both spectra display high enough accuracy and
resolution to detect a point mutation if one were present.
[0076] These unique properties of mass spectrometry, MALDI-TOF MS
in particular, to separate nucleic acid fragments and identify
their mass exactly and the methods taught herein provide novel
methods for the screening of target nucleic acids and
identification of changes in base composition that might result
from genetic mutation.
Improving Mass Accuracy By Internal Calibration and Internal
Self-Calibration
[0077] Mass spectrometers are typically calibrated using analytes
of known mass. A mass spectrometer can then analyze an analyte of
unknown mass with an associated mass accuracy and precision.
However, the calibration, and associated mass accuracy and
precision, for a given mass spectrometry system (including
MALDI-TOF MS) can be significantly improved if analytes of known
mass are contained within the sample containing the analyte(s) of
unknown mass(es). The inclusion of these known mass analytes within
the sample is referred to as use of internal calibrants. External
calibrants, i.e. analytes of known mass that are not mixed in with
the set of nonrandom length fragments of unknown mass and
simultaneously analyzed in a mass spectrometer, are analyzed
separately. External calibrants can also be used to improve mass
accuracy, but because they are not analyzed simultaneously with the
set of fragments of unknown mass, they will not increase mass
accuracy as much as internal calibrants do. Another disadvantage of
using external calibrants is that it requires an extra sample to be
analyzed by the mass spectrometer. For MALDI-TOF MS, generally only
two calibrant molecules are needed for complete calibration,
although sometimes three or more calibrants are used. All of the
embodiments of the invention described herein can be performed with
the use of internal calibrants to provide improved mass
accuracy.
[0078] Using the methods described herein, one can obtain a mass
spectrum with numerous mass peaks corresponding to the set of
nonrandom length fragments of the gene or target nucleic acid under
study. If no mutation is present in the target nucleic acid, all of
the mass peaks corresponding to the nonrandom length fragments will
be at mass-to-charge ratios associated with the set of NLFs from
the wild type target nucleic acid. However, if the target nucleic
acid contains a mutation, usually no more than one or two of the
mass peaks will be shifted in mass, leaving the majority of mass
peaks at unaltered locations. In a preferred embodiment of the
invention, a self-calibration algorithm uses these unmutated or
nonpolymorphic NLFs for internal calibration to optimize the mass
accuracy for analysis of the NLFs containing a mutation, thus
requiring no added calibrant(s), simplifying the calibration, and
avoiding potential spectral overlaps. In a given sample, however,
it will not be known a priori which mass peaks, if any, are altered
or shifted from their expected masses for the wild type NLFs.
[0079] The self-calibration algorithm begins by dividing up the
observed mass peaks into subsets, each subset consisting of all but
one or two of the observed mass peaks. Each data subset has a
different one or two mass peaks deleted from consideration. For
each subset, the algorithm divides the subset further into a first
group or two or three masses which are then used to generate a new
set of calibration constants, and a second group which will serve
as an internal consistency check on those new constants. The
internal consistency check begins by calculating the mass
difference between the m/z values calculated for the second group
of mass peaks and the values corresponding to reasonable choices
for the associated wild-type NLFs. The internal consistency check
can thus take the form of a chi-square minimization where the key
parameter is this mass difference. The algorithm finds which data
subset has the lowest sum of the squares of these mass differences
resulting in a choice of optimized calibration constants associated
with group one of this data subset.
[0080] After new self-optimized calibration constants are obtained,
the mass-to-charge ratios are determined for the mass peaks omitted
from the data subset; these are the nonrandom length fragments
suspected to contain a mutation. The differences from the observed
mass peaks for the wild type NLFs are then used to determine
whether a mutation has occurred, and if so, what the nature of this
mutation is (e.g. the exact type of deletion, insertion, or point
mutation). This self-calibration procedure should yield a mass
accuracy of approximately 1 part in 10,000.
Fragmentation of Target Nucleic Acids
[0081] Fragmentation of a target nucleic acid is important for
several reasons. First, fragmentation allows direct analysis of
large segments of a gene or other target nucleic acid using a
single PCR amplification, eliminating the need to multiplex or run
separately many smaller-segment PCR reactions.
[0082] Second, sequencing of thousands of bases of a gene or other
target nucleic acid, by mass spectrometry or otherwise, is a
complex and expensive process. With current capabilities in MALDI
and ESI, it is impractical to sequence nucleic acids greater than
50-100 bases in length. Consequently, in order to rapidly screen
large genetic regions or target nucleic acids using mass
spectrometric nucleic acid sequencing, an impractical and
cumbersome number of independent sequencing reactions are necessary
to cover the entire genetic region of interest. Therefore, for
screening large genetic regions or target nucleic acids for a wide
range of potential mutations using mass spectrometry, fragmentation
of amplified target nucleic acids ranging from 100 to 1000 base
pairs (bp) facilitates faster screening of larger target nucleic
acids or genetic regions of interest.
[0083] Sequencing can identify the exact location and nature of a
genetic mutation in a target nucleic acid, but requires the use of
many primers in many separate reactions. Mutations, especially for
heterozygous samples analyzed using fluorescence-based systems, are
often difficult to identify with confidence. Using the
fragmentation methods described herein, a heterozygous sample would
yield two distinct mass spectral peaks, correlating to the
different masses of the mutant and wild type nucleic acids.
Accordingly, the methods described herein can be used to detect a
mutation in a target nucleic acid unambiguously.
[0084] Third, mass spectrometric analysis of smaller nucleic acid
fragments, ranging in size from 2 to 300 bases, more preferably
from 10 to 100 bases in length, is desirable because the smaller
nucleic acid fragments result in:
[0085] (a) more specific localization of any mutations than for
larger sized nucleic acid fragments,
[0086] (b) superior mass accuracy and resolution of nucleic acid
fragments in this mass range, and
[0087] (c) a multiplicity of mass peaks that can be used as
internal self-calibration standards, further improving the mass
accuracy.
[0088] For analysis with MALD-TOF MS, the goal of fragmentation is
to produce a set of nonrandom length fragments ranging in length
from 2-300 bases, preferably from 10-100 bases in length. The range
of lengths serves to better separate and resolve the fragment peaks
in the resulting mass spectrum.
[0089] Fragmentation of target nucleic acids larger than 100 bases
in length can be accomplished using a number of means, including
cleavage with one or more DNA restriction endonucleases targeting
specific sequences within double-stranded DNA, chemical cleavage at
structure-specific and/or base-specific locations, polymerase
incorporation of modified nucleotides that create cleavage sites
when incorporated, and targeted structure-specific and/or
sequence-specific nuclease treatment.
[0090] An exemplary case is where a larger target nucleic acid,
e.g. 500 bases in length, is nonrandomly fragmented to produce 10
to 30 nonrandom length fragments that can all be individually
resolved by MALDI-TOF mass spectrometry. Two different nonrandom
length fragments having the same number of bases can still be
resolved from each other by mass spectrometry when they differ in
base composition and consequently in mass. Gel electrophoresis
methods typically cannot resolve equivalent length fragments.
[0091] For example, for a 5 kilobase pair (kb) target nucleic acid
to be fully analyzed, using nonrandom length fragments with an
average size of 30 bases, approximately 170 nonrandom length
fragments would need to be screened. Typically, the target nucleic
acid would be amplified by a number of DNA amplifications,
.about.10-20, in order to reduce the number of fragments to be
analyzed in any given sample. Each amplified target nucleic acid
product would be digested using restriction endonucleases, often
with four-base recognition sites to produce the optimal size
fragments. It is preferable that the fragments vary in size to
simplify the mass spectral data, e.g. 32 bp+28 bp+27 bp+37 bp+. . .
, although, as stated above, nonrandom length fragments of the same
size could potentially be analyzed if their base compositions vary
enough to minimize spectral overlap.
[0092] A schematic of the process along with a hypothetical mass
spectrum is shown in FIG. 2. FIG. 2 illustrates a 161 base target
nucleic acid that has been PCR amplified and fragmented using
restriction endonucleases. The resulting 6 nonrandom length
fragments are produced. When the laser desorption process occurs,
during MALDI-TOF mass spectrometric analysis, the 6 double-stranded
fragments are mostly denatured and the resulting 12 single-stranded
nonrandom length fragments are ionized and detected. Shown at the
bottom of FIG. 2 is a simulated mass spectral data plot with all
the mass peaks resolved.
[0093] As can be seen in FIG. 2 it is very common that restriction
endonuclease treatment will produce a number of complementary
fragments with the same number of bases, e.g. two at 19 and two at
32. The presence of these equal-length fragments places higher
constraints on the required resolution for distinguishing all of
the different peaks. It is also not uncommon for the two
equal-length, complementary fragments to have identical or nearly
identical mass values, leaving the possibility that two
complementary fragments will not be resolvable.
[0094] Often samples will be heterozygous, containing a 50% mixture
of both the normal wild type nucleic acid and the mutated target
nucleic acid. In the case where the target nucleic acid carries a
mutation in a heterozygous mix, one would observe a splitting of
peaks within the nonrandom length fragments containing the
mutation. An example of this splitting is shown in FIG. 3 where an
A-T to T-A transversion or base flip has occurred in one copy of
the gene. The expected peaks would be half normal height since
their concentrations are halved relative to homozygous
concentrations. In this case, the difference between mutant and
wild type peaks would be .about.9 Da which can be resolved in the
32 base long fragment. The presence of wild type peaks provides
internal self-calibrants allowing highly accurate mass differences
(as opposed to absolute mass) to be used to determine the base
composition change.
[0095] The methods described herein permit MALDI-TOF MS analysis of
nonrandom length fragments which has a mass accuracy of
approximately 1 part in 10,000. The use of internal self-calibrants
makes it possible to extend this level of accuracy up to and
potentially beyond 30,000 Da or 100 bases. This mass accuracy
enables exact sizing of nucleic acid fragments and the
determination of the presence and nature of any mutation, including
point mutations, insertions and deletions, even in a heterozygous
environment. Further described herein are methods for improving the
resolution of individual fragments by means including elimination
of equal-length complementary pairs through the use
single-strand-targeted fragmentation and/or isolation procedures,
and the incorporation of mass-modified nucleotides to enhance the
mass difference between similar sized fragments and/or mutant and
wild type fragments. In addition, these methods provide for removal
of salts and other deleterious materials as well as a means for the
removal of unwanted nucleic acid fragments prior to mass
spectroscopic analysis.
Mass Resolution, Mass Accuracy, and the Use of Mass-Modified
Nucleotides
[0096] Any of the embodiments of the invention described herein
optionally include nonrandom length fragments having one or more
nucleotides replaced with mass-modified nucleotides, wherein the
mass-modified nucleotides comprise nucleotides or nucleotide
analogs having modifications that change their mass relative to the
nucleotides that they replace. The mass-modified nucleotides
incorporated into the nonrandom length fragments of the invention
must bekamenable to the enzymatic and nonenzymatic processes used
for the production of nonrandom length fragments. For example, the
mass-modified nucleotides must be able to be incorporated by DNA or
RNA polymerase during amplification of the target nucleic acid.
Moreover, the mass-modified nucleotides must not inhibit the
processes used to produce nonrandom length fragments, including,
inter alia, specific cleavage by restriction endonucleases or
structure-specific endonucleases and digestion by single-stand
specific endonucleases, whenever such steps are used.
Mass-modifications can also be incorporated in the nonrandom length
fragments of the invention after the enzymatic steps have been
concluded. For example, a number of small chemicals can react to
modify specific bases, such as kethoxal or formaldehyde.
[0097] Any or all of the nucleotides in the nonrandom length
fragments can be mass-modified, if necessary, to increase the
spread between their masses. It has been shown that modifications
at the C5 position in pyrimidines or the N7 position in purines do
not prevent their incorporation into growing nucleic acid chains by
DNA or RNA polymerase. [L. Lee et al. "DNA Sequencing with
Dye-Labeled Terminators and T7 DNA Polymerase: Effect of Dyes and
dNTPs on Incorporation of Dye-Terminators and Probability Analysis
of Termination Fragments" Nuc. Acids. Res. 20, 2471 (1992)] For
example, an octynyl moiety can be used in place of methyl on
thymidine to alter the mass by 94 Da.
[0098] Mass-modifying groups can be, for example, halogen, alkyl,
ester or polyester, ether or polyether, or of the general type XR,
wherein X is a linking group and R is a mass-modifying group. The
mass-modifying group can be used to introduce defined mass
increments into the nonrandom length fragments. One of skill in the
art will recognize that there are numerous possibilities for
mass-modifications useful in modifying nucleic acid fragments or
oligonucleotides, including those described in Oligonucleotides and
Analogues: A Practical Approach, Eckstein ed. (Oxford 1991) and in
PCT/US94/00193, which are both incorporated herein by
reference.
[0099] At larger mass ranges (30,000-90,000 Da), the mass
resolution and mass accuracy of current MALDI-TOF mass
spectrometers will not be sufficient to identify a single base
change. For this reason, it may be preferable to increase the
useful mass range artificially by substituting standard nucleotides
within either a target nucleic acid or a nonrandom length fragment
with mass-modified nucleotides having significantly larger mass
differentials. Use of mass-modified nucleotides applies as well to
the mass range below 30,000 Da. Mass modification can generally
increase the quality of the mass spectra by enlarging the mass
differences between NLFs of similar size and composition. For
example, mass-modified nucleotides can increase the minimum mass
difference between two nonrandom length fragments that are
identical in base composition except for a single base which is an
A in one NLF and is a T in the other. Normally, these two NLFs will
differ in mass by only 9 Da. By incorporating a single
mass-modified nucleotide into one of the bases, the mass difference
can be >20 Da. The spectra in FIG. 4 depict the influence
mass-modified nucleotides can have on fragment resolution. One
example of the many possible mass modifications useful in this
invention is the use of 5-(2-heptynyl)-deoxyuridine in place of
thymidine. The replacement of a methyl group by heptynyl changes
the mass of this particular nucleotide by 65 Da. An A to T
transversion in a nucleic acid fragment in which all thymidine
bases have been replaced with 5-(2-heptynl)-deoxyuridine would
produce a peak shift of 56 Da as opposed to 9 Da for the same
nucleic acid fragments without the mass-modified nucleotides. The
use of mass-modified nucleotides is especially important in the
analysis of NLFs derived from RNA. Normally, the masses of C and U
vary by only 1 Da, making it practically impossible to detect C to
U or U to C point mutations within a given fragment.
Benefits of Analyzing Single-Stranded Nucleic Acids
[0100] The goal of this invention is the accurate determination of
the masses of a set of resolved nonrandom length fragments and
correlation of this data to the characterization of any mutation,
if present. The embodiments of this invention include mass
spectrometric determination of masses of the members of a set of
single-stranded nonrandom length fragments as well as mass
determination of the members of a set of mass-modified,
double-stranded nonrandom length fragments. the preferred
embodiment is to detect mutations in a target nucleic acid
comprising obtaining a set of nonrandom length fragments in
single-stranded form, wherein the single-stranded nonrandom length
fragments are derived from one of either the positive or the
negative strand of the target nucleic acid or where the set is a
subset of fragments derived from both the positive and the negative
strands of the target nucleic acid. The examples of single-stranded
methods described herein focus on fragments derived from the
positive strand.
[0101] FIGS. 2 and 3 illustrate that each double-stranded nonrandom
length fragment, comprising two complementary strands, produces two
peaks in the mass spectrum corresponding to the denatured single
strands. The additional peaks from double-stranded nonrandom length
fragments as compared to single-stranded nonrandom length fragments
add to congestion of mass peaks in the mass spectra, as well as
introducing the possibility that it may be extremely difficult, if
not impossible, to resolve the complementary fragments if they have
nearly or exactly identical base compositions. Furthermore, some
portion of the double-stranded nonrandom length fragments do not
fully denature, and mass peaks corresponding to the double-stranded
products increase the spectral congestion.
[0102] Because spectra using both strands contain a two-fold
redundancy in data, since any mutation in one strand will be
present within its complement, it is reasonable to remove one
strand prior to mass spectrometric analysis and still produce all
of the data necessary for complete mutation analysis. For these
reasons, it is the preferred embodiment to analyze a set of single
strands where only one of the two complementary sets nucleic acid
fragments representing the full target sequence is used.
[0103] FIG. 5 shows the expected spectrum if only the nonrandomly
fragmented positive strand of a target nucleic acid from FIG. 3 is
analyzed by mass spectrometry. Analysis of one of the two
complementary strands of the double-stranded nonrandom length
fragments halves the number of expected peaks within the mass
spectra, allowing more total fragments to be resolved and the
possibility that longer total sized target nucleic acids can be
analyzed at one time. Removal of one of the two strands from each
nonrandom length fragment eliminates the greatest source of
complication for each spectra. A number of methods for isolating
and preparing both single-stranded and double-stranded nonrandom
length fragments for mass spectrometry are described herein.
Method of Nonrandom Fragmentation of Target Nucleic Acid
[0104] The methods of the invention all involve obtaining from a
target nucleic acid a set of resolvable, nonrandom-length fragments
and determining the mass of the members of that set using mass
spectrometry without sequencing the target nucleic acid. All of the
methods described herein involving mass spectrometry include inter
alia two types of mass spectrometry, electrospray ionization (ESI)
and matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF). In addition to the restriction endonuclease approach
to nonrandomly fragmenting a target nucleic acid, there are a
number of other approaches which are described below.
Nonrandom Fragmentation Using Restriction Site Probes
[0105] Target nucleic acid can be nonrandomly fragmented using
hybridization to nucleic acid, restriction site probes followed by
cleavage with one or more restriction endonucleases the recognition
sequences of which are contained in the restriction site probes
used. "Restriction site probes" are oligonucleotides that when
hybridized to single-stranded target nucleic acid at specific
sequences form a complete double-stranded recognition site
cleavable using restriction endonucleases. The use of restriction
site probes is illustrated in FIG. 6.
[0106] The sequence of a wild type target nucleic acid can be
analyzed to determine which restriction sites would result in an
ideal spread of members of a set of NLFs. The restriction site
probes are then made using well-known synthetic techniques. The
restriction site probes can range from 6-100 nucleotides in length,
preferably from 10-30 nucleotides in length. One advantage of using
very short restriction site probes is that after cleavage with the
selected restriction endonucleases, the mass of the members of the
set of NLFs having cleaved restriction site probes attached can be
directly determined in the mass spectrometer without requiring an
isolating step to remove the cleaved restriction site probes. On
the other hand, if the cleaved restriction site probes are intended
to be used also as capture probes, then the restriction site probes
must either have a first binding moiety that is capable of binding
to a second binding moiety attached to a solid support or the
restriction site probes must have at least one additional
nucleotide sequence that is complementary to another probe that is
bound to a solid support. A "capture probe" is an oligonucleotide
that comprises a portion capable of hybridizing to a nucleic acid,
such as a target nucleic acid or a nonrandom length fragment, and a
binding moiety that binds the capture probe to a solid phase,
either through covalent binding or affinity binding, or a mixture
thereof. A capture probe can itself bind to a solid support via
binding moieties (direct capture) or can bind to a solid support
via another capture probe that binds to a solid support (indirect
capture). Also, when the restriction site probe is also used as a
capture probe, the preferred range is from 30-50 nucleotides in
length, to stabilize the hybridization of the capture probe. By
using larger restriction site probes complementary to singular
locations on the target nucleic acid it is possible to prevent a
restriction enzyme from cutting at all possible locations in a
target nucleic acid where restriction sites for a particular
restriction endonuclease appear, e.g. cutting at only 5 or 10
restriction sites within a single-stranded target. This is another
tool that can be used to produce the optimal nonrandom length
fragment set of subset.
[0107] An alternative form of restriction site probe is the
universal restriction probe as described by Szybalski. [W.
Szybalski "Universal Restriction Endonucleases: Designing Novel
Cleavage Specificities by Combining Adapter Oligodeoxynucleotide
and Enzyme Moieties," Gene 40, 169 (1985) (incorporated by
reference herein)] These universal restriction probes comprise two
regions, the first region being single-stranded and complementary
to a specific sequence within the target nucleic acid, and the
second region being double-stranded and containing the restriction
recognition site for a particular class IIS restriction
endonuclease. Class IIS restriction endonucleases cleave
double-stranded DNA at a specific distance from their recognition
sequence. By using this property, and the universal restriction
site probe design, it is possible to nonrandomly fragment a
single-stranded DNA target at virtually any sequence, providing the
means to better control the selection of fragment sizes. It is also
possible to mix standard restriction site probes and universal
restriction probes in a single reaction.
[0108] In this approach, a positive single-stranded target nucleic
acid is hybridized to one or more restriction site probes that are
complementary to one or more restriction endonuclease recognition
sequences within the target nucleic acid. Upon hybridization of the
restriction site probes to the target nucleic acid, hybridized
target nucleic acids are formed, comprising double-stranded regions
where the restriction site probes have hybridized to the target
nucleic acid and at least one single-stranded region where the
target nucleic acid remains unhybridized to a restriction site
probe. The double-stranded regions of the hybridized target nucleic
acids are recognition sites for cleavage by one, two or more
restriction endonucleases, the recognition sequences of which are
contained within the double-stranded regions.
[0109] The resulting nonrandom length fragments have at least one
cleaved restriction site oligonucleotide probe annealed. In some
cases, these cleaved probes will be of a size too small to remain
hybridized to the target fragments. These nonrandom length
fragments can either be purified with the cleaved restriction site
oligonucleotide probes attached, or the NLFs can be purified from
the cleaved oligonucleotide restriction site probes. Both types of
purification can be accomplished using a variety of techniques
known in the art, including filtration, precipitation, or dialysis.
The preferred approach is to capture the NLFs to a solid support.
The set of nonrandom length fragments can be directly captured to a
solid support themselves using a number of means including a
binding moiety such as biotin incorporated at numerous base
positions throughout the NLFs. Or the NLFs can be indirectly
captured to a solid support via hybridization to one or more
capture probes that is itself bound to a solid support. The capture
probe can comprise the full-length strand of the target nucleic
acid that is complementary to the strand from which the nonrandom
length fragments were derived. Alternatively, the capture probes
can be a set of capture probes each containing at least one
sequence complementary to the nonrandom length fragments.
[0110] By combining an asymmetric amplification method to produce
single-stranded target nucleic acids with the use of restriction
site probes, as described herein, one can produce predominantly the
desired set of single-stranded NLFs. The restriction site probes
used to produce the recognition sites may copurify with the NLFs
but can be designed so that they do not interfere with the majority
of the mass spectra. For example, the restriction site probes can
be designed so that after cleavage their final sizes are less than
20 bases in length and the nonrandom length fragments can have
sizes in the range of 20 to 100 bases.
[0111] The methods described above can also be modified with the
use of uncleavable restriction probes. These uncleavable probes,
synthesized with a restriction endonuclease resistant backbone such
as phosphorothioate, boranophosphate, or methyl phosphonate, can be
used to keep the target nucleic acid NLFs tethered together
following restriction digest and can provide a different approach
to purification of the NLFs.
Fragmentation Using Fragmenting Probes and Single-Strand-Specific
Cleavage
[0112] While the use of restriction endonucleases in various
combinations and in multiple digests can be an effective approach
to fragmentation of the target nucleic acid, when a target presents
long sequence lengths (>100 bases) that do not contain any
restriction sites, alternative nonrandom fragmentation techniques
are preferred. Long>100 base fragments will be difficult to
probe with sufficient mass accuracy to determine if a base change
mutation has occurred. One way to control the size of fragments is
through the use of fragmenting probes and single-stranded-specific
endonucleases.
[0113] Fragmenting probes are defined as nonrandom length,
single-stranded oligonucleotides complementary to selected regions
of a single-stranded target nucleic acid, and are used through
hybridization to define and differentiate within the target nucleic
acid regions that are double-stranded versus regions that remains
single-stranded. Following differentiation by hybridization the
single-stranded regions are subjected to cleavage. As is the case
for all of the methods described here that utilize
oligonucleotides, the fragmenting probes may be comprises on DNA,
RNA or modified forms of nucleic acid such as phosphorothioates,
methyl prosphonates or peptide nucleic acids. Three examples of
single-strand-specific nucleases that can be used in these methods
are Mung bean nuclease, Nuclease S1, and RNase A. These enzymes cut
single-strand-specific DNA or RNA exclusively and act as both exo-
and endonucleases.
[0114] An example of how these probes and enzymes are used follows.
A set of fragmenting probes of defined size and sequence are
designed to hybridize to complementary regions of the target
nucleic acid. It is preferable that the target nucleic acid be
primarily if not entirely single-stranded. Use of a T7 or SP6 RNA
polymerase transcription system for final amplification is a simple
approach to producing the required single-stranded target nucleic
acid. Asymmetric PCR can also be utilized to produce primarily
single-stranded target.
[0115] FIG. 7 shows how different portions of the single-stranded
target nucleic acid are hybridized to the oligonucleotide probes.
Following hybridization, any regions of the target nucleic acid
that remain single-stranded are cleaved using a
single-strand-specific endo/exonuclease, such as S1 Nuclease, Mung
bean nuclease, or RNase A. The size of the single-stranded region
can be as small as a single phosphodiester bridge, i.e. the
phosphodiester bond across from a nick. S1 nuclease is capable of
cleaving across from nicks. The end products are double-stranded
hybrids comprised of two equal length stands; one strand is a
member of the set of nonrandom length fragments derived from the
target nucleic acid and the other stand is a member of the set of
fragmenting probes, wherein the NLFs are hybridized to the
fragmenting probes. Either these double-stranded hybrids or
isolated single-stranded nonrandom length fragments derived from
the target nucleic acid can be used for MALDI-TOF mass
spectrometric analysis. Preferably, the analysis of the
single-stranded nonrandom length fragments derived from the target
nucleic acid provides a simpler mass spectrum. It should be noted
that when the complementary strands are a mixed DNA/RNA hybrid
there will be a significant mass difference between the two strands
in all cases, making each strand more easily resolvable in the mass
spectrum.
[0116] Unlike the restriction endonuclease nonrandom fragmentation
approach, with this method it is possible to use a DNA/RNA hybrid
providing a convenient route toward digesting the fragmenting
probes after nonrandomly fragmenting the target nucleic acid.
Isolation of the set of NLFs from the set of fragmenting probes is
another means to simplify the mass spectra. Because of the
different chemical nature of the two strands of the hybrid, it is
possible to utilize DNA- or RNA-specific enzymes to digest the
fragmenting probes. As an example, DNase can be used to digest
fragmenting probes comprised of DNA while leaving nonrandom length
RNA fragments intact or RNase can be used to digest RNA probes
while leaving nonrandom length DNA fragments intact. It is also
possible to utilize different chemistries to specifically digest
one strand or the other. These chemistries include the use of acid
to digest DNA or base to digest RNA as well as a multiplicity of
other chemistries that can be used to cut modified versions of DNA
or RNA. This differential cutting can be exploited to purify and
analyze only one of the two strands as described in a later
section.
[0117] Thus, another embodiment of this invention is a method of
detecting a mutation in a DNA fragment from a DNA/RNA hybrid
nucleic acid comprising obtaining a DNA/RNA hybrid wherein the
DNA/RNA hybrid comprises a single-strand of a DNA fragment
hybridized to a single-strand of a RNA fragment, digesting the
single-strand of RNA using a RNA-specific reagent, including RNase
or a base, determining the mass of the single-stranded DNA fragment
using mass spectrometry, and comparing the mass to a mass of a wild
type single-stranded DNA fragment. Another embodiment is a method
of detecting a mutation in a RNA fragment from a DNA/RNA hybrid
nucleic acid comprising obtaining a DNA/RNA hybrid wherein the
DNA/RNA hybrid comprises a single-strand of a DNA fragment
hybridized to a single-strand of a RNA fragment, digesting the
single-strand of DNA using a DAN-specific reagent, including DNase
or an acid, determining the mass of the single-stranded RNA
fragment using mass spectrometry, and comparing the mass to a mass
of a wild type single-stranded RNA fragment. These embodiments can
also be applied to a set of DNA/RNA hybrids, and using the
DNA-specific or RNA-specific digestion to leave a set of nonrandom
length fragments consisting of DNA fragments or a set of nonrandom
length fragments consisting of RNA fragments.
[0118] Complete digestion using restriction endonucleases produces
a series of fragments that can be aligned end to end but do not
overlap. With the use of fragmenting probes and
single-strand-specific cleaving reagents described herein, one can
design a set of sequence and size specific fragmenting probes that
can be used to produce a set of nonrandom length fragments such
that one or more members of the set comprise a nonoverlapping
nucleotide sequence and a nucleotide sequence that overlaps with a
nucleotide sequence of another member of the set. The example shown
in FIG. 7 uses a set of sequence and size specific fragmenting
probes that overlap (e.g. split into two sets of hybridization
reactions) to produce an overlapping set of nonrandom length
fragments. The set of nonrandom length fragments that overlap could
be nested. By using a set of overlapping nonrandom length fragments
to screen for a mutation, one can more narrowly localize the region
containing a mutation. If two overlapping nonrandom length
fragments both contain the mutation, as is the case in FIG. 7, it
is then known that the mutation exists within the small region of
overlap. Conversely, if only one of the overlapping fragments
contains a mutation, it is known that the mutation cannot be in an
overlapping region. This approach plus the ability to design
certain fragmenting probes to be very small in size, e.g. 10 to 20
bases (typical fragmenting probes will be anywhere between 10 and
100 bases in length), allows one to probe genetic regions that are
known hot spots for mutation with greater detail.
[0119] One variant of this method is to use single-strand-specific
chemical reagents as a means for cleaving a target nucleic acid
target into a set of nonrandom length fragments. Several
base-specific cleavage chemistries have been identified that cleave
the nucleic acid backbone at base-specific sites that are
single-stranded and, under optimal conditions, demonstrate zero or
extremely reduced cleavage levels at base-specific sites that are
double-stranded. As an option the target nucleic acid can be
synthesized using one or more modified nucleotides in order to make
the backbone more vulnerable to chemical cleavage. By using
fragmenting probes to hybridize to a target nucleic acid at all
sites except the specific locations where cleavage is desired, it
is possible to limit cleavage to these single-stranded sites and
create a sequence-specific set of nonrandom length fragments. The
method, schematized in FIG. 8, can utilize one of a number of
different chemistries that are known to be single-strand specific
including hydrogen peroxide cleavage and/or
2-hydroperoxytetrahydrofuran cleavage at C. [P. Richterich et al.
"Cytosine specific DNA sequencing with hydrogen peroxide" Nuc.
Acids Res. 23, 4922 (1995); G. Liang, P. Gannet & B. Gold "The
Use of 2-Hydroperoxytetrahydrofuran as a Reagent to Sequence
Cytosine and to Probe Non-Watson-Crick DNA Structures" Nuc. Acids
Res 23, 713 (1995)]. Target nucleic acids that contain
cleavage-modified nucleotides can be made by incorporation of
modified nucleotide triphosphates during an amplification or
polymerization step.
[0120] A second variant of this method is to create heterozygous
hybrids between the wild type fragmenting probes and the target
nucleic acid. By using fragmenting probes comprised of wild type
sequence, any hybrids that form with mutant sequence containing a
point mutation will create a base mismatch or bulge. If the
mutation is a small insertion or deletion, a looped out sequence
will occur. With this heterozygous hybrid, it is possible to use
one of the structure-specific enzymes or chemistries described in
the following section to create a mutation-specific cleavage at the
site of a mutation. An example of the pattern of nonrandom length
fragments produced is shown in FIG. 9. This approach permits
determination of the type and location of the mutation that has
occurred. Also as will be described, performance of a
mutation-specific cleavage relaxes the mass accuracy and resolution
constraints, thus increasing the useful size range for the
nonrandom length fragments to be analyzed with MALDI-TOF mass
spectrometry to a range of several hundred bases.
Mutation-Specific Cleavage Using Structure-Specific
Endonucleases
[0121] Another nonrandom fragmentation technique involves the use
of mutation-specific cleavage at base mismatch regions, if present,
using structure-specific endonucleases or single-strand-specific
cleavage. Creation of mismatch regions requires hybridization
between a mutation containing, single-stranded target nucleic acid
and a set of one or more single-stranded complementary wild type
probes derived from wild type sequence. Wild type probes can be
restriction site probes, fragmenting probes, or capture probes
comprising wild type nucleotide sequence that when hybridized to a
complementary mutation-containing region of a target nucleic acid
results in a base mismatch bulge or loop structure. A base mismatch
will be created at the location of the mutation. In one embodiment,
the mutation containing positive strand is hybridized to a
complementary wild-type probe that comprises the entire negative
strand. In the preferred embodiment, the complex of mutation
containing positive strand hybridized to one or more complementary,
wild type nucleic acid probes is fragmented using either
restriction endonucleases, or fragmenting probes coupled with a
single-strand-specific cleavage reagent. Any base mismatch regions
between the set of wild type probes and the set of NLFs can be
specifically cleaved using one or more mismatch-specific cleaving
reagents. Examples of these reagents include: structure-specific
endonucleases such as T4 endonuclease VII, RuvC, MutY, or the
endonucleolytic activity from the 5'-3' exonuclease subunit of
thermostable DNA polymerases, single-strand-specific enzymes such
as Mung bean nuclease, S1 nuclease or RNase A, and
single-strand-specific chemistries such as hydroxylamine, osmium
tetroxide, potassium permanganate, or peroxide modification of
unpaired bases followed by a backbone cleaving oxidation step.
[0122] This mismatch-specific cleavage is used to cleave the
mutation-containing nonrandom length fragment at the site of the
mutation, thus producing two smaller fragments from the larger
mutation-containing fragment. This approach is an efficient and
simple way to identify the exact location of a mutation as well as
its type. This mismatch-specific cleavage used in combination with
one of the nonrandom fragmentation methods described herein can be
used to fragment a large (>200 bases), single-stranded target
nucleic acid into a set of smaller, mass resolvable nonrandom
length fragments.
[0123] Like EMC and CCM, the mismatch-specific cleavage approach
utilizes a mismatch targeting reagent to cut at the point of
mutation. The approach described herein improves upon the gel
electrophoresis-based methods by focusing on relatively small
fragments that take maximum advantage of the mass spectrometer's
ability to detect the exact size of a fragment leading to the
identification of the exact location and nature of a mutation. The
EMC and CCM methods must be followed by DNA sequencing in order to
fully characterize a mutation. Using the methods described herein,
a mutation in a target nucleic acid can be detected and its
location and nature determined without any sequencing.
[0124] An example of how a structure-specific enzyme like T4
endonuclease VII can be used for mismatch-specific cleavage is
shown in FIG. 10. The first step involves two amplification
reactions. First, a target nucleic acid suspected of containing a
mutation is amplified. Second, the corresponding wild type target
nucleic acid is amplified to create wild type proves. These two
amplification reactions can be performed together in one tube if
the target nucleic acid is a heterozygous mixture of mutant and
wild type. For certain diagnostic procedures, it may be more
efficient to produce the wild type probes separately prior to the
screening process. The next steps involve fragmentation of the
target nucleic acid, e.g. a multiple digest of the target nucleic
acid using more than one restriction endonuclease, and a step in
which the fragments are mixed, denatured, and then annealed. The
fragmentation and denaturing/annealing steps can occur in either
order. The purpose of the denaturing/annealing step is to produce a
mixture of hybrid target nucleic acids. In a 50:50 mixture of
mutant target and wild type nucleic accidents, four different
products result: 25% homozygous mutant double-stranded nonrandom
length fragments, 25% homozygous wild type double-stranded
nonrandom length fragments, and 25% each of the two forms of
heterozygous mutant/wild type hybrid nonrandom length fragments.
See FIG. 10 (illustrating the use of wild type NLFs as wild type
probes to generate a base mismatch with mutant NLFs). The
heterozygous nonrandom length fragments contain at least one base
mismatch at the site of mutation, i.e. the point(s) of sequence
variation between mutant and wild type. The next step involves
treatment of the nonrandom length fragments with a
mismatch-specific reagent that cleaves at the site of the base
mismatch in the heterozygous mutant/wild type nonrandom length
fragments. These new cleavages (the number of cleavage events will
depend on the particular enzyme used) typically reduce the
nonrandom length fragment containing the mutation into two smaller
nonrandom length fragments. The 50% of the mixture that contains
the homozygous double-stranded nucleic acid fragments with no
mismatches will not be cleaved during the mutation-specific
cleavage.
[0125] Example schematic mass spectral plots are shown in FIG. 10B.
An expected spectrum would show a reduction in the peak size of the
nonrandom length fragment containing the base mismatch that is
cleaved by the structure-specific endonuclease (e.g. peaks
32+(Mut), 32 +(Wt), 32-(Wt) and 32-(Mut) and the introduction of
several smaller peaks at lower masses than the mutant peaks
representing the set of heterozygous mutant/wild type NLFs that
contain base mismatches (see peaks 8+(Mut), 8+(Wt), 11-, 21-(Wt),
21-(Mut), and 24+). These peaks corresponding to the heterozygous
NLFs containing base mismatches are reduced in intensity but
continue to be present since only 50% of the molecules exist in the
heterozygous form that can undergo'the mutation-specific
cleavage.
[0126] It is possible to bias the population of the different
heterozygous/homozygous forms by performing the amplifications of
the target nucleic acid asymmetrically. Thus, one can maximize the
types of nonrandom length fragments yielding mutational data with
the majority of the duplex formed during the annealing process
being heterozygous positive (+) strand mutant and negative (-)
strand wild type.
[0127] While it is possible to observe similar patterns using gel
electrophoresis techniques, the mass accuracy obtained by mass
spectrometry provides the advantage of accurate determination of
the nature of the mutation and the ability to determine the size
and order of the two nonrandom length fragments created by the
mutation-specific cleavage. In the example in FIG. 10B, the
resulting mismatch-specific cleavage fragments are represented by
sizes 8, 11, 21, and 24 nucleotides in length. Using
electrophoretic techniques, it would be impossible to differentiate
the two mutant forms at 8 and 21 (fragments 24+ and 12- do not
possess the mutant base and are identical in heterozygous forms C
and D), nor would it be possible to directly determine which
fragment is upstream (toward the 5' end) and which fragment is
downstream (toward the 3' end), e.g. in the positive strand it is
8+ that is upstream from 24+. By providing exact mass values, mass
spectrometry allows these strands to be ordered based on mass value
database comparison with the fragments expected from the known
sequence of the wild type target nucleic acid. By completely
identifying the location and nature of the mutation this mass
spectrometric method eliminates any need for sequencing the target
nucleic acid.
[0128] FIG. 10B shows how the mismatch-specific cleavage event adds
complexity to the mass spectra. In the example shown, there are
several locations where 2, 3, and even 4 different NLFs have the
potential to overlap in the mass spectrum, making the full spectrum
difficult to resolve. As discussed previously, and shown in FIG. 5,
the mass spectra can be greatly simplified by performing the mass
spectrometric analysis on only the +or the -strands of the
nonrandom length fragments. For example, FIG. 11 shows the set of
nonrandom length fragments that are derived by analyzing only the
+positive strand of the mutant target nucleic acid. By eliminating
the homozygous nonrandom length fragments that are not
mutation-specifically cleaved and removing the negative strand from
the mass spectrometric analysis, the total number of nonrandom
length fragments to be analyzed can be reduced from 20 to 7, with
no two mass peaks having the same number of nucleotides. Of course,
in other situations, two peaks may be from nonrandom length
fragments of the same length depending on the type of mutation
present, but such situations will be infrequent.
[0129] This mismatch-specific cleavage, like the incorporation of
mass-modified nucleotides, extends the usable mass range of the
initial target nucleic acid for mass spectrometric analysis since
the primary mass accuracy needs are in determining the reduced mass
of the nonrandom length fragments created by the mutation-specific
cleavage and not in determining the mass of the other nonrandom
length fragments that are unaffected by the mutation-specific
cleavage.
[0130] It is not always necessary to fragment the target nucleic
acid in tandem with mismatch-specific cleavage if the size of the
nonrandom length fragments created by the mismatch-specific
cleavage is small enough to fall into the usable mass range with
the necessary mass resolution and accuracy. Target nucleic acids as
large as 200 base pairs will yield at least one nonrandom length
fragment created by the mutation-specific cleavage wherein the
nonrandom length fragments can be a size less than 100 base pairs,
e.g. a 200 bp target nucleic acid with a mutation at position 135
will produce nonrandom length fragments of 65 and 135 after
cleavage at the site of base mismatch.
Fragmentation Using Structure-Specific Endonucleases to Cleave a
Folded Target Nucleic Acid
[0131] Another nonrandom fragmentation method of the invention
involves providing a target nucleic acid that is either a positive
or a negative single-strand; providing conditions permitting
folding of the single-stranded target nucleic acid to form a
three-dimensional structure having intramolecular secondary and
tertiary interactions, and nonrandomly fragmenting the folded
target nucleic acid with at least one structure-specific
endonuclease to form a set of single-stranded nonrandom length
fragments. A diagram of this procedure is provided in FIG. 12. An
example of conditions that permit folding of the single-stranded
target nucleic acid are heating to denaturation followed by slow
cooling to permit annealing to form a thermodynamically favored
secondary and tertiary structure. The structure-specific
endonucleases include: T4 endonuclease VII, RuvC, MutY, and the
endonucleolytic activity from the 5'-3' exonuclease subunit of
thermostable DNA polymerases.
[0132] An alternative to the use of structure-specific
endonucleases is the use of some of the same single-strand-specific
chemical cleavage procedures describe earlier in the text. Because
of the higher frequency with which these reagents might cleave
relative to the structure-specific endonucleases, it is necessary
that the secondary and tertiary structures formed by the
single-stranded target be more compact, limiting the access of the
chemical reagents to the various reactive nucleotides. Approaches
to forming these more compact structures include performance of the
reactions at lower temperature, under higher salt conditions, or
the use of RNA versus DNA since RNA is known to form more complete
secondary and tertiary structures. Using this method, the cleavage
reaction can be run to completion to produce a standard set of
nonrandom length fragments or run only partially with the potential
of producing a nested set of products that can be analyzed by mass
spectrometry or by electrophoresis methods.
Purification Methods
[0133] When analyzing nucleic acids, including nonrandom length
fragments, by mass spectrometry, there are several requirements
that need to be met.
[0134] First, as has been described earlier, is the need to produce
fragments within the resolvable range and high mass accuracy range
of the mass spectrometer.
[0135] Second, is to eliminate from the sample, nucleic acid
fragments that do not contribute to the analysis and may
unnecessarily convolute the mass spectra. With analysis methods
such as gel electrophoresis, a mixture of specifically labeled
nucleic acid fragments (radioactive or by fluorescent tagged) can
be visualized in the presence of other unlabeled nucleic acid
fragments that comigrate but are invisible and therefore do not
convolute analysis of the gel data. The mass spectrometric methods
described herein do not use any form of labeling that could render
certain fragments invisible, e.g. the negative strand in a
double-stranded product, and it is therefore necessary to remove
such fragments prior to analysis.
[0136] Third, is the need to produce samples of relatively high
purity prior to introduction to the mass spectrometer. The presence
of impurities, especially salts, greatly affects the resolution,
accuracy and intensity of the mass spectrometric signal.
Contaminating primers, residual sample genomic DNA, and proteins,
all can affect the quality of the mass spectra.
[0137] In addition to the three requirements listed above it is
also desirable for the methods to be amenable to automation, fast
and inexpensive, providing an effective approach for detecting
genetic mutations,.
[0138] Existing purification methods are all designed to work with
labeled molecules that were typically analyzed by gel
electrophoresis. As well as utilizing labels, electrophoresis is,
to a certain degree, tolerant of impurities including salts and
proteins. For mass spectrometric analysis, prior art purification
methods such as precipitation combined with vigorous alcohol
washes, filtering and dialysis, and ion exchange chromatography are
unsatisfactory because they cannot eliminate unwanted nucleic acid
fragments and normally do not remove all salts from a sample. Solid
phase approaches such as glass bead capture under high salt
conditions, biotin/streptavidin binding, direct solid-phase
covalent linkage, and capture via hybridization to solid phase
bound oligonucleotide probes can be used to eliminate unwanted
nucleic acid fragments but typically require high levels of salt
during many of the wash steps, rendering the products less pure and
compromised for mass spectrometric analysis.
[0139] The purifications methods of the present invention are
better suited to mass spectrometric analysis of nucleic acids than
the prior art methods. First, the methods herein physically isolate
selected sets of nucleic acids from a multiplicity of impurities
including undesirable nucleic acid fragments, proteins, salts, that
would result in a poor quality mass spectrum. Second, the methods
optionally use a solution comprising volatile salts such as
ammonium bicarbonate, dimethyl ammonium bicarbonate or trimethyl
ammonium bicarbonate in any of the steps, including hybridization,
endonuclease digestion or washing. These two differences are
significant advantages over the prior art because: (1) physical
separation of the desired set of nucleic acid fragments for mass
spectrometric analysis is better than the labelling methods of the
prior art that do not physically separate the target nucleic acids
from a variety of other impurities that interfere with an accurate
mass spectrum; and (2) the use of volatile salts in any of the
steps precludes the need for any wash step known in the prior art
to merely remove salts or inorganic ions.
Double Strand Fragment Capture Approaches
[0140] There are a number of basic ways to purify DNA restriction
products from salts and other small molecules including
precipitation, filtering, dialysis, and ion exchange
chromatography. While all of these methods are effective, they are
not all equally useful for removing amplification primers residual
DNA, i.e., genomic DNA, or any proteins used. In addition, none of
the basic approaches meets all of the requirements of automation,
speed and cost. The approach that comes closest is the use of small
ion exchange spin columns, which are somewhat expensive and not
simple to integrate into an automated setup. These small ion
exchange spin columns can, however, produce high quality nucleic
acids for mass spectrometric analysis. A better alternative is the
use of (magnetic) glass beads to capture/precipitate nucleic acids
of a specific size range and allow them to be rigorously washed.
However, this method, like all of the other prior art methods
described above, does not allow for the removal of unincorporated
DNA primer since they are of the same size as the nonrandom length
fragments to be analyzed and cannot be simply differentiated.
[0141] Another general approach to purification of double-stranded
fragments is to directly capture the target nucleic acid and/or a
set of nonrandom length fragments by one of three means: (A)
hybridization to capture probes comprising a first binding moiety
that specifically binds to a second binding moiety attached to a
solid phase; (B) binding the target nucleic acid or the members of
the set of NLFs each comprising a nucleotide sequence and a first
binding moiety to a second binding moiety attached to a solid
phase; or (C) direct covalent attachment of the target nucleic acid
or the members of the set of NLFs to the solid support. Each of
these methods has advantages and disadvantages.
[0142] (A) Hybridization to solid support bound capture probes is
straightforward, specific, and can be made thermodynamically and
kinetically favored by optimizing the size and concentration of the
capture probes. Optimization is necessary since the set of NLFs
would generally prefer to hybridize to their complements rather
than to the capture probes. (This approach also works well for
single-strand isolation as described in the following section.) A
variation is to bind the probes to the solid phase after
hybridization to target. Both biotin/streptavidin and covalent
approaches for linking the probes to the solid phase are feasible.
The principal concern with this approach is that maintenance of the
hybridization, especially during wash steps, requires relatively
high level of salts and makes it more difficult to produce a
salt-free product for mass spectrometric analysis. Solutions to
this problem include the use of relatively long capture probes to
increase melting temperatures or the use of volatile salts that can
be removed prior to mass spectrometric analysis. The use of
volatile salts is described in more detail elsewhere.
[0143] (B) Biotin coupling to streptavidin (or avidin) requires
that any target nucleic acid or nonrandom length fragment to be
captured contain a biotin. It is straightforward to capture the
target nucleic acid because biotinylated primers can be used in the
PCR amplification. In order to capture all of the fragments after a
restriction digest, it is necessary to incorporate biotin into all
of the fragments. Three possible routes for biotin labeling are,
(1) the inclusion of a biotinylated nucleoside triphosphate during
fragment synthesis, (2) the use of a DNA polymerase to fill in at
5' restriction overhangs using a biotinylated nucleotide
triphosphate, and (3) the use of ligase to ligate a biotinylated
oligonucleotide at the restricted ends of the nonrandom length
fragments, where the oligonucleotides are either complementary to
the restriction sequence overhangs or are capable of blunt end
ligation.
[0144] Each of the three approaches have their problems but are
feasible. Biotins incorporated in method (1) may inhibit the
restriction endonucleases to be used and prevent the use of
structure-specific nucleases in a second mutation-specific step
since the biotin may be recognized as DNA modifications to be
excised. Method (2) is more feasible but requires a preliminary
cleanup step to exchange the normal triphosphates for biotinylated
ones. Restriction sites are limited to enzymes that produce 5'
overhangs. Method (3) is more generalizable than (2); its principal
weakness is competition with larger fragments that will want to
relegate. However, this competition can be overcome by using an
excess of the biotinylated linkers.
[0145] (C) The approach of direct covalent attachment of NLFs or
target to a solid support faces many of the same challenges as the
biotin/streptavidin approach but also includes the need to design
specific, "hot" (i.e. fast an efficient) binding chemistry working
with low concentrations of material.
[0146] The target or members of a set of NLFs can be covalently
attached to a solid support using any of the number of methods
commonly employed in the art to immobilize an oligonucleotide or
polynucleotide on a solid support. The target or NLFs covalently
attached to the solid support should be stable and accessible for
base hybridization.
[0147] Covalent attachment of the target of NLFs to the solid
support may occur by reaction between a reactive site or a binding
moiety on the solid support and a reactive site or another binding
moiety attached to the target or NLFs or via intervening linkers or
spacer molecules, where the two binding moieties can react to form
a covalent bond. Coupling of a target or NLF to a solid support may
be carried out through a variety of covalent attachment functional
groups. Any suitable functional group may be used to attach the
target or NLF to the solid support, including disulfide, carbamate,
hydrazone, ester, N-functionalized thiourea, functionalized
maleimide, streptavidin or avidin/biotin, mercuric-sulfide,
gold-sulfide, amide, thiolester, azo, ether and amino.
[0148] The solid support may be made from the following materials:
cellulose, nitrocellulose, nylon membranes, controlled-pore glass
beads, acrylamide gels, polystyrene, activated dextran, agarose,
polyethylene, functionalized plastics, glass, silicon, aluminum,
steel, iron, copper, nickel and gold. Some solid support materials
may require functionalization prior to attachment of an
oligonucleotide or capture probe. Solid supports that may require
such surface modification include wafers of aluminum, steel, iron,
copper, nickel, gold, and silicon. Solid support materials for use
in coupling to a capture probe include functionalized supports such
as the 1,1'-carbonyidiimidazole activated supports available from
Pierce (Rockford, Ill.) or functionalized supports such as those
commercially available from Chiron Corp. (Emeryville, Calif.).
Binding of a target or NLF to a solid support can be carried out by
reacting a free amino group of an amino-modified target or NLF 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 the target or NLFs and the support.
[0149] The target or NLFs may also be bound to a solid support
comprising a gold surface. The target or NLFs can be modified at
their 5'-end with a linker arm terminating in a thiol group, and
the modified target or NLFs can be chemisorbed with high affinity
onto gold surfaces (Hegner, et al., Surface Sci. 291:39-46
(1993b)).
[0150] In all of the methods in which a solid-phase approach is
used, the double-stranded nonrandom length fragments can be
rigorously washed to remove deleterious contaminants. Following
washing it is necessary to release these fragments from the solid
support for mass spectrometric analysis. The isolation of a set of
NLFs may be performed on the same plate that is used within the
mass spectrometer. Both the capture probe hybridization and
biotin/streptavidin approaches can use heat and/or pH denaturation
to disrupt the noncovalent interactions and afford release of the
set of NLFs bound to the solid support. Alternatively, a cleavable
linkage can be incorporated between the first binding moiety and
the NLFs. Any covalent coupling chemistry will need to be either
reversible or it will be necessary to include a separate chemically
cleavable linkage somewhere within the bound product. It may also
be useful to use a chemically cleavable linkage approach with the
biotin/streptavidin strategies so that release of the
double-stranded fragments can be performed under relatively mild
conditions. In all cases the cleavable linkage can be located
within the linker molecule connecting the biotin and the base (e.g.
a disulfide bond in the linker), within the base itself (e.g. a
more labile glycosidic linkage), or within the phosphate backbone
linkage (e.g. replacement of phosphate with a phosphoramidate).
[0151] One alternative to these solid-phase approaches described
above is to capture the target nucleic acids prior to nonrandom
fragmentation with one or more restriction endonucleases. Rigorous
washes to remove polymerase, salts, primers and triphosphates
required for amplification are followed by treatment with minimal
amounts of restriction enzyme under very low salt conditions. This
mixture is then directly analyzed in the mass spectrometer. Mass
spectrometry can tolerate salts if their concentrations are low
enough and a limited class of restriction enzymes can work under
very low salt conditions.
[0152] The low salt approach does limit the restriction sites that
can be cleaved as part of the methods of detecting mutations. Many
restriction endonucleases require a significant level of salt. An
attractive alternative to limiting the restriction endonuclease
cleavage reactions to low levels of salt is to replace the salts
normally used with volatile salts. These salts, such as ammonium
bicarbonate, dimethylammonium bicarbonate or trimethylammonium
bicarbonate, can be removed prior to mass spectrometric analysis
through simple evaporation. Evaporation can be accelerated by
placement of the sample in a vacuum, such as the mass spectrometer
sample chamber, or by heating the sample.
Approaches to Capturing Single-Stranded Fragments
[0153] As described earlier, analysis of single-stranded nonrandom
length fragments is generally preferable since it provides a
complete set of data with the minimal number of fragments and
therefore simplifies the spectra and facilitates an increase in the
total length of nucleic acid that can be analyzed in a single
assay. A number of approaches, as described above, can be taken
toward the production of single-stranded fragments and their
purification which includes the elimination of undesired
fragments.
[0154] If DNA restriction endonucleases are used to produce the
nonrandom length fragments, it is necessary that the target nucleic
acid have a double-stranded form prior to restriction, or more
specifically, that the restriction endonuclease recognition sites
be located in double-stranded DNA. The alternative to having fully
double-stranded DNA prior to restriction is to hybridize
restriction site probes to single-stranded DNA, wherein the
restriction site probes are complementary to the restriction sites
for selected restriction endonucleases.
[0155] The basic known methods for DNA isolation--precipitation,
dialysis, filtration and chromatography do not isolate
single-stranded from double-stranded DNA. If these purification
methods are employed it is necessary to add a separate step where
single-strand isolation is performed.
[0156] Isolation of a set of single-stranded NLFs can be
accomplished using a set of capture probes. "Capture probes" are
oligonucleotides or polynucleotides comprising a single-stranded
region complementary to at least one nucleotide sequence of the
single-stranded NLFs to be isolated and a first binding moiety. The
first binding moiety is capable of covalent or noncovalent binding
to a second binding moiety attached to a solid support. The capture
probes can comprise a set of capture probes, each of which contains
single-stranded regions complementary to a corresponding member of
a set of NLFs. A capture probe can also comprise a full-length
single-stranded target nucleic acid that is complementary to the
nucleotide sequences of the members of a set of NLFs. The capture
probes can be bound to a solid support using the methods described
above for binding a target or set of NLFs to a solid support.
[0157] If restriction endonucleases are used to produce nonrandom
length fragments from DNA, the preferred method for isolating
single-strand fragments from these products is to use a select set
of capture probes. In one embodiment the capture probe consists of
either full length positive or full length negative strand where
the strand has been modified to contain a solid-phase binding
moiety. The process using full length negative strand modified to
contain a biotin at the 5' end is illustrated in FIG. 13. The
capture probe is made and the target nucleic acid is fragmented in
two separate reactions. Following inactivation of the restriction
enzymes the probe and double-stranded fragments are mixed,
denatured and annealed producing a hybrid product of positive
strand fragments annealed to full length negative strand capture
probe. The capture probe can be bound to the solid phase via a
biotin-streptavidin interaction prior to or following of the
probe/fragment hybrid. Following the necessary wash steps the
fragments are released and analyzed by mass spectrometry.
Optionally, the fragments can be probed for a mutation-specific
base-base mismatch and fragmented using one of the mismatch
specific reagents described earlier. Illustrations of the different
spectra produced without and with the optional second step are
shown in FIG. 13. Note that after mutation-specific,
mismatch-specific cleavage fragments that are distal from the solid
phase binding site will be released into solution and washed away,
therefore, not analyzed. Lose of these fragments can enhance the
ability for mass spectrometry to quickly and easily identify the
site of mutation.
[0158] An alternative approach to using restriction endonucleases
is the use of fragmenting probes. These have been described in
detail above, and allow the use of a target nucleic acid consisting
of either DNA or RNA. The final products, using fragmenting probes
and single-strand-specific nucleases, are double-stranded and thus
without any additional steps do not themselves produce the set of
single-stranded, nonrandom length fragments necessary for analysis.
However, there are several approaches that can be used to yield
single-stranded nonrandom length fragments.
[0159] The first approach for producing single-stranded nonrandom
length fragments is useful when the target is RNA and the probes
are DNA or visa versa. In this case, the double-stranded products
are RNA/DNA hybrids and can be selectively treated with either a
DNA or RNA specific nuclease to yield the opposite NLF intact. Acid
or base treatments are also an option. These single-stranded
products can then be isolated using a number of conventional
methods described above.
[0160] A second approach to producing single-stranded products for
mass spectrometry is to attach the size and sequence specific
capture probes to a solid support before or after hybridization to
the target nucleic acid and the single-strand-specific cleavage.
Since the probes are bound to the solid phase it becomes possible
to capture, wash, and then selectively release the nonrandom length
target fragments as single-stranded molecules. Following any wash
steps, the nonrandom length target fragments are removed from the
solid support by denaturation of the double-stranded complex. Once
released, the single-stranded fragments can be directly analyzed by
the mass spectrometer.
[0161] One of skill in the art will know how to use capture probes
to capture single-strands of a set of NLFs to a solid support in
all the embodiments of this invention. For example biotinylated
capture probes can be used to capture single-stranded fragments
following cleavage of the target nucleic acid with restriction
endonucleases (optionally after neutralizing the restriction
endonucleases). The use of capture probes provides a relatively
high level of flexibility to select which set of NLFs to analyze at
any given time. Large capture probes, capable of hybridizing to all
or several different fragments, can be used to capture the
fragments correlating to one strand of a target nucleic acid, e.g.
a capture probe that is full length negative strand. A short
capture probe or combinations of shorter capture probes can be used
to selectively choose particular fragments from either strand to
analyze in a given mass spectrometric sample. For example, if
several fragments share similar sizes it might be preferable to
analyze them separately.
[0162] As another embodiment, a full length target nucleic acid can
be captured before restriction digestion using a capture probe that
is nuclease resistant. In this case it is necessary to modify the
capture probe, typically by changing the backbone composition from
phosphate to a phosphorothioate, methyl phosphonate or
borano-phosphate. [Uhlmann and Peyman, "Antisense Oligonucleotides:
A New Therapeutic Principle," Chemical Reviews 90(4):543-584 (1990)
(incorporated by reference herein)] These forms of modification
limit cutting on the probe strand, resulting only in the nicking of
the target molecule to create sequence-specific, nonrandom
length,fragments without creating any double stranded breaks. By
leaving the modified probe strand intact, it is possible to quickly
capture the nonrandom length fragments to the solid phase and
purify for mass spectrometric analysis.
[0163] All of these isolation or purification methods can be
utilized in cases where a mutation-specific cleavage event is
utilized. In order to present a base mismatch mutation for
cleavage, a heterozygous, double-stranded molecule must be present.
Typically this means that the fragmenting probe is composed of the
wild type sequence and is hybridized to the target nucleic acid
fragments containing the potentially mutated target nucleic
acid.
Volatile Salts
[0164] The methods of this invention include the use of volatile
salts, which is an innovative alternative to NaCl, MgCl.sub.2, or
other commonly used salts. Volatile salts are any salts that
completely evaporate, leaving little or no salt residue in the
sample to be analyzed in the mass spectrometer, for example, the
isolated set of NLFs. Volatile salts useful in the methods
described herein include ammonium bicarbonate, dimethyl ammonium
bicarbonate and trimethyl ammonium bicarbonate. These volatile
salts are useful in many different aspects of the methods described
herein, including use in hybridizing of nucleic acids, washing
nucleic acids to remove impurities, and digestion of nucleic acids
with endonucleases or other enzymes. Rather than performing washes
at reduced levels of nonvolatile salts, which might cause the
nonrandom length target fragments to denature from a solid support
bound oligonucleotide probe, it is a preferred embodiment to wash
support-bound nonrandom length fragments in the presence of
relatively high levels of NH.sub.4HCO.sub.3, e.g. 100 mM, and then
to evaporate the volatile salt prior to analysis by mass
spectrometry. Volatile salts are useful for buffer exchange in all
cases where nucleic acids are to be analyzed by mass
spectrometry.
[0165] Solid phase purification schemes involving DNA hybridization
commonly described in the literature do not focus on the removal of
salts since gel electrophoresis techniques are much more tolerant
of salts than mass spectrometry. [S. Wang, M. Krinks & M. Moos
"DNA Sequencing from Single Phage Plaques using Solid-Phase
Magnetic Capture" Biotechniques 18, 130 (1995); R. Sandaltzopoulos
& P. Becker "Solid-Phase DNase I Footprinting" Boehringer
Mannheim Biochemica 4, 25 (1995); both incorporated by reference
herein] These methods are primarily focus on the removal of strands
complementary to template prior to enzymatic reaction and/or
enzymes and unincorporated labeled nucleotides or primers following
reaction. In such schemes residual salt levels can be as high as
100 mM NaCl and 25 mM MgCl.sub.2. Mass spectrometry is intolerant
of salt concentrations of this level. [T. Shaler et al., "Effect of
Impurities on the Matrix-Assisted Laser Desorption Mass Spectra of
Single-Stranded Oligodeoxynucleotides: Anal. Chem. 68, 576 (1996)]
The methods described herein using volatile salts provide an
innovative approach to isolating and handling target nucleic acids
and/or nonrandom length fragments for mass spectrometric
analysis.
[0166] The volatile salts can be removed from the sample prior to
mass spectrometric analysis by evaporation. Evaporation of the
volatile salts can be enhanced using a variety of methods,
including use of vacuum, heating, laminar flow of a dry gas over
the sample, or, in the case of ammonium bicarbonate (or dimethyl-
or trimethylammonium bicarbonate), reduction of the pH by addition
of an acid, including 3-HPA, can speed up the decomposition of the
salt into ammonia (or dimethyl- or trimethylammonia) and carbon
dioxide. Volatile salts can be used in a variety of methods, beyond
those described here, for preparing samples of any number of
organic molecules, including proteins, polypeptides, and
polynucleotides, for mass spectrometric analysis.
[0167] Each of the nonrandom fragmentation techniques described
herein can be used in combination with any of the isolation methods
also described herein. Moreover the nonrandom fragmentation
techniques can be used in combination with each other, as one of
ordinary skill in the art using the techniques described herein how
to combine the different aspects of the invention. For example, the
mutation-specific cleavage technique can be combined with a set of
restriction endonuclease-cleaved NLFs. All of these methods and
combinations thereof can optionally include use of mass-modified
nucleotides, internal calibrants and volatile salts.
[0168] The kits described above for nonrandomly fragmenting target
nucleic acids and detecting mutations in one or more target nucleic
acids can also contain a combination of different means of
nonrandomly fragmenting the target nucleic acids as well as
different means of isolating the nonrandom length fragments that
are to be analyzed by mass spectrometry.
[0169] The following examples are provided to illustrate
embodiments of the invention, but do not limit the scope of the
invention.
EXAMPLES
Example 1
PCR Amplification of Source Nucleic Acids
[0170] PCR methods have been extensively developed during the last
decade. An example protocol is as follows. A sample containing
10-10,000 copies of a source DNA molecule is mixed with two
antiparallel DNA primers that surround a targeted sequence, e.g.
the coding region for a gene involved in carcinogenesis. The PCR
mix is composed of: 8 .mu.l 2.5 mM deoxynucleoside triphosphates,
10 .mu.l 10.times.PCR buffer, 10 .mu.l 25 mM MgCl.sub.2, 3 .mu.l 10
.mu.M forward primer, 3 .mu.l 10 .mu.M reverse primer, 0.3 .mu.l
thermostable Taq DNA polymerase, 64.7 .mu.l H.sub.2O, and 1 .mu.l
source DNA. The sample tube is sealed and placed into a thermal
cycling device. A typical cycling protocol is as follows:
1 Step 1 95.degree. C. 2 min. Step 2 95.degree. C. 15 sec. Step 3
55.degree. C. 15 sec. Step 4 72.degree. C. 1 min. Step 5 repeat
Steps 2-4 35 times Step 6 72.degree. C. 5 min. Step 7 stop
Example 2
Production of Single-Stranded Nucleic Acids by Asymmetric PCR
[0171] The basic PCR procedure can be modified in order to produce
predominantly one of the two strands. These asymmetric procedures
involve modifying the ratios of the two primers, a typical ratio is
10:1.
Example 3
Production of Single-Stranded DNA via Biotinylated PCR Products
[0172] For the preparation of capture probes one of the two primers
can be synthesized with a biotin moiety internally or at the 5' end
of the oligonucleotide. Following a standard PCR, the
double-stranded product can be bound to a solid-phase surface
coated with streptavidin. For example, 10 pmol of double-stranded
PCR product is mixed with 5 .mu.l MPG [10 mg/ml] paramagnetic
streptavidin-coated beads in a binding/washing buffer of 2.0 M
NaCl, 10 mM TrisCl, 1 mM EDTA, pH 8.0. The solution is incubated
for 15 min. at room temperature with mixing. Following incubation
the tube is placed next to a high field, rare earth magnet and the
paramagnetic beads with the bound biotinylated PCR product are
precipitated to the wall of the tube. The supernatant is removed,
and the particles, outside the influence of the magnetic field, are
resuspended into binding/washing buffer. The beads and wash
solution are mixed and then subjected once again to the magnetic
field to precipitate the magnetic particles. The supernatant is
once again removed and either the wash step is repeated or the
alkaline denaturation step commences. In order to release the
unbiotinylated strand from the double-stranded product the beads
are mixed with an alkaline denaturation solution, 0.1 M NaOH. The
beads are incubated at room temperature for 10 min. which denatures
the PCR product and releases the unbiotinylated product into
solution. The biotinylated strand, bound to the magnetic beads is
precipitated from the solution under the magnetic field and
unbiotinylated strand, now single-stranded, is transferred to a new
tube with the supernatant. In an optional secondary step, the now
single-stranded biotinylated strand can be freed from the magnetic
beads by boiling the beads in water for 10 min and transferred with
the new supernatant after magnetic precipitation of the magnetic
beads.
Example 4
Mass Modification of Target Nucleic Acids
[0173] Mass modification of the target nucleic acid is performed
during the amplification step. One or more standard deoxynucleoside
triphosphates are replaced with modified deoxynucleoside
triphosphates. As an example thymidine is replaced with a
5-alkynyl-substituted-2'-deoxy- uridine triphosphate. Because the
modified nucleotides may not be efficient substrates for DNA
polymerase it may be necessary to increase the concentration of the
corresponding triphosphate by a factor of 2 to 100 over normal
levels.
Example 5
Nonrandom Fragmentation of Double-Stranded Target Nucleic Acids
Using Restriction Endonucleases
[0174] Specifically-sized, double-strand DNA products produced, for
example, by PCR are subjected to sequence-specific fragmentation
using restriction endonucleases. As an example, 10 pmoles of a 500
base pair PCR product is treated with one unit each of the
frequently cutting enzymes Mnl I and HinP I in the buffer
recommended by the enzyme supplier. The reaction is incubated at
37.degree. C. for 1 hour, followed by an enzyme-denaturing
incubation at 65.degree. C. for 15 min.
Example 6
Nonrandom Fragmentation of Single-Stranded Target Nucleic Acids
Using Small Oligonucleotide Restriction Site Probes in Combination
with Restriction Endonucleases
[0175] Single-stranded DNA target, produced, for example, by
asymmetric PCR or by the solid phase methods described in Example
3, is mixed with small oligonucleotide restriction probes
complementary to selected restriction site locations. As an
example, a set of 10 base long probes targeting the Hae III
recognition sequence, are synthesized with the sequence (SEQ ID NO:
1) 5' NNNGGCCNNN 3', where the N's are chosen to allow the
restriction site probes to fully complement the single-stranded
target DNA at the sites where the Hae III recognition site (e.g.
the probe (SEQ ID NO: 2) 5' GACGGCCAAA 3' to complement the target
sequence (SEQ ID NO: 3) 5' . . . TTTGGCCGTC . . . 3'). The mixture
of target and probes, dissolved in the restriction buffer to be
used in the cleavage step, is denatured at 95.degree. C. and then
incubated at 32.degree. C. (the average T.sub.m melting temperature
for the probes) for 15 min. allowing the probes to anneal to target
and producing a mixture of single-stranded and double-stranded
regions within the target nucleic acid. The hybridized product is
then cleaved at the double-stranded sites using one or more
specific restriction endonucleases (e.g. Hae III), under conditions
similar to those described in Example 3.
Example 7
Nonrandom Fragmentation of Single-Stranded Target Nucleic Acids
Using Fragmentation Probes in Combination with
Single-Strand-Specific Endonucleases
[0176] Single-stranded DNA target, produced, for example, by
asymmetric PCR or by the solid phase methods described in Example
3, are mixed with fragmenting probes complementary to the target
DNA. As an example, a mixture of probes with sizes of 24, 26, 28,
30, 32 and 34 each with sequences complementary to different,
nonoverlapping regions of the single-stranded target DNA. The
mixture of target and probes, dissolved in S1 nuclease digest
buffer comprised of 50 mM NaAcetate pH 4.5, 280 mM NaCl, 50 mM
MgCl.sub.2, and 4.5 mM ZnSO.sub.4, are denatured at 95.degree. C.
and then incubated at 55.degree. C. (the average T.sub.m for the
probes) for 15 min. allowing the probes to anneal to target and
producing a mixture of single-stranded and double-stranded regions
within the target nucleic acid. The hybridized product is then
digested in the single-stranded regions using 1 U S1 nuclease per
.mu.g target DNA, incubated at room temperature for 30 min.
Example 8
Nonrandom Fragmentation of Single-Stranded Target Nucleic Acids
Using Mismatch-Specific Cleavage
Example 8.1
Chemical Cleavage at Mismatched Cytosine
[0177] A heterozygous, mutation-containing DNA target is produced,
either by PCR of a heterozygous source nucleic acid or by
hybridization of wild-type probes to a mutation-containing
single-stranded target DNA. For solid phase capture and
purification protocols the DNA probes are synthesized either
chemically or enzymatically in such a way as to contain biotin
moieties. By either route, when a mutation is present a mismatch
forms between the target and wild type. A cleavage solution of
hydroxylamine is prepared by dissolving 1.39 g of hydroxylamine
hydrochloride in 1.6 mL of warm H.sub.2O followed by the dropwise
addition of 1.75 mL of diethylamine to yield a solution of pH 6. A
6 mL sample of double-stranded DNA containing a mismatch site is
mixed with a 20 mL of hydroxylamine solution and the resulting
solution is incubated at 37.degree. C. for 30 minutes. The reaction
is stopped by the addition of 374 mL of H.sub.2O and the solution
is removed either by solid phase capture of the reaction products
using magnetic beads with washes performed in a similar manner to
that described in Example 3 or by multistep centrifugation in a
Microcon-30 ultrafiltration unit (Amicon). The reaction products
are redissolved in 45 mL of H.sub.2O and 5 mL of piperidine is
added. The solution is incubated at 90.degree. C. for 30 minutes
and then placed on ice to cool. A 300 mL portion of H.sub.2O is
added and samples are either evaporated to dryness or purified by
one of the two methods described in Examples 9 and 10.
[0178] A typical mass spectrum obtained from the hydroxylamine
fragmentation at a point mutation is shown in FIG. 14. The source
DNA in this case is a section of the coding sequence for the p53
gene. A 134 base long PCR product is produced as in Example 1,
amplifying p53 from codon 188 to 233 containing a heterozygous
point mutation in codon 213, CGA.fwdarw.TGA. The forward primer
containing a 5'-biotin and a chemically labile linker within the
primer, the reverse primer being a standard oligonucleotide. The
mismatch containing PCR product is treated with hydroxylamine as
described above, cleaving the mismatch at C in codon 213. The
product is purified as described in Example 10, and analyzed as
described in example 11. A strong peak appears at the mass
correlating to a product 75 bases in size identifying that a C is
present in a mismatch in the first position of codon 213. An
analysis of mutation-free wild type, shown in FIG. 15, contains no
mismatch and therefore no cleavage occurs.
Example 8.2
Chemical Cleavage at Mismatched Thymine
[0179] DNA is obtained in a similar manner to Example 8.1. The
modification reagent is a 20 mM solution of KMnO.sub.4 in deionized
H.sub.2O. To 6 mL of double-stranded DNA containing a mismatch site
is added 14 mL of the modification reagent. The solution is mixed
gently at room temperature over the course of two minutes during
which time the solution turns slightly brown. A 20 mL portion of a
solution consisting of 1.25 M sodium acetate pH 8.5 and containing
1 M 2-mercaptoethanol is added to stop the reaction, which results
in the solution becoming immediately colorless. A 360 mL portion of
H.sub.2O is added and the solution is either spun through a
Microcon-30 ultrafiltration unit 2X, collected, and then evaporated
to dryness or taken through a solid phase capture and wash
protocol. The DNA is redissolved in 45 mL of H.sub.2O and 5 mL of
piperidine is added. The resulting solution is heated to 90.degree.
C. for 30 minutes and then placed on ice to cool. After it cools,
the solution is diluted by the addition of 300 mL of H.sub.2O and
then evaporated to dryness. As an alternative the cleavage products
can be purified by one of the two methods described in Examples 9
and 10.
[0180] A typical mass spectrum obtained from the KMnO.sub.4
fragmentation at a point mutation is shown in FIG. 16. The source
DNA in this case is a section of the coding sequence for the p53
gene. A 134 base long PCR product is produced as in Example 1,
amplifying p53 from codon 188 to 233 containing a heterozygous
point mutation in codon 213, CGA.fwdarw.TGA. The forward primer
containing a 5'-biotin and a chemically labile linker within the
primer, the reverse primer being a standard oligonucleotide. The
mismatch containing PCR product is treated with KMnO.sub.4 as
described above, cleaving the mismatch at C in codon 213. The
product is purified as described in example 10, and analyzed as
described in Example 11. A strong peak appears at the mass
correlating to a product 75 bases in size identifying that a T is
present in a mismatch in the first position of codon 213. Based on
the data from the analysis in FIG. 14 and FIG. 16 it is possible to
confirm that a C.fwdarw.T mutation has occurred in this p53
sample.
Example 9
Purification of Nonrandom Length Fragments Using Capture Probes
[0181] Nonrandom fragments are purified by annealing to a capture
probes. The capture probe or probes consists of a sequence or
sequences complementary to the selected target nonrandom length
fragments. One method uses the a full length capture probe prepared
as described in Example 3, another uses a number of chemically
synthesized capture probes prepared with biotin covalently
attached. For either method the procedure is identical. A 10 .mu.L
sample containing a single full-length biotinylated capture probe
or a mixture of smaller, synthetic, biotinylated capture probes is
mixed with 10 .mu.L of nonrandom fragments in an annealing buffer
consisting of 300 mM NaCl, 10 mM Tris, and 1 mM EDTA pH 7.5. The
mixture is heated in a boiling-H.sub.2O bath for 10 min. and then
quickly placed in an ice-H.sub.2O bath. The mixture is then
transferred to a pre-heated thermal block at 42.degree. C. (the
temperature is adjusted depending on the T.sub.m of the capture
probe or probes) and incubated for 1 hour. The solution is then
allowed to cool and then mixed with streptavidin-coated magnetic
beads. Binding to the beads takes place according to the procedure
described in Example 3. After the binding step, in place of the
alkaline denaturation step, the bound, hybridized nonrandom
fragments are washed with a volatile buffer such as 1 M
NH.sub.4HCO.sub.3. After 6 cycles of resuspension in 1 M
NH.sub.4HCO.sub.3, magnetic precipitation, and removal of the
supernatant, the beads are resuspended in 10.mu.L of deionized
H.sub.2O and heated to 65.degree. C. for 5 min. in order to release
the nonrandom fragments from the bound biotinylated strand. The
beads are quickly precipitated from the warm solution and the
supernatant containing the nonrandom fragments is transferred to
another tube. The solution of nonrandom fragments is dried to
remove excess volatile buffer and then analyzed by mass
spectrometry as described in Example 11.
[0182] An example of capture and analysis of nonrandom length
fragments is shown in FIG. 1 7. The source DNA in this case is a
section of the coding sequence for the p53 gene. A 184 base long
PCR product is produced as in Example 1, amplifying p53 from codon
232 to 292 containing a heterozygous point mutation in codon 248,
CGG.fwdarw.CAG. The double-stranded PCR product is digested using
the restriction enzyme Mnl I under conditions described in Example
5. A full length capture probe of the negative strand is produced
as in Example 3, and the nonrandom length fragments derived from
the positive strand are captured and purified as described above.
The purified single-stranded fragments are analyzed as described in
Example 11. Shown in FIG. 16 are the 5 single-stranded positive
fragments produced from an Mnl I digest of the wild type 184 base
long PCR product. By performing single-stranded isolation the five
similarly sized negative strand fragments are eliminated from the
spectra and all of the fragments are fully resolved.
[0183] Shown in FIG. 18 is a magnification of the spectra examining
the 26 base long fragment that, in the heterozygous mutation case,
contains the G.fwdarw.A mismatch. Shown are two clearly resolved
peaks with a mass difference of 16 Da, exactly the difference
between G and A and thus confirming the presence of a mutation. The
third smaller peak correlates to a salt adduct of the high mass 26
base product and emphasizes the need for a process that stringently
removes salt prior to analysis.
Example 10
Alternative Purification Method for Mismatch-Specific Nonrandom
Length Fragments
[0184] The purification of nonrandom fragments that were produced
by a mutation-specific cleavage, e.g. chemical cleavage at mismatch
sites, can be achieved in an alternative way. In this case the
fragmentation is performed on a PCR product that has one
solid-phase capturable strand, e.g. containing biotin, and that is
also able to be cleaved from the solid support, e.g. a bridging
phosphorothioate linkage contained in the primer region [Mag et
al., Nucleic Acids Res. 19(7):1437-1441 (1991)]. As an example of
this method, a PCR reaction is performed as described in Example 1,
but with one of the primers containing a 5'-end biotin modification
and also a bridging phosphorothioate linkage located 3-5 bases from
the 3'-end, and the other primer a normal one. After amplification
the PCR product is subjected to a mutation-specific fragmentation
method directly since, for heterozygous mutations,
mismatch-containing heteroduplexes are formed in situ during the
PCR. In order to check for the possibility of a homozygous
mutation, the sample is mixed with an equal amount of wild type
control, annealed and then subjected to the fragmentation reaction.
The material recovered from the fragmentation reactions is purified
and made single-stranded by the method described in Example 3. In
this case, after the denaturing step, the products are released
from the magnetic beads after several H.sub.2O washes by treatment
with 5 .mu.L of 0.02 mM AgNo.sub.3 and incubating at 45.degree. C.
for 15 min. The Ag+ ions are sequestered by the addition of 1 .mu.L
of 100 mM DTT. The samples are dried to remove excess DTT and then
analyzed by mass spectrometry by the method described in Example
11.
Example 11
Mass Spectrometry Analysis
[0185] The nucleic acid sample to be analyzed is typically mixed
with an equal volume of matrix solution consisting of 0.5 M
3-hydroxypicolinic acid (3-HPA) and 50 mM diammonium hydrogen
citrate. Typically a 1 .mu.L portion of the sample is applied to
the mass spectrometer sample stage and allowed to dry under a
gentle stream of nitrogen gas at room temperature. When the sample
has completely dried to form crystals (typically 5 min.) the sample
is inserted into the mass spectrometer for analysis. The usual
analysis conditions employ the use of a Nd:YAG laser operating at
266 nm with an average pulse energy of 50 mJ/cm.sup.2. An average
of 100 laser shots is typically used to obtain a spectrum.
[0186] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0187] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the invention and the appended claims.
Sequence CWU 1
1
* * * * *