U.S. patent application number 10/358989 was filed with the patent office on 2004-06-10 for primer extension methods for detecting nucleic acids.
This patent application is currently assigned to EXACT SCIENCES CORPORATION. Invention is credited to Lapidus, Stanley N., Shuber, Anthony P..
Application Number | 20040110162 10/358989 |
Document ID | / |
Family ID | 22074469 |
Filed Date | 2004-06-10 |
United States Patent
Application |
20040110162 |
Kind Code |
A1 |
Shuber, Anthony P. ; et
al. |
June 10, 2004 |
Primer extension methods for detecting nucleic acids
Abstract
Methods are provided for selective nucleic acid sequence
detection in single base primer extension reactions of high
sensitivity. These methods are useful for detecting small amounts
of mutant nucleic acid in a heterogeneous biological sample. These
methods are particularly useful for identifying individuals with
gene mutations indicative of early colorectal cancer.
Inventors: |
Shuber, Anthony P.; (Mendon,
MA) ; Lapidus, Stanley N.; (Bedford, NH) |
Correspondence
Address: |
WEINGARTEN, SCHURGIN, GAGNEBIN & LEBOVICI LLP
TEN POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Assignee: |
EXACT SCIENCES CORPORATION
|
Family ID: |
22074469 |
Appl. No.: |
10/358989 |
Filed: |
February 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10358989 |
Feb 5, 2003 |
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09067212 |
Apr 27, 1998 |
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6566101 |
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09067212 |
Apr 27, 1998 |
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08877333 |
Jun 16, 1997 |
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5888778 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 1/6869 20130101; C12Q 1/6883 20130101; C12Q 1/6869 20130101;
C12Q 1/6827 20130101; C12Q 1/6858 20130101; C12Q 1/6869 20130101;
C12Q 2525/204 20130101; C12Q 2525/107 20130101; C12Q 2535/125
20130101; C12Q 2565/501 20130101; C12Q 2525/107 20130101; C12Q
2565/501 20130101; Y10T 436/143333 20150115; C12Q 2525/204
20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method for identifying a single nucleotide in a nucleic acid
sample, the method comprising the steps of: (a) annealing an
oligonucleotide primer to a nucleic acid sample under conditions
that promote exact complementary hybridization between said primer
and a portion of a nucleic acid in said sample; (b) extending said
primer by a single base; (c) separating said extended primer from
said portion; (d) repeating steps (a) through (c); and (e)
identifying the base incorporated into said extended primer,
thereby to identify said single nucleotide.
2. The method of claim 1, wherein said base in step (b) is a
chain-terminating nucleotide.
3. The method of claim 1, wherein said extension reaction in step
(b) is performed by addition of one or more chain-terminating
nucleotides.
Description
[0001] This patent application is a continuation-in-part of U.S.
Ser. No. 08/877,333, filed Jun. 16, 1997, the disclosure of which
is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to oligonucleotide
primer extension methods for identifying a single nucleotide in a
nucleic acid sample. Methods of the invention are useful for
disease diagnosis by detecting and identifying the presence of
genetic mutations or disease-causing microorganisms in biological
samples.
BACKGROUND OF THE INVENTION
[0003] The knowledge of molecular defects causative of diseases,
such as inherited disorders and cancer, is increasing rapidly.
Inherited diseases thought to be caused by genetic mutations
include sickle cell anemia, .alpha.- and .beta.-thalassemias,
phenylketonuria, hemophilia, .alpha..sub.i-anti-trypsin deficiency,
and cystic fibrosis. Sickle cell anemia, for example, is reported
to result from homozygosity resulting from a single base pair
substitution in the sixth codon of the .beta.-globin gene.
Antonarakis, New England J. Med., 320: 153-163 (1989). Mutations in
the insulin receptor gene and in the insulin-responsive glucose
transporter gene have been detected in insulin-resistant diabetes.
Krook et al., Human Molecular Genetics, 1: 391-396 (1992).
[0004] Cancer has been associated with genetic mutations in a
number of oncogenes and tumor suppressor genes. Duffy, Clin. Chem.,
41: 1410-1413 (1993). For example, point mutations in the ras genes
have been shown to convert those genes into transforming oncogenes.
Bos et al., Nature, 315: 726-730. Mutations and the loss of
heterozygosity at the p53 tumor suppressor locus have been
correlated with various types of cancer. Ridanpaa et al., Path.
Res. Pract., 191: 399402 (1995); Hollstein et al., Science, 253:
49-53 (1991). In addition, the loss or other mutation of the apc
and dcc tumor suppressor genes has also been associated with tumor
development. Blum, Europ. J. Cancer, 31A: 1369-1372 (1995). Those
mutations can serve as markers for early stages of disease and for
predisposition thereto. Early diagnosis is not only important for
successful treatment, but can also lead to prevention or treatment
before chronic symptoms occur.
[0005] Colorectal cancer is an example of a disease that is highly
curable if detected early. With early detection, colon cancer may
be effectively treated by, for example, surgical removal of the
cancerous tissue. Surgical removal of early-stage colon cancer is
usually successful because colon cancer begins in cells of the
colonic epithelium and is isolated from the general circulation
during its early stages. Thus, detection of early mutations in
colorectal cells would greatly increase survival rate. Current
methods for detection of colorectal cancer focus on extracellular
indicia of the presence of cancer, such as the presence of fecal
occult blood or carcinoembryonic antigen circulating in serum. Such
extracellular indicia typically occurs only after the cancer has
become invasive. At that point, colorectal cancer is very difficult
to treat.
[0006] Methods have been devised to detect the presence of
mutations within disease-associated genes. One such method is to
compare the complete nucleotide sequence of a sample genomic region
with the corresponding wild type region. See, e.g., Engelke et al.,
Proc. Natl. Acad. Sci, U.S.A., 85: 544-548 (1988); Wong et al.,
Nature, 330: 384-386 (1988). However, such methods are costly, time
consuming, and require the analysis of multiple clones of the
targeted gene for unambiguous detection of low-frequency mutations.
As such, it is not practical to use extensive sequencing for
large-scale screening of genetic mutations.
[0007] A variety of detection methods have been developed which
exploit sequence variation in DNA using enzymatic and chemical
cleavage techniques. A commonly-used screen for DNA polymorphisms
consists of digesting DNA with restriction endonucleases and
analyzing the resulting fragments by means of southern blots, as
reported by Botstein et al., Am. J. Hum. Genet., 32: 314-331 (1980)
and White et al, Sci. Am., 258: 40-48 (1988). Mutations that affect
the recognition sequence of the endonuclease will preclude
enzymatic cleavage at that site, thereby altering the cleavage
pattern of the DNA. Sequences are compared by looking for
differences in restriction fragment lengths. A problem with this
method (known as restriction fragment length polymorphism mapping
or RFLP mapping) is its inability to detect mutations that do not
affect cleavage with a restriction endonuclease. One study reported
that only 0.7% of the mutational variants estimated to be present
in a 40,000 base pair region of human DNA were detected using RFLP
analysis. Jeffreys, Cell, 18: 1-18 (1979).
[0008] Single base mutations have been detected by differential
hybridization techniques using allele-specific oligonucleotide
(ASO) probes. Saiki et al., Proc. Natl. Acad. Sci. USA, 86:
6230-6234 (1989). Mutations are identified on the basis of the
higher thermal stability of the perfectly-matched probes as
compared to mismatched probes. Disadvantages of this approach for
mutation analysis include: (1) the requirement for optimization of
hybridization for each probe, and (2) the nature of the mismatch
and the local sequence impose limitations on the degree of
discrimination of the probes. In practice, tests based only on
parameters of nucleic acid hybridization function poorly when the
sequence complexity of the test sample is high (e.g., in a
heterogeneous biological sample). This is partly due to the small
thermodynamic differences in hybrid stability generated by single
nucleotide changes. Therefore, nucleic acid hybridization is
generally combined with some other selection or enrichment
procedure for analytical and diagnostic purposes.
[0009] In enzyme-mediated ligation methods, a mutation is
interrogated by two oligonucleotides capable of annealing
immediately adjacent to each other on a target DNA or RNA molecule,
one of the oligonucleotides having its 3' end complementary to the
point mutation. Adjacent oligonucleotide sequences are only
covalently attached when both oligonucleotides are correctly
base-paired. Thus, the presence of a point mutation is indicated by
the ligation of the two adjacent oligonucleotides. Grossman et al.,
Nucleic Acid Research, 22: 4527-4534 (1994). However, the
usefulness of this method for detection is compromised by high
backgrounds which arise from tolerance of certain nucleotide
mismatches or from non-template directed ligation reactions.
Barringer et al., Gene, 89:117-122 (1990).
[0010] A number of detection methods have been developed which are
based on a template-dependent, primer extension reaction. These
methods fall essentially into two categories: (1) methods using
primers which span the region to be interrogated for the mutation,
and (2) methods using primers which hybridizes proximally and
upstream of the region to be interrogated for the mutation.
[0011] In the first category, Caskey and Gibbs [U.S. Pat. No.
5,578,458] report a method wherein single base mutations in target
nucleic acids are detected by competitive oligonucleotide priming
under hybridization conditions that favor the binding of the
perfectly-matched primer as compared to one with a mismatch. Vary
and Diamond [U.S. Pat. No. 4,851,331] described a similar method
wherein the 3' terminal nucleotide of the primer corresponds to the
variant nucleotide of interest. Since mismatching of the primer and
the template at the 3' terminal nucleotide of the primer inhibits
elongation, significant differences in the amount of incorporation
of a tracer nucleotide result under normal primer extension
conditions.
[0012] It has long been known that primer-dependent DNA polymerases
have, in general, a low replication error rate. This feature is
essential for the prevention of genetic mistakes which would have
detrimental effects on progeny. Methods in a second category
exploit the high fidelity inherent in this enzymological reaction.
Detection of mutations is based on primer extension and
incorporation of detectable, chain-terminating nucleoside
triphosphates. The high fidelity of DNA polymerases ensures
specific incorporation of the correct base labeled with a reporter
molecule. Such single nucleotide primer-guided extension assays
have been used to detect aspartylglucosaminuria, hemophilia B, and
cystic fibrosis; and for quantifying point mutations associated
with Leber Hereditary Optic Neuropathy (LHON). See. e.g.,
Kuppuswamy et al., Proc. Natl. Acad. Sci. USA, 88: 1143-1147
(1991); Syvanen et al, Genomics, 8: 684-692 (1990); Juvonen et al.,
Human Genetics, 93:16-20 (1994); Ikonen et al., PCR Meth.
Applications, 1: 234-240 (1992); Ikonen et al., Proc. Natl. Acad.
Sci. USA, 88: 11222-11226 (1991); Nikiforov et al., Nucleic Acids
Research, 22: 4167-4175 (1994). An alternative primer extension
method involving the addition of several nucleotides prior to the
chain terminating nucleotide has also been proposed in order to
enhance resolution of the extended primers based on their molecular
weights. See e.g., Fahy et al, WO/96/30545 (1996).
[0013] Strategies based on primer extension require considerable
optimization to ensure that only the perfectly annealed
oligonucleotide functions as a primer for the extension reaction.
The advantage conferred by the high fidelity of the polymerases can
be compromised by the tolerance of nucleotide mismatches in the
hybridization of the primer to the template. Any "false" priming
will be difficult to distinguish from a true positive signal.
[0014] The selectivity and sensitivity of an oligonucleotide primer
extension assay are related to the length of the oligonucleotide
primer, and to the reaction conditions. In general, primer lengths
and reaction conditions that favor high selectivity result in low
sensitivity. Conversely, primer lengths and reaction conditions
that favor high sensitivity result in low selectivity.
[0015] Under typical reaction conditions, short primers (i.e. less
than about a 15-mer) exhibit transient, unstable hybridization.
Therefore, the sensitivity of a primer extension assay is low when
a short primer is used, because a transient, unstable
oligonucleotide hybrid does not readily prime the extension
reaction, resulting in a low yield of extended oligonucleotide.
Moreover, in a complex heterogeneous biological sample, short
primers exhibit non-specific binding to a wide variety of
perfectly-matched complementary sequences. Thus, because of their
low stability and high non-specific binding, short primers are not
very useful for reliable identification of a mutation at a known
location. Therefore, detection methods based on primer extension
assays use oligonucleotide primers ranging in length from 15-mer to
25-mer. See e.g., PCT Patent Publications WO 91/13075; WO 92/15712;
and WO 96/30545. Lengthening the probe to increase stability,
however, has the effect of diminishing selectivity. A single base
mismatch usually has less effect on the binding efficiency of a
longer oligonucleotide primer than it does on that of a shorter
primer, because of the relatively smaller thermodynamic difference
between a mismatched primer and a perfectly matched primer. This
higher tolerance of nucleotide mismatches in the hybridization of
the longer primer to the template can result in higher levels of
non-specific "false" priming in complex heterogeneous biological
samples.
[0016] The reaction conditions of a primer extension reaction can
be optimized to reduce "false" priming due to a mismatched
oligonucleotide. However, optimization is labor intensive and
expensive, and often results in lower sensitivity due to a reduced
yield of extended primer. Moreover, since considerable optimization
is required to ensure that only the perfectly annealed
oligonucleotide functions as a primer for the extension reaction,
only limited multiplexing of the primer extension assays is
possible. Krook et al., supra report that multiplexing can be
achieved by using primers of different lengths and by monitoring
the wild-type and mutant nucleotide at each mutation site in two
separate single nucleotide incorporation reactions. However, given
that the selectivity and stability of the oligonucleotide primer
extension assay is determined by the length of the oligonucleotide
primer and the reaction conditions, the number of primers that can
be tested simultaneously in a given reaction mixture is very
limited.
[0017] Methods in the art reduce the possibility of false priming
by decreasing the sequence complexity of the test sample. Thus,
genomic DNA is isolated from the biological sample and/or amplified
with PCR using primers which flank the region to be interrogated.
The primer extension analysis is then conducted on the purified PCR
products. See PCT Patent Publications WO 91/13075; WO 92/15712; and
WO 96/30545. However, these methods are time consuming and
expensive, because they involve additional steps of sample
processing. Furthermore, these methods are not adapted for multiple
primer extension reactions in a single sample.
[0018] Therefore, there is a need in the art for a selective and
sensitive nucleic acid detection method, and for reliable
large-scale screening methods for a large number of genomic
mutations in heterogeneous biological samples. Such methods are
provided herein.
SUMMARY OF THE INVENTION
[0019] The invention provides methods of mutation detection having
high sensitivity and high selectivity. In a general embodiment, the
invention comprises a single base extension reaction that is
repeated at least once Methods of the invention are useful to
detect and identify genetic mutations or the presence of
disease-causing microorganisms in an heterogeneous biological
sample.
[0020] Methods of the invention comprise conducting multiple cycles
of a single-base extension reaction, thereby increasing the
sensitivity of the primer extension assay without compromising the
selectivity. In a preferred embodiment, methods of the invention
comprise between 2 and 100 cycles of primer extension. More
preferably, between 10 and 50 cycles are performed. Most
preferably, approximately 30 cycles are performed.
[0021] In a preferred embodiment, an excess of primer is used, to
ensure that additional extended primer products are produced in
each extension cycle. The oligonucleotide primer length is
preferably between about 10 to about 100 nucleotides, more
preferably between about 15 and about 35 nucleotides, and most
preferably about 25 nucleotides.
[0022] In a preferred embodiment, each extension reaction includes
conditions that promote hybridization of the primer only to nucleic
acids with a perfect complementary sequence (i.e. mismatched base
pairs are not tolerated). In one embodiment, the hybridization is
performed at about the Tm for the primer in the assay. In a more
preferred embodiment, the hybridization is performed above the Tm
for the primer.
[0023] In one embodiment, a hybridized oligonucleotide primer is
extended with a labeled terminal nucleotide. Labeled ddNTPs or
dNTPs preferably comprise a "detection moiety" which facilitates
detection of the extended primer. Detection moieties are selected
from the group consisting of fluorescent, luminescent or
radioactive labels, enzymes, haptens, and other chemical tags such
as biotin which allow for easy detection of labeled extension
products by, for example, spectrophotometric methods.
[0024] In a preferred embodiment, a further cycle of primer
extension is started by denaturing the hybridized and extended
primer, annealing nonextended primer, and extending the newly
hybridized primer. The presence of excess primer in the reaction
promotes annealing of nonextended primer in each cycle of the
reaction.
[0025] In a further embodiment, methods of the invention comprise
conducting at least two cycles of a single-base extension reaction
using segmented primers. Methods of the invention comprise
hybridizing two probes adjacent to a site of suspected mutation,
wherein neither probe alone is capable of being a primer for
template-dependent extension, but when the probes hybridize
adjacent to each other, they are capable of priming extension. In a
preferred embodiment, methods of the invention comprise hybridizing
to a target nucleic acid a probe having a length from about 5 bases
to about 10 bases, wherein the probe hybridizes immediately
upstream of a suspected mutation. Methods of the invention further
comprise hybridizing a second probe upstream of the first probe,
the second probe having a length from about 15 to about 100
nucleotides and having a 3' non-extendible nucleotide. The second
probe is substantially contiguous with the first probe. Preferably,
substantially contiguous probes are between 0 and about 1
nucleotide apart. A linker is preferably used where the first and
second probes are separated by two or more nucleotides, provided
the linker does not interfere with the nucleic acid extension
reaction. Such linkers are known in the art and include, for
example, peptide nucleic acids, DNA binding proteins, and
ligation.
[0026] In an alternative embodiment, segmented primers comprise a
series of first oligonucleotide probes. No member of the series of
the first probes is capable of being a primer for nucleic acid
polymerization unless every member of said series hybridize
simultaneously to substantially contiguous portions of the target
nucleic acid, thereby forming a contiguous primer. In one
embodiment, the segmented primers comprise three 8-mer first
probes. In another embodiment, the segmented primers comprise four
6-mer first probes.
[0027] In each cycle of the extension assay, an extension reaction
adds nucleotides to the segmented primer resulting from
co-hybridization of the above-described probes in a
template-dependent manner. In a preferred embodiment, first probes
hybridized to a target nucleic acid are extended with a labeled
terminal nucleotide whereas first probes hybridized to a wild-type
or non-target nucleic acid are extended with an unlabeled terminal
nucleotide. Labeled ddNTPs or dNTPs preferably comprise a
"detection moiety" which facilitates detection of the short probes
that have been extended with a labeled terminal nucleotide.
Detection moieties are selected from the group consisting of
fluorescent, luminescent or radioactive labels, enzymes, haptens,
and other chemical tags such as biotin which allow for easy
detection of labeled extension products by, for example,
spectrophotometric methods.
[0028] In a preferred embodiment, several cycles of extension
reactions are conducted in order to amplify the assay signal.
Extension reactions are conducted in the presence of an excess of
first and second probes, labeled dNTPs or ddNTPs, and heat-stable
polymerase. Once an extension reaction is completed, the first and
second probes bound to target nucleic acids are dissociated by
heating the reaction mixture above the melting temperature of the
hybrids. The reaction mixture is then cooled below the melting
temperature of the hybrids and first and second probes are
permitted to associate with target nucleic acids for another
extension reaction. In a preferred embodiment, 10 to 50 cycles of
extension reactions are conducted. In a most preferred embodiment,
30 cycles of extension reactions are conducted.
[0029] Methods disclosed herein may be used to detect single
nucleotide polymorphisms (SNPs), mutations such as insertions,
deletions, and substitutions. Nucleic acid samples that can be
screened with the methods of the present invention include human
nucleic acid samples. A primer (or segmented primer) is designed so
that the 3' end of the hybridized primer is immediately upstream of
the position that is complementary to the nucleotide position being
assayed. The nucleotide position being assayed is identified as the
nucleotide that is complementary to the nucleotide incorporated in
the single-base primer extension reaction. For example, if a G is
incorporated in the reaction, a C is present at the complementary
position on the nucleic acid in the biological sample. In a
preferred embodiment, a primer extension reaction is performed in
the presence of four nucleotides, preferably chain terminating
nucleotides, for example the dideoxynucleotides ddATP, ddCTP,
ddGTP, and ddTTP. In a more preferred embodiment, the nucleotides
are detectably labeled, preferably differentially labeled. In
alternative embodiments, the extension reaction is performed in the
presence of one, two, or three different nucleotides. If the
biological sample is heterogeneous at the nucleotide position being
assayed, the complementary nucleotides (if they are included in the
primer extension reaction) will be incorporated in the primer
extension assay.
[0030] Methods disclosed herein may be used to detect mutations
associated with diseases such as cancer. Additionally, methods of
the invention may be used to detect a deletion or a base
substitution mutation causative of a metabolic error, such as
complete or partial loss of enzyme activity.
[0031] In another embodiment, the specific nucleic acid sequence
comprises a portion of a particular gene or genetic locus in the
patient's genomic nucleic acid known to be involved in a
pathological condition or syndrome. Non-limiting examples include
cystic fibrosis, Tay-Sachs disease, sickle-cell anemia,
.beta.-thalassemia, and Gaucher's disease.
[0032] In yet another embodiment, the specific nucleic acid
sequence comprises part of a particular gene or genetic locus that
may not be known to be linked to a particular disease, but in which
polymorphism is known or suspected.
[0033] In yet another embodiment, the specific nucleic acid
sequence comprises part of a foreign genetic sequence e.g. the
genome of an invading microorganism. Non-limited examples include
bacteria and their phages, viruses, fungi, protozoa, and the like.
The present methods are particularly applicable when it is desired
to distinguish between different variants or strains of a
microorganism in order to choose appropriate therapeutic
interventions.
[0034] Genomic nucleic acid samples are isolated from a biological
sample. Once isolated, the nucleic acids may be employed in the
present invention without further manipulation. Alternatively, one
or more specific regions present in the nucleic acids may be
amplified by, for example, PCR. Amplification at this step provides
the advantage of increasing the concentration of specific nucleic
acid sequences within the target nucleic acid sequence population.
In another embodiment, genomic nucleic acids are fragmented before
further analysis.
[0035] In one embodiment, the nucleic acids are bound to a
solid-phase support. This allows the simultaneous processing and
screening of a large number of samples. Non-limiting examples of
supports suitable for use in the present invention include
nitrocellulose or nylon filters, glass beads, magnetic beads coated
with agents for affinity capture, treated or untreated microtiter
plates, and the like. In a preferred embodiment, the support is a
microtiter dish, having a multiplicity of wells. The use of such a
support allows the simultaneous determination of a large number of
samples and controls, and thus facilitates the analysis. Moreover,
automated systems can be used to provide reagents to such
microtiter dishes. In an alternative embodiment, methods of the
invention are conducted in an aqueous phase.
[0036] In one embodiment of the invention, the extended primers or
probes are enumerated. The primers or probes are preferably
extended with a nucleotide labeled with an impedence bead, and the
number of impedence beads is counted (using for example a Coulter
counter). The number of labeled primers is then determined from the
number of impedence beads. The label is more preferably a
radioactive isotope, and the amount of radioactive decay associated
with the labeled primer or probe is determined. The number of
labeled primers or probes is calculated from the amount of
radioactive decay. The numbers of extended primers or probes are
useful for a statistical analysis of the cycled extension
reaction.
[0037] Finally, methods of the invention further comprise isolating
and sequencing the extended primers or first probes. Primers or
first probes preferably comprise a "separation moiety" that
facilitates their isolation. Non-limiting examples of separation
moieties include hapten, biotin, and digoxigenin. In a preferred
embodiment, primers or first probes comprising a separation moiety
are immobilized to a solid support having affinity for the
separation moiety (e.g., coated with anti-hapten, avidin,
streptavidin, or anti-digoxigenin). The solid support is selected
from the group consisting of glass, plastic, and paper. The support
is fashioned as a column, bead, dipstick, or test tube. In a
preferred embodiment, the separation moiety is incorporated in the
labeled ddNTPs or dNTPs and only first probes extended with a
labeled ddNTP or dNTP are immobilized to the support. As such,
labeled primers or first probes are isolated from unextended
primers or first probes and second probes. In an alternative
preferred embodiment, the separation moiety is incorporated in all
the first probes, provided the separation moiety does not interfere
with the first probe's ability to hybridize with template and to be
extended. By incorporating the separation moiety in the first
probes, all first probes are immobilized to a solid support. First
probes are isolated from second probes by one or more washing
steps.
[0038] Labeled primers or first probes are then sequenced to
identify a mutation or disease-causing microorganism. In one
embodiment, the immobilized primers or probes are directly
subjected to sequencing, using for example, chemical methods
standard in the art. In other embodiments, the labeled first probes
are removed from the solid support and sequencing of labeled first
probes is performed in aqueous solution. The isolated first probes
are contacted with a multiplicity of complementary
oligonucleotides. In one embodiment, enzymatic sequencing is
performed using the isolated first probes as primers and the
complementary oligonucleotides as templates. In an alternative
embodiment, a single base extension reaction is performed using the
isolated first probes as primers and the complementary
oligonucleotides as templates. The sequence of the extension
product is determined by enzymatic sequencing. The sequence of the
extended labeled first probes identifies the genetic mutations or
the disease-causing microorganisms present in the sample.
[0039] Further aspects and advantages of the invention are apparent
upon consideration of the following detailed description
thereof.
DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a diagram depicting the use of a segmented primer
in a single base extension reaction for the detection of single
base polymorphisms. The white bar represents the template, the dark
gray bar represents second probe which hybridizes to a region on
the template that is substantially contiguous with the first probe
(light gray). The site suspected to be a single base mutation is
labeled A. The detectable label is marked B.
[0041] FIGS. 2A and 2B are model Gaussian distributions showing
regions of low statistical probability.
[0042] FIG. 3 is graph showing the probable values of N for a
heterogeneous population of cells in which 1% of the cells are
mutated.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention is the first to provide a single base
extension assay with both high selectivity and high sensitivity.
The present invention provides methods for detecting specific
nucleic acids in a biological sample with both high sensitivity and
high selectivity. The present methods provide the high selectivity
of stringent hybridization condition, without losing sensitivity
due to low yield of extended product. In general, methods of the
invention comprise performing multiple cycles of a single base
extension reaction in a biological sample. By cycling, extended
product yield is high, and there is no significant loss of
selectivity because hybridization conditions for the primer are
kept stringent relative to those typically applied during a
single-base extension reaction. Methods of the invention are useful
to detect and identify mutations associated with diseases such as
cancer, deletions or a base substitution mutations causative of a
metabolic error, such as complete or partial loss of enzyme
activity, portions of a particular gene or genetic locus in the
patient's genomic nucleic acid known to be involved in a
pathological condition or syndrome, single nucleotide polymorphisms
(SNPs), or part of a foreign genetic sequence e.g. the genome of an
invading disease-causing microorganism.
[0044] A single base primer extension reaction is performed by
annealing an oligonucleotide primer to a complementary nucleic
acid, and by extending the 3' end of the annealed primer with a
chain terminating nucleotide that is added in a template directed
reaction catalyzed by a DNA polymerase. The selectivity and
sensitivity of a single base primer extension reaction are affected
by the length of the oligonucleotide primer and the reaction
conditions (e.g. annealing temperature, salt concentration).
[0045] The selectivity of a primer extension reaction reflects the
amount of exact complementary hybridization between an
oligonucleotide primer and a nucleic acid in a sample. A highly
selective reaction promotes primer hybridization only to nucleic
acids with an exact complementary sequence (i.e. there are no base
mismatches between the hybridized primer and nucleic acid). In
contrast, in a non selective reaction, the primer also hybridizes
to nucleic acids with a partial complementary sequence (i.e. there
are base mismatches between the hybridized primer and nucleic
acid). In general, parameters which favor selective primer
hybridization (for example shorter primers and higher annealing
temperatures) result in a lower level of hybridized primer.
Therefore, parameters which favor a selective single-base primer
extension assay result in decreased sensitivity of the assay.
[0046] Methods of the invention comprise conducting at least two
cycles of a single-base extension reaction. By repeating the
single-base extension reaction, methods of the invention increase
the signal of a single-base primer extension assay, without
reducing the selectivity of the assay. The cycling increases the
signal, and the extension reaction can therefore be performed under
highly selective conditions (for example, the primer is annealed at
about or above its Tm).
[0047] In a preferred embodiment, methods of the invention are
performed by annealing an excess of primer under conditions which
favor exact hybridization, extending the hybridized primer,
denaturing the extended primer, and repeating the annealing and
extension reactions at least once. In a most preferred embodiment,
the reaction cycle comprises a step of heat denaturation, and the
polymerase is temperature stable (for example, Taq polymerase or
Vent polymerase).
[0048] Preferred primer lengths are between 10 and 100 nucleotides,
more preferably between 10 and 50 nucleotides, and most preferably
about 30 nucleotides. Useful primers are those that hybridize
adjacent a suspected mutation site, such that a single base
extension at the 3' end of the primer incorporates a nucleotide
complementary to the mutant nucleotide if it is present on the
template.
[0049] Preferred hybridization conditions comprise annealing
temperatures about or above the Tm of the oligonucleotide primer in
the reaction. The Tm of an oligonucleotide primer is determined by
its length and GC content, and is calculated using one of a number
of formulas known in the art. Under standard annealing conditions,
a preferred formula for a primer approximately 25 nucleotides long,
is
Tm (.degree. C.)=4.times.(Number of Gs+Number of
Cs)+2.times.(Number of As+Number of Ts).
[0050] In a preferred reaction, the annealing and denaturation
steps are performed by changing the reaction temperature. In one
embodiment of the invention, the primer is annealed at about the Tm
for the primer, the temperature is raised to the optimal
temperature for extension, the temperature is then raised to a
denaturing temperature. An example of annealing, extension and
denaturing temperatures and times is described in Example 2. In a
more preferred embodiment of the invention, the reaction is cycled
between the annealing temperature and the denaturing temperature,
and the single base extension occurs during transition from
annealing to denaturing conditions.
[0051] In a preferred embodiment of the invention, two or more
cycles of extension are performed. In a more preferred embodiment,
between 5 and 100 cycles are performed. In a further embodiment,
between 10 and 50 cycles, and most preferably about 30 cycles are
performed.
[0052] In a preferred embodiment of the invention, the nucleotide
added to the 3' end of the primer in a template dependent reaction
is a chain terminating nucleotide, for example a dideoxynucleotide.
In a more preferred embodiment, the nucleotide is detectably
labeled as discussed infra.
[0053] I. Cycled Extension Reactions with Segmented Primers
[0054] In one embodiment, methods of the invention comprise
conducting at least two cycles of single-base extension with a
segmented primer. In a preferred embodiment, the segmented primer
comprises a short first probe and a longer second probe capable of
hybridizing to substantially contiguous portions of the target
nucleic acid. The two probes are exposed to a sample under
conditions that do not favor the hybridization of short first probe
in the absence of longer second probe. Factors affecting
hybridization are well known in the art and include temperature,
ion concentration, pH, probe length, and probe GC content. A first
probe, because of its small size, hybridizes numerous places in an
average genome. For example, any given 8-mer occurs about 65,000
times in the human genome. However, an 8-mer has a low melting
temperature (T.sub.m) and a single base mismatch greatly
exaggerates this instability. A second probe, on the other hand, is
larger than the first probe and will have a higher T.sub.m. A
20-mer second probe, for example, typically hybridizes with more
stability than an 8-mer. However, because of the small
thermodynamic differences in hybrid stability generated by single
nucleotide changes, a longer probe will form a stable hybrid but
will have a lower selectivity because it will tolerate nucleotide
mismatches. Accordingly, under unfavorable hybridization conditions
for the first probe (e.g., 10-40.degree. C. above first probe
T.sub.m), the first probe hybridizes with high selectivity (i.e.,
hybridizes poorly to sequence with even a single mismatch), but
forms unstable hybrids when it hybridizes alone (i.e., not in the
presence of a second probe). The second probe will form a stable
hybrid but will have a lower selectivity because of its tolerance
of mismatches.
[0055] The extension reaction in the present invention will not
occur absent contiguous hybridization of the first and second
probes. A first (proximal) probe alone is not a primer for
template-based nucleic acid extension because it will not form a
stable hybrid under the reaction conditions used in the assay.
Preferably, the first probe comprises between about 5 and about 10
nucleotides. The first probe hybridizes adjacent to a nucleic acid
suspected to be mutated. A second (distal) probe in mutation
identification methods of the invention hybridizes upstream of the
first probe and to a substantially contiguous region of the target
(template). The second probe alone is not a primer of
template-based nucleic acid extension because it comprises a 3'
non-extendible nucleotide. The second probe is larger than the
first probe, and is preferably between about 15 and about 100
nucleotides in length.
[0056] According to methods of the invention, template-dependent
extension takes place only when a first probe hybridizes next to a
second probe. When this happens, the short first probe hybridizes
immediately adjacent to the site of the suspected single base
mutation. The second probe hybridizes in close proximity to the 5'
end of the first probe. The presence of the two probes together
increases stability due to cooperative binding effects. Together,
the two probes are recognized by polymerase as a primer. This
system takes advantage of the high selectivity of a short probe and
the hybridization stability imparted by a longer probe in order to
generate a primer that hybridizes with the selectivity of a short
probe and the stability of a long probe. Accordingly, there is
essentially no false priming with segmented primers. Since the
tolerance of mismatches by the longer second probe will not
generate false signals, several segmented primers can be assayed in
the same reaction, as long as the hybridization conditions do not
permit the extension of short first probes in the absence of the
corresponding longer second probes. Moreover, due to their
increased selectivity for target, methods of the invention may be
used to detect and identify a target nucleic acid that is available
in small proportion in a sample and that would normally have to be
amplified by, for example, PCR in order to be detected.
[0057] By requiring hybridization of the two probes, false positive
signals are reduced or eliminated. As such, the use of segmented
oligonucleotides eliminates the need for careful optimization of
hybridization conditions for individual probes, as presently
required in the art, and permits extensive multiplexing. Several
segmented oligonucleotides can be used to probe several target
sequences assayed in the same reaction, as long as the
hybridization conditions do not permit stable hybridization of
short first probes in the absence of the corresponding longer
second probes.
[0058] The first and second probes hybridize to substantially
contiguous portions of the target. For purposes of the present
invention, substantially contiguous portions are those that are
close enough together to allow hybridized first and second probes
to function as a single probe (e.g., as a primer of nucleic acid
extension). Substantially contiguous portions are preferably
between zero (i.e., exactly contiguous so there is no space between
the portions) nucleotides and about one nucleotide apart. A linker
is preferably used where the first and second probes are separated
by two or more nucleotides, provided the linker does not interfere
with the assay (e.g., nucleic acid extension reaction). Such
linkers are known in the art and include, for example, peptide
nucleic acids, DNA binding proteins, and ligation. It has now been
realized that the adjacent probes bind cooperatively so that the
longer, second probe imparts stability on the shorter, first probe.
However, the stability imparted by the second probe does not
overcome the selectivity (i.e., intolerance of mismatches) of the
first probe. Therefore, methods of the invention take advantage of
the high selectivity of the short first probe and the hybridization
stability imparted by the longer second probe.
[0059] Thus, in a preferred embodiment, first and second probes are
hybridized to substantially contiguous regions of target, wherein
the first probe is immediately adjacent and upstream of a site of
suspected mutation, for example, a single base mutation. The sample
is then exposed to dideoxy nucleic acids that are complements of
possible mutations at the suspected site. For example, if the
wild-type nucleic acid at a known site is adenine, then dideoxy
adenine, dideoxy cytosine, and dideoxy guanine are placed into the
sample. Preferably, the dideoxy nucleic acids are labeled.
Deoxynucleotides may alternatively be used if the reaction is
stopped after the addition of a single nucleotide. Polymerase,
either endogenously or exogenously supplied, catalyzes
incorporation of a dideoxy base on the first probe. Detection of
label indicates that a non-wild-type (i.e., mutant) base has been
incorporated, and there is a mutation at the site adjacent the
first probe. Alternatively, methods of the invention may be
practiced when the wild-type sequence is unknown. In that case, the
four common dideoxy nucleotides are differentially labeled.
Appearance of more than one label in the assay described above
indicates a mutation may exist.
[0060] In an alternative preferred embodiment, a segmented
oligonucleotide comprises a series of first probes, wherein
sufficient stability is only obtained when all members of the
segmented oligonucleotide simultaneously hybridize to substantially
contiguous portions of a nucleic acid. It has now been realized
that, although short probes exhibit transient, unstable
hybridization, adjacent short probes bind cooperatively and with
greater stability than each individual probe. Together, a series of
adjacently-hybridized first probes will have greater stability than
individual probes or a subset of probes in the series. For example,
in an extension reaction with a segmented primer comprising a
series of three first probes (i.e., three short probes with no
terminal nucleotide capable of hybridizing to a substantially
contiguous portion of a nucleic acid upstream of the target nucleic
acid), the concurrent hybridization of the three probes will
generate sufficient cooperative stability for the three probes to
prime nucleic acid extension and the short probe immediately
adjacent to a suspected mutation will be extended. Thus, segmented
probes comprising a series of short first probes offer the high
selectivity (i.e., intolerance of mismatches) of short probes and
the stability of longer probes.
[0061] In a preferred embodiment, several cycles of extension
reactions are conducted in order to amplify the assay signal.
Extension reactions are conducted in the presence of an excess of
first and second probes, labeled dNTPs or ddNTPs, and heat-stable
polymerase. Once an extension reaction is completed, the first and
second probes bound to target nucleic acids are dissociated by
heating the reaction mixture above the melting temperature of the
hybrids. The reaction mixture is then cooled below the melting
temperature of the hybrids and first and second probes permitted to
associate with target nucleic acids for another extension reaction.
In a preferred embodiment, 10 to 50 cycles of extension reactions
are conducted. In a most preferred embodiment, 30 cycles of
extension reactions are conducted.
[0062] II. Detection of Extended Primers
[0063] Labeled ddNTPs or dNTPs preferably comprise a "detection
moiety" which facilitates detection of the extended primers, or
extended short first probes in a segmented primer reaction.
Detection moieties are selected from the group consisting of
fluorescent, luminescent or radioactive labels, enzymes, haptens,
and other chemical tags such as biotin which allow for easy
detection of labeled extension products. Fluorescent labels such as
the dansyl group, fluorescein and substituted fluorescein
derivatives, acridine derivatives, coumarin derivatives,
pthalocyanines, tetramethylrhodamine, Texas Red.RTM.,
9-(carboxyethyl)-3-hydroxy-6-oxo-6H-xanthenes, DABCYL.RTM. and
BODIPY.RTM. (Molecular Probes, Eugene, Oreg.), for example, are
particularly advantageous for the methods described herein. Such
labels are routinely used with automated instrumentation for
simultaneous high throughput analysis of multiple samples.
[0064] In a preferred embodiment, primers or first probes comprise
a "separation moiety." Such separation moiety is, for example,
hapten, biotin, or digoxigenin. These primers or first probes,
comprising a separation moiety, are isolated from the reaction
mixture by immobilization on a solid-phase matrix having affinity
for the separation moiety (e.g., coated with anti-hapten, avidin,
streptavidin, or anti-digoxigenin). Non-limiting examples of
matrices suitable for use in the present invention include
nitrocellulose or nylon filters, glass beads, magnetic beads coated
with agents for affinity capture, treated or untreated microtiter
plates, and the like.
[0065] In a preferred embodiment, the separation moiety is
incorporated in the labeled ddNTPs or dNTPs. By denaturing
hybridized primers or probes, and immobilizing primers or first
probes extended with a labeled ddNTP or dNTP to a solid matrix,
labeled primers or labeled first probes are isolated from
unextended primers or unextended first probes and second probes,
and primers or first probes extended with an unlabeled ddNTPs by
one or more washing steps.
[0066] In an alternative preferred embodiment, the separation
moiety is incorporated in the primers or first probes, provided the
separation moiety does not interfere with the first primer's or
probe's ability to hybridize with template and be extended. Eluted
primers or first probes are immobilized to a solid support and can
be isolated from eluted second probes by one or more washing
steps.
[0067] Alternatively, the presence of primers or first probes that
have been extended with a labeled terminal nucleotide may be
determined without eluting hybridized primers or probes. The
methods for detection will depend upon the label or tag
incorporated into the primers or first probes. For example,
radioactively labeled or chemiluminescent first probes that have
bound to the target nucleic acid can be detected by exposure of the
filter to X-ray film. Alternatively, primers or first probes
containing a fluorescent label can be detected by excitation with a
laser or lamp-based system at the specific absorption wavelength of
the fluorescent reporter.
[0068] In an alternative embodiment, the bound primers or first and
second probes are eluted from a matrix-bound target nucleic acid
(see below). Elution may be accomplished by any means known in the
art that destabilizes nucleic acid hybrids (i.e., lowering salt,
raising temperature, exposure to formamide, alkali, etc.). In a
preferred embodiment, the bound oligonucleotide probes are eluted
by incubating the target nucleic acid-segmented primer complexes in
water, and heating the reaction above the melting temperature of
the hybrids.
[0069] Deoxynucleotides may be used as the detectable single
extended base in any of the reactions described above that require
single base extension. However, in such methods, the extension
reaction must be stopped after addition of the single
deoxynucleotide. Such methods may be employed regardless of whether
a specific mutation is known (i.e., C.fwdarw.G). Moreover, the
extension reaction need not be terminated after the addition of
only one deoxynucleotide if only one labeled species of
deoxynucleotide is made available in the sample for detection of
the single base mutation. This method may actually enhance signal
if there is a nucleotide repeat including the interrogated single
base position.
[0070] In a preferred embodiment, target nucleic acids are
immobilized to a solid support prior to exposing the target nucleic
acids to primers or segmented primers and conducting an extension
reaction. Once the nucleic acid samples are immobilized, the
samples are washed to remove non-immobilized materials. The nucleic
acid samples are then exposed to one or more set of primers or
segmented primers according to the invention. Once the single-base
extension reaction is completed, the primers or first probes
extended with a labeled ddNTP or dNTP are preferably isolated from
unextended probes and probes extended with an unlabeled ddNTPs or
dNTP. Bound primers or first and second probes are eluted from the
support-bound target nucleic acid. Elution may be accomplished by
any means known in the art that destabilizes nucleic acid hybrids
(i.e., lowering salt, raising temperature, exposure to formamide,
alkali, etc.). In a preferred embodiment, the first and second
probes bound to target nucleic acids are dissociated by incubating
the target nucleic acid-segmented primer complexes in water, and
heating the reaction above the melting temperature of the hybrids
and the extended first probes are isolated. In an alternative
preferred embodiment, the extension reaction is conducted in an
aqueous solution. Once the single-base extension reaction is
completed, the oligonucleotide probes are dissociated from target
nucleic acids and the extended first probes are isolated. In an
alternative embodiment, the nucleic acids remain in aqueous
phase.
[0071] Finally, methods of the invention comprise isolating and
sequencing the extended first probes. A "separation moiety" such
as, for example, hapten, biotin, or digoxigenin is used for the
isolation of extended first probes. In a preferred embodiment,
first probes comprising a separation moiety are immobilized to a
solid support having affinity for the separation moiety (e.g.,
coated with anti-hapten, avidin, streptavidin, or
anti-digoxigenin). Non-limiting examples of supports suitable for
use in the present invention include nitrocellulose or nylon
filters, glass beads, magnetic beads coated with agents for
affinity capture, treated or untreated microtiter plates, and the
like.
[0072] In a preferred embodiment, the separation moiety is
incorporated in the labeled ddNTPs or dNTPs. By immobilizing eluted
primers or first probes extended with a labeled ddNTP or dNTP to a
solid support, labeled primers or first probes are isolated from
unextended first probes and second probes, and primers or first
probes extended with an unlabeled ddNTPs by one or more washing
steps.
[0073] In an alternative preferred embodiment, the separation
moiety is incorporated in the primers or first probes, provided the
separation moiety does not interfere with the first primer's or
probe's ability to hybridize with template and to be extended.
Eluted primers or first probes are immobilized to a solid support
and can be isolated from eluted second probes by one or more
washing steps.
[0074] The labeled primers or first probes are then sequenced to
identify the detected mutation or disease-causing microorganism. In
one embodiment, the immobilized probes are directly subjected to
sequencing, using a chemical method standard in the art. In other
embodiments, the immobilized labeled first probes are removed from
the solid support and sequencing of labeled first probes is
performed in aqueous solution.
[0075] III. Enumerative Detection Methods
[0076] Methods of the invention are useful in any context in which
enumeration of nucleic acids is necessary or desirable. Primarily,
detection methods discussed above are useful for detecting
nucleotide mutations in biological samples. Accordingly, methods of
the invention are useful for enumeration of a nucleic acid (e.g.,
an allele, a single nucleotide polymorphism or a mutation)
associated, or suspected to be associated, with a disease. Once a
number of a target nucleic acid has been determined in a patient
sample, that number is compared to the number expected to be
present if the sample were obtained from a healthy individual. A
statistically-significant difference exists between the number of a
nucleic acid in the patient sample and the number expected in a
healthy patient (which number may be determined from pooled samples
of healthy individuals), the patient is diagnosed as having a
disease or the propensity therefor. Methods of the invention are
also useful for detecting nucleic acids in biological samples,
which are often heterogeneous, and mutated nucleic acids are often
present in small amounts relative to wild-type nucleic acids. In
stool samples for example, mutant nucleic-acids from transformed
cells shed onto the stool are rare relative to wild-type nucleic
acids from normal cells shed onto the stool, especially in the
early stages of colorectal cancer. Methods of the invention
comprise statistical analysis to determine whether the results from
a single-base extension assay of the invention are indicative of
the presence of mutant nucleic acid in a biological sample. In a
preferred embodiment, methods of the invention comprise enumeration
of the single-base extended primers or probes. In a more preferred
embodiment, the number of extended primers or probes is analyzed to
determine whether a statistically significant amount of mutant
nucleic acid sequence is present in the biological sample.
[0077] In one embodiment of the invention, primers or probes are
preferably extended, as discussed herein, with a labeled
nucleotide. The number of labeled primers is then determined. The
label is more preferably a radioactive isotope, and the amount of
radioactive decay associated with the labeled primer or probe is
determined. The number of labeled primers or probes is calculated
from the amount of radioactive decay. The number of molecules is
counted by measuring a number X of radioactive decay events (e.g.
by measuring the total number of counts during a defined interval
or by measuring the time it takes to obtain a predetermined number
of counts) specifically associated with the labeled primer or
probe. The number X is used to calculate the number X1 of
radionucleotides which are specifically associated with the labeled
primer or probe. The number X1 is used to calculate the number X2
of labeled primer or probe molecules, knowing the number of
radionucleotide molecules associated with each labeled molecule in
the assay, as disclosed in co-owned, co-pending patent application
Ser. No. ______ (Attorney docket No. EXT-005), incorporated by
reference herein. The numbers of extended primers or probes present
in the assay are useful for subsequent statistical analysis.
[0078] Methods of the present invention are useful for detecting
loss of heterozygosity in a small number of cells in an impure
cellular population, because such methods do not rely upon knowing
the precise deletion end-points and such methods are not affected
by the presence in the sample of heterogeneous DNA. For example, in
loss of heterozygosity, deletions occur over large portions of the
genome and entire chromosome arms may be missing. Methods of the
invention comprise counting a number of molecules of a target
nucleic acid suspected of being deleted and comparing it to a
reference number. In a preferred embodiment the reference number is
the number of molecules of a nucleic acid suspected of not being
deleted in the same sample. All that one needs to know is at least
a portion of the sequence of a target nucleic acid suspected of
being deleted and at least a portion of the sequence of a reference
nucleic acid suspected of not being deleted. Methods of the
invention, while amenable to multiple mutation detection, do not
require multiple mutation detection in order to detect indicia of
cancer in a heterogeneous sample.
[0079] Accordingly, methods of the present invention are useful for
the detection of loss of heterozygosity in a subpopulation of cells
or debris therefrom in a sample. Loss of heterozygosity generally
occurs as a deletion of at least one wild-type allelic sequence in
a subpopulation of cells. In the case of a tumor suppressor gene,
the deletion typically takes the form of a massive deletion
characteristic of loss of heterozygosity. Often, as in the case of
certain forms of cancer, disease-causing deletions initially occur
in a single cell which then produces a small subpopulation of
mutant cells. By the time clinical manifestations of the mutation
are detected, the disease may have progressed to an incurable
stage. Methods of the invention allow detection of a deletion when
it exists as only a small percentage of the total cells or cellular
debris in a sample.
[0080] Methods of the invention comprise a comparison of the number
of molecules of two nucleic acids that are expected to be present
in the sample in equal numbers in normal (non-mutated) cells. In a
preferred embodiment, the comparison is between (1) an amount of a
genomic polynucleotide segment that is known or suspected not to be
mutated in cells of the sample (the "reference") and (2) an amount
of a wild-type (non-mutated) genomic polynucleotide segment
suspected of being mutated in a subpopulation of cells in the
sample (the "target"). A statistically-significant difference
between the amounts of the two genomic polynucleotide segments
indicates that a mutation has occurred.
[0081] In a preferred embodiment, the reference and target nucleic
acids are alleles of the same genetic locus. Alleles are useful in
methods of the invention if there is a sequence difference which
distinguishes one allele from the other. In a preferred embodiment,
the genetic locus is on or near a tumor suppressor gene. Loss of
heterozygosity can result in loss of either allele, therefore
either allele can serve as the reference allele. The important
information is the presence or absence of a statistically
significant difference between the number of molecules of each
allele in the sample. Also in a preferred embodiment, the reference
and target nucleic acids are different genetic loci, for example
different genes. In a preferred embodiment, the reference nucleic
acid comprises both alleles of a reference genetic locus and the
target nucleic acid comprises both alleles of a target genetic
locus, for example a tumor suppressor gene. Specifically, in the
case of a deletion in a tumor suppressor gene, the detected amount
of the reference gene is significantly greater than the detected
amount of the target gene. If a target sequence is amplified, as in
the case of certain oncogene mutations, the detected amount of
target is greater than the detected amount of the reference gene by
a statistically-significant margin.
[0082] Methods according to the art generally require the use of
numerous probes, usually in the form of PCR primers and/or
hybridization probes, in order to detect a deletion or a point
mutation. However, because methods of the present invention involve
enumerative detection of nucleotide sequences and enumerative
comparisons between sequences that are known to be stable and those
that are suspected of being unstable, only a few probes must be
used in order to accurately assess cancer risk. In fact, a single
set (pair) of primers or probes is all that is necessary to detect
a single large deletion. The risk of cancer is indicated by the
presence of a mutation in a genetic region known or suspected to be
involved in oncogenesis. Patients who are identified as being at
risk based upon tests conducted according to methods of the
invention are then directed to other, typically invasive,
procedures for confirmation and/or treatment of the disease.
[0083] According to methods of the invention, the target and
reference nucleic acids are differentially labeled using cycled
single-base extension reactions that incorporate differently
labeled nucleotides at the 3' ends of the primers or probes that
selectively hybridize to the target and reference nucleic acids.
For example, the primers or probes are designed such that template
directed single-base extension of the primer or probe hybridized to
the target nucleic acid results in addition of a T, whereas
template directed single-base extension of the primer or probe
hybridized to the reference nucleic acid results in addition of a
G. The extension reactions are performed, for example, in the
presence of .sup.35S-labeled chain terminating T. and
.sup.32P-labeled chain terminating G. Alternatively, the two chain
terminating nucleotides are labeled with large and small impedence
beads, respectively. These chain terminating nucleotides can be
labeled with any detectably different markers that allow
enumeration of the extended primers or probes, as discussed
herein.
[0084] Enumerative sampling of a nucleotide sequence that is
uniformly distributed in a biological sample typically follows a
Poisson distribution. For large populations, such as the typical
number of genomic polynucleotide segments in a biological sample,
the Poisson distribution is similar to a normal (Gaussian) curve
with a mean, N, and a standard deviation that may be approximated
as the square root of N.
[0085] Statistically-significance between numbers of target and
reference genes obtained from a biological sample may be determined
by any appropriate method. See, e.g., Steel, et al., Principles and
Procedures of Statistics, A Biometrical Approach (McGraw-Hill,
1980), the disclosure of which is incorporated by reference herein.
An exemplary method is to determine, based upon a desired level of
specificity (tolerance of false positives) and sensitivity
(tolerance of false negatives) and within a selected level of
confidence, the difference between numbers of target and reference
genes that must be obtained in order to reach a chosen level of
statistical significance. A threshold issue in such a determination
is the minimum number, N, of genes (for each of target and
reference) that must be available in a population in order to allow
a determination of statistical significance. The number N will
depend upon the assumption of a minimum number of mutant alleles in
a sample containing mutant alleles (assumed herein to be at least
1%) and the further assumption that normal samples contain no
mutant alleles. It is also assumed that a threshold differences
between the numbers of reference and target genes must be at least
0.5% for a diagnosis that there is a mutation present in a
subpopulation of cells in the sample. Based upon the foregoing
assumptions, it is possible to determine how large N must be so
that a detected difference between numbers of mutant and reference
alleles of less than 0.5% is truly a negative (i.e. no mutant
subpopulation in the sample) result 99.9% of the time.
[0086] The calculation of N for specificity, then, is based upon
the probability of one sample measurement being in the portion of
the Gaussian distribution covering the lowest 3.16% of the
population (the area marked "A" in FIG. 2A) and the probability
that the other sample measurement is in the portion of the Gaussian
distribution covering the highest 3.16% of the population (the area
marked "B" in FIG. 2B). Since the two sample measurements are
independent events, the probability of both events occurring
simultaneously in a single sample is approximately 0.001 or 0.1%.
Thus, 93.68% of the Gaussian distribution (100%-2.times.3.16%) lies
between the areas marked A and B in FIG. 3. Statistical tables
indicate that such area is equivalent to 3.72 standard deviations.
Accordingly, 0.5% N is set equal to 3.72 sigma. Since sigma (the
standard deviation) is equal to {square root}{square root over
(N)}, the equation may be solved for N as 553,536. This means that
if the lower of the two numbers representing reference and target
is at least 553,536 and if the patient is truly normal, the
difference between the numbers will be less than 0.5% about 99.9%
of the time.
[0087] To determine the minimum N required for 99% sensitivity a
similar analysis is performed. This time, one-tailed Gaussian
distribution tables show that 1.28 standard deviations (sigma) from
the mean cover 90% of the Gaussian distribution. Moreover, there is
a 10% (the square root of 1%) probability of one of the numbers
(reference or target) being in either the area marked "A" in FIG. 3
or in the area marked "B" in FIG. 3. If the two population means
are a total of 1% different and if there must be a 0.5% difference
between the number of target and reference genes, then the distance
from either mean to the threshold for statistical significance is
equivalent to 0.25% N (See FIG. 3) for 99% sensitivity. As shown in
FIG. 3, 0.25% N corresponds to about 40% of one side of the
Gaussian distribution. Statistical tables reveal that 40% of the
Gaussian distribution corresponds to 1.28 standard deviations from
the mean. Therefore, 1.28 sigma is equal to 0.0025N, and N equals
262,144. Thus, for abnormal samples, the difference will exceed
0.5% at least 99% of the time if the lower of the two numbers is at
least 262,144. Conversely, an erroneous negative diagnosis will be
made only 1% of the time under these conditions.
[0088] In order to have both 99.9% specificity (avoidance of false
positives) and 99% sensitivity (avoidance of false negatives), a
sample with DNA derived from at least 553,536 (or roughly greater
than 550,000) cells should be counted. A difference of at least
0.5% between the numbers obtained is significant at a confidence
level of 99.0% for sensitivity and a difference of less than 0.5%
between the numbers is significant at a confidence level of 99.9%
for specificity. As noted above, other standard statistical tests
may be used in order to determine statistical significance and the
foregoing represents one such test.
[0089] Based upon the foregoing explanation, the skilled artisan
appreciates that methods of the invention are useful to detect
mutations in a subpopulation of a polynucleotides in any biological
sample. For example, methods disclosed herein may be used to detect
allelic loss (the loss of heterozygosity) associated with diseases
such as cancer. Additionally, methods of the invention may be used
to detect a deletion or a base substitution mutation causative of a
metabolic error, such as complete or partial loss of enzyme
activity. For purposes of exemplification, the following provides
details of the use of methods according to the present invention in
colon cancer detection. Inventive methods are especially useful in
the early detection of a mutation (and especially a large deletion
typical of loss of heterozygosity) in a tumor suppressor gene.
Accordingly, while exemplified in the following manner, the
invention is not so limited and the skilled artisan will appreciate
its wide range of applicability upon consideration thereof.
[0090] Methods according to the invention preferably comprise
comparing a number of a target polynucleotide known or suspected to
be mutated to a number of a reference polynucleotide known or
suspected not to be mutated. In addition to the alternative
embodiments using either alleles or genetic loci as reference and
target nucleic acids, the invention comprises a comparison of a
microsatellite repeat region in a normal allele with the
corresponding microsatellite region in an allele known or suspected
to be mutated. Exemplary detection means of the invention comprise
determining whether a difference exists between the number of
counts of each nucleic acid being measured. The presence of a
statistically-significant difference is indicative that a mutation
has occurred in one of the nucleic acids being measured.
EXAMPLES
[0091] For purposes of exemplification, the following provides
details of the use of methods according to the present invention in
colon cancer detection. Inventive methods are especially useful in
the early detection of a mutation. Accordingly, while exemplified
in the following manner, the invention is not so limited and the
skilled artisan will appreciate its wide range of applicability
upon consideration thereof.
Exemplary Methods for Detection of Colon Cancer or Precancer
Example 1
Sample Preparation
[0092] In accordance with the present invention, the target nucleic
acid represents a sample of nucleic acid isolated from a patient.
This nucleic acid may be obtained from any cell source or body
fluid. Non-limiting examples of cell sources available in clinical
practice include blood cells, buccal cells, cervicovaginal cells,
epithelial cells from urine, fetal cells, or any cells present in
tissue obtained by biopsy. Body fluids include blood, urine,
cerebrospinal fluid, and tissue exudates at the site of infection
or inflammation.
[0093] In a preferred embodiment, the sample is a cross-sectional
or circumferential portion of stool. Preferred methods for
preparing a cross-sectional or circumferential portion of stool are
provided in co-owned U.S. Pat. No. 5,741,650, and in co-owned
co-pending patent application Ser. No. ______ (Attorney docket No.
EXT-015), incorporated by reference herein. As stool passes through
the colon, it adheres cells and cellular debris sloughed from
colonic epithelial cells. Similarly, cells and cellular debris are
sloughed by a colonic polyp (comprising mutated DNA). However, only
the portion of stool making contact with the polyp will adhere
sloughed cells. It is therefore necessary to obtain at least a
cross-sectional or circumferential portion of stool in order to
ensure that the stool sample contains a mixture of all sloughed
cells, including those sloughed by presumptive cancer cells (e.g.,
polyps).
[0094] After sample preparation, the sample is homogenized in an
appropriate buffer, such as phosphate buffered saline comprising a
salt, such as 20-100 mM NaCl or KCl, and a detergent, such as 1-10%
SDS or Triton.TM., and/or a proteinase, such as proteinase K. An
especially-preferred buffer is a Tris-EDTA-NaCl buffer as disclosed
in co-owned, co-pending U.S. patent application Ser. No. ______,
[Attorney Docket No.: EXT-006], incorporated by reference herein.
The buffer may also contain inhibitors of DNA and RNA degrading
enzymes. Double-stranded DNA in the sample is melted (denatured to
form single-stranded DNA) by well-known methods See, e.g.,
Gyllensten et al., in Recombinant DNA Methodology II, 565-578 (WNu,
ed., 1995), incorporated by reference herein. DNA is then isolated
from the cell source or body fluid using any of the numerous
methods that are standard in the art. See, Smith-Ravin et al., Gut,
36: 81-86 (1995), incorporated by reference herein. It will be
understood that the particular method used to extract DNA will
depend on the nature of the source.
[0095] Once extracted, the target nucleic acid may be employed in
the present invention without further manipulation. Alternatively,
one or more specific regions present in the target nucleic acid may
be amplified by PCR. In this case, the amplified regions are
specified by the choice of particular flanking sequences for use as
primers. Amplification at this step provides the advantage of
increasing the concentration of specific nucleic acid sequences
within the target nucleic acid sequence population.
[0096] In one embodiment, the target nucleic acid, with or without
prior amplification of particular sequences, is bound to a
solid-phase support. This allows the simultaneous processing and
screening of a large number of patient samples. Non-limiting
examples of supports suitable for use in the present invention
include nitrocellulose or nylon filters, glass beads, magnetic
beads coated with agents for affinity capture, treated or untreated
microtiter plates, and the like. The conventional 96-well
microtiter dishes used in diagnostic laboratories and in tissue
culture are a preferred support. In a preferred embodiment, the
support is a microtiter dish, having a multiplicity of wells. The
use of such a support allows the simultaneous determination of a
large number of samples and controls, and thus facilitates the
analysis. Moreover, automated systems can be used to provide
reagents to such microtiter dishes. It will be understood by a
skilled practitioner that the method by which the target nucleic
acid is bound to the support will depend on the particular matrix
used. For example, binding of DNA to nitrocellulose can be achieved
by simple adsorption of DNA to the filter, followed by baking the
filter at 75-80.degree. C. under vacuum for 15 min-2 h.
Alternatively, charged nylon membranes can be used that do not
require any further treatment of the bound nucleic acid. Beads and
microtiter plates that are coated with avidin or streptavidin can
be used to bind target nucleic acid that has had biotin attached
(via e.g. the use of biotin-conjugated PCR primers). In addition,
antibodies can be used to attach target nucleic acids to any of the
above solid supports by coating the surfaces with the antibodies
and incorporating an antibody-specific hapten into the target
nucleic acids. The target nucleic acids can also be attached
directly to any of the above solid supports by epoxide/amine
coupling chemistry. See Eggers et al. Advances in DNA Sequencing
Technology, SPIE conference proceedings (1993). Once the nucleic
acid samples are immobilized, the samples are washed to remove
non-immobilized materials. The nucleic acid samples are then
exposed to one or more set of segmented primers according to the
invention. In an alternative embodiment, the nucleic acids remain
in aqueous phase.
Example 2
Multiple cycles of a Single Base Extension Reaction
[0097] a) Primer Selection
[0098] Genomic regions suspected to contain one or more mutations
are identified by reference to a nucleotide database, such as
GenBank, EMBL, or any other appropriate database or publication, or
by sequencing. For cancer detection, genetic mutations in a number
of oncogenes and tumor suppressor genes are known. Duffy, Clin.
Chem., 41: 1410-1413 (1993). Preferred genes for use in mutation
detection methods of the invention include one or more oncogenes
and/or one or more tumor suppressor genes. Specifically preferred
genes include the ras oncogenes, p53, dcc, apc, mcc, and other
genes suspected to be involved in the development of an oncogenic
phenotype.
[0099] As will be described below, methods of the invention permit
the detection of a mutation at a locus in which there is more than
one nucleotide to be interrogated. Moreover, methods of the
invention may be used to interrogate a locus in which more than one
single base mutation is possible. Once regions of interest are
identified, at least one primer is prepared to detect the presence
of a suspected mutation. A primer of the invention preferably has a
length from about 10 to about 100 nucleotides, more preferably
between about 15 and about 35 nucleotides, and most preferably
about 25 nucleotides.
[0100] The primer may be natural or synthetic, and may be
synthesized enzymatically in vivo, enzymatically in vitro, or
non-enzymatically in vitro. Primers for use in methods of the
invention are preferably selected from oligodeoxyribonucleotides,
oligoribonucleotides, copolymers of deoxyribonucleotides and
ribonucleotides, peptide nucleic acids (PNAs), and other functional
analogues. Peptide nucleic acids are well-known. See Pluskal, et
al., The FASEB Journal, Poster #35 (1994). They are synthetic
oligoamides comprising repeating amino acid units to which adenine,
cytosine, guanine, thymine or uracil are attached. See Egholm, et
al., Nature, 365: 566-568 (1993); Oerum, et al. Nucl. Acids Res.,
23: 5332-36 (1993); Practical PNA: Identifying Point Mutations by
PNA Directed PCR Clamping, PerSeptive Biosystems Vol. 1, Issue 1
(1995). Peptide nucleic acid synthons and oligomers are
commercially available form PerSeptive Biosystems, Inc.,
Framingham, Mass. See, e.g., PCT publications EP 92/01219, EP
92/01220, US92/10921. In many applications, PNA probes are
preferred to nucleic acid probes because, unlike nucleic
acid/nucleic acid duplexes, which are destabilized under conditions
of low salt, PNA/nucleic acid duplexes are formed and remain stable
under conditions of very low salt Additionally, because PNA/DNA
complexes have a higher thermal melting point than the analogous
nucleic acid/nucleic acid complexes, use of PNA probes can improve
the reproducibility of blotting assays.
[0101] For exemplification, a primer designed to detect a mutation
in the K-ras gene is provided below. According to methods of the
invention, primers complementary to either portions of the coding
strand or to portions of the non-coding strand may be used. For
illustration, a primer useful for detection of mutations in the
coding strand are provided below. Mutations in K-ras frequently
occur in the codon for amino acid 12 of the expressed protein.
[0102] The wild-type codon 12 of the K-ras gene and its upstream
nucleotides are:
[0103] wild-type template 3'-TATTTGAACACCATCAACCTCGAC-5' (SEQ ID
NO: 1)
[0104] The three nucleotides encoding amino acid 12 are underlined.
A primer (Primer 1) capable of interrogating the first nucleotide
position in the codon encoding amino acid 12 of the K-ras gene is
provided below.
[0105] Primer 1 5'-ATAAACTTGTGGTAGTTGGAGCT-3' (SEQ ID NO:9)
[0106] b) Multiple Cycles of Primer Extension
[0107] Primer 1 is hybridized to a nucleic acid sample under
conditions (see Tables 1 and 2) that promote selective binding of
Primer 1 to the complementary sequence in the K-ras gene. The
extension reaction is performed in the presence of the 4 different
dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTTP, each labeled
with a different detectable label. The extension reaction is cycled
30 times as indicated in Table 2
1TABLE 1 Reaction mixture for a single base extension cycling
reaction Component Amount H2O 25.5 10X seq Buffer 4 ddNTP (50 uM) 5
Primer (5 uM) 5 Thermo Sequenase 0.5 DNA sample 10
[0108]
2TABLE 2 Temperature profile for a cycled single base extension
reaction Step Temp. (C.) Time (Sec.) 1 94 5 2 94 30 3 64 10 4 72 10
5 Goto step 2, 29 times 6 4 hold
[0109] The reaction products are assayed for the incorporation of
labeled ddNTPs. A nucleic acid sample containing wild-type DNA
should only have labeled ddGTP incorporated. The incorporation of
any other ddNTP in a statistically significant amount is indicative
of the presence of a mutant K-ras nucleic acid in the sample.
Example 3
Preparation of Segmented Primers
[0110] a) Primer Selection
[0111] Once regions of interest are identified, at least one
segmented primer is prepared to detected the presence of a
suspected mutation. A segmented primer comprises at least two
oligonucleotide probes, a first probe and a second probe, which are
capable hybridizing to substantially contiguous portions of a
nucleic acid.
[0112] A first probe of the invention preferably has a length of
from about 5 to about 10 nucleotides, more preferably between about
6 and about 8 nucleotides, and most preferable about 8 nucleotides.
A second probe of the invention has a preferable length of between
about 15 and 100 nucleotides, more preferably between about 15 and
30 nucelotides, and most preferably about 20 nucleotides. Further,
a second probe is incapable of being a primer for
template-dependent nucleic acid synthesis absent a first probe
because it has a 3' terminal nucleotide that is non-extendible.
Preferred non-extendible 3' terminal nucleotides include dideoxy
nucleotides, C3 spacers, a 3' inverted base, biotin, or a modified
nucleotide. Although, longer probes have a lower selectivity
because of their tolerance of nucleotide mismatches, second probes
are non-extendible and will not produce false priming in the
absence of the proximal probe.
[0113] In an alternative embodiment, a segmented primer comprises a
series of first probes, wherein each member of the series has a
length of from about 5 to about 10 nucleotides, and most preferable
about 6 to about 8 nucleotides. Although the first probes do not
have a terminal nucleotide, nucleic acid extension will not occur
unless all members of the series are hybridized to substantially
contiguous portions of a nucleic acid.
[0114] The oligonucleotide probes of the segmented primer may be
natural or synthetic, and may be synthesized enzymatically in vivo,
enzymatically in vitro, or non-enzymatically in vitro. Probes for
use in methods of the invention are preferably selected from
oligodeoxyribonucleotides, oligoribonucleotides, copolymers of
deoxyribonucleotides and ribonucleotides, peptide nucleic acids
(PNAs), and other functional analogues. Peptide nucleic acids are
well-known. See Pluskal, et al., The FASEB Journal, Poster #35
(1994). They are synthetic oligoamides comprising repeating amino
acid units to which adenine, cytosine, guanine, thymine or uracil
are attached. See Egholm, et al., Nature, 365: 566-568 (1993);
Oerum, et al. Nucl. Acids Res., 23: 5332-36 (1993); Practical PNA:
Identifying Point Mutations by PNA Directed PCR Clamping,
PerSeptive Biosystems Vol. 1, Issue 1 (1995). Peptide nucleic acid
synthons and oligomers are commercially available form PerSeptive
Biosystems, Inc., Framingham, Mass. See, e.g., PCT publications EP
92/01219, EP 92/01220, US92/10921. In many applications, PNA probes
are preferred to nucleic acid probes because, unlike nucleic
acid/nucleic acid duplexes, which are destabilized under conditions
of low salt, PNA/nucleic acid duplexes are formed and remain stable
under conditions of very low salt. Additionally, because PNA/DNA
complexes have a higher thermal melting point than the analogous
nucleic acid/nucleic acid complexes, use of PNA probes can improve
the reproducibility of blotting assays.
[0115] For exemplification, segmented primers designed to detect
mutations in the K-ras gene are provided below. According to
methods of the invention, probes complementary to either portions
of the coding strand or to portions of the non-coding strand may be
used. For illustration, probes useful for detection of mutations in
the coding strand are provided below Mutations in K-ras frequently
occur in the codon for amino acid 12 of the expressed protein.
Several of the possible probes for detection of mutations at each
of the three positions in codon 12 are shown below.
[0116] The wild-type codon 12 of the K-ras gene and its upstream
nucleotides are:
[0117] wild-type template 3'-TATTTGAACACCATCAACCTCGAC-5' (SEQ ID
NO: 1)
[0118] The three nucleotides encoding amino acid 12 are underlined.
First probes and second probes capable of interrogating the three
nucleotides coding for amino acid 12 of the K-ras gene are provided
below. First probe A is a first probe as described generally above,
and has a sequence complementary to the nucleotides immediately
upstream of the first base in codon 12 (i.e., immediately adjacent
to the cytosine at codon position 1). Second probe A is a second
probe as generally described above. It is complementary to a
sequence that is substantially contiguous (here, exactly
contiguous) with the sequence to which the first probe A is
complementary. The bolded nucleotide in each of the second probes
shown below is the nonextendible 3' terminal nucleotide.
Hybridization of first and second probes suitable for detection of
a mutation in the first base of K-ras codon 12 are shown below:
3 (SEQ ID NO:2) second probe A 5'-ATAAACTTGTGGTAG (SEQ ID NO:3)
first probe A TTGGAGCT (SEQ ID NO:1) wild-type template
3'-TATTTGAACACCATCAACCTCGACCA-5'
[0119] Detection of a mutation in the second base in codon 12 may
be performed by using the same second probe as above (second probe
A), and a first probe, identified as first probe B below, that is
complementary to a sequence terminating immediately adjacent (3')
to the second base of codon 12. Hybridization of probes suitable
for detection of a mutation in the second base of codon 12 are
shown below:
4 (SEQ ID NO:2) second probe A 5'-ATAAACTTGTGGTAG (SEQ ID NO:4)
first probe B TGGAGCTG +TL, 32 (SEQ ID NO:1) wild-type template
3'-TATTTGAACACCATCAACCTCGACCA-5'
[0120] Detection of a mutation at the third position in codon 12 is
accomplished using the same second probe as above, and first probe
C, which abuts the third base of codon 12. Hybridization of probes
suitable for detection of a mutation in the third base of codon 12
are shown below
5 (SEQ ID NO:2) second probe A 5'-ATAAACTTGTGGTAG (SEQ ID NO:6)
first probe C GGAGCTGG (SEQ ID NO:1) wild-type template
3'-TATTTGAACACCATCAACCTCGACCA-5'
[0121] In methods for detection of mutations at the second and
third nucleotides of codon 12 described above, the second probe is
1 and 2 nucleotides, respectively, upstream of the region to which
the first probe hybridizes. Alternatively, second probes for
detection of the second and third nucleotides of codon 12 may
directly abut (i.e., be exactly contiguous with) their respective
first probes. For example, an alternative second probe for
detection of a mutation in the third base of codon 12 in K-ras
is:
[0122] 5'-ATAAACTTGTGGTAGTT (SEQ ID NO: 5)
[0123] The detection of mutations can also be accomplished with a
segmented primer comprising a series of at least three first
probes. A series of first probes suitable for detection of a
mutation in the third base of codon 12 is shown below:
6 (SEQ ID NO:7) first probe X 5'-ATAAACTT (SEQ ID NO:8) first probe
Y TGGTAGTT (SEQ ID NO:6) first probe Z GGAGCTGG (SEQ ID NO:1)
wild-type template 3'-TATTTGAACACCATCAACCTC- GACCA-5'
[0124] b) Multiple Cycles of Primer Extension
[0125] First and second probes are exposed to sample under
hybridization conditions that do not favor the hybridization of the
short first probe in the absence of the longer second probe.
Factors affecting hybridization are well known in the art and
include raising the temperature, lowering the salt concentration,
or raising the pH of the hybridization solution. Under unfavorable
hybridization conditions (e.g., at a temperature 30-40 .degree. C.
above first probe T.sub.m), first probe forms an unstable hybrid
when hybridized alone (i.e., not in the presence of a second probe)
and will not prime the extension reaction. The longer, second
probe, having a higher T.sub.m, will form a stable hybrid with the
template and, when hybridized to substantially contiguous portions
of the nucleic acid, the second probe will impart stability to the
shorter first probe, thereby forming a contiguous primer.
[0126] In a preferred embodiment, a modification of the dideoxy
chain termination method as reported in Sanger, Proc. Nat'l Acad.
Sci. (USA), 74: 5463-5467 (1977), incorporated by reference herein,
is then used to detect the presence of a mutation. The method
involves using at least one of the four common 2', 3'-dideoxy
nucleoside triphosphates (ddATP, ddCTP, ddGTP, and ddTTP). A
detectable detection moiety can be attached to the dideoxy
nucleoside triphosphates (ddNTPs) according to methods known in the
art. A DNA polymerase, such as Sequenase.TM. (Perkin-Elmer), is
also added to the sample mixture. In a preferred embodiment, a
thermostable polymerase, such as Taq or Vent DNA polymerase is
added to the sample mixture. Using the substantially contiguous
first and second probes as a primer, the polymerase adds one ddNTP
to the 3' end of the first probe, the incorporated ddNTP being
complementary to the nucleotide that exists at the single-base
polymorphic site. Because the ddNTPs have no 3' hydroxyl, further
elongation of the hybridized probe will not occur. Chain
termination will also result where there is no available
complementary ddNTP (or deoxynucleoside triphosphates) in the
extension mixture. After completion of the single base extension
reaction, extension products are isolated and detected.
[0127] Also in a preferred embodiment, labeled deoxynucleotides may
be used for detection if either the extension reaction is stopped
after addition of only one nucleotide or if only one labeled
nucleotide, corresponding to the complement of the expected
mutation, is exposed to the sample.
[0128] In the simplest embodiment of the invention, exemplified in
Examples 2 and 3, the nucleoside triphosphate mixture contains just
the labeled ddNTP or dNTP complementary to the known mutation. For
example, to interrogate a sample for a C.fwdarw.A mutation in the
first nucleotide of codon 12 of the K-ras gene, second probe A and
first probe A are exposed to an extension reaction mixture
containing labeled ddTTP or dTTP. The incorporation of a labeled
ddTTP or dTTP in first probe A indicates the presence of a
C.fwdarw.A mutation in the first nucleotide of codon 12 of the
K-ras gene in the sample tested. First probe A co-hybridized with
second probe A to a wild-type template will not be extended or,
alternatively, will be extended with unlabeled ddGTP or dGTP if
available in the reaction mixture.
[0129] Given the large number of mutations that have been
associated with colorectal cancer, a detection method for this
disease preferably screens a sample for the presence of a large
number of mutations simultaneously in the same reaction (e.g., apc,
K-ras, p53, dcc, MSH2, and DRA). As described above, only very
limited multiplexing is possible with detection methods of the
prior art. Since methods of the present invention eliminate false
positive signals resulting from the tolerance of mismatches of the
longer second probes, the use of segmented oligonucleotide avoids
the need for optimization of hybridization conditions for
individual probes and permits extensive multiplexing. Several
segmented primers can be assayed in the same reaction, as long as
the hybridization conditions do not permit stable hybridization of
short first probes in the absence of the corresponding longer
second probes.
[0130] In a preferred embodiment, the primer extension reactions
are conducted in four separate reaction mixtures, each having an
aliquot of the biological sample, a polymerase, and the three
labeled complementary non-wild-type ddNTPs (or dNTPs). Optionally,
the reaction mixtures may also contain the unlabeled complementary
wild-type ddNTP (or dNTP). The segmented primers are multiplexed
according to the wild-type template. In the present
exemplification, the first two nucleotides coding for amino acid 12
of the K-ras gene are cysteines. Accordingly, second probe A and
first probes A and B are added to a reaction mixture containing
labeled ddATP (or dATP), ddTTP (or dTTP), and ddCTP (or dCTP).
Second probe C and first probe C are added to a reaction mixture
containing labeled ddATP (or dATP), ddCTP (or dCTP), and ddGTP (or
dGTP). Any incorporation of a labeled ddNTP in a first probe
indicates the presence of a mutation in codon 12 of the K-ras gene
in the sample. This embodiment is especially useful for the
interrogation of loci that have several possible mutations, such as
codon 12 of K-ras.
[0131] In an alternative preferred embodiment, the primer extension
reactions are conducted in four separate reaction mixtures, each
containing only one labeled complementary non-wild-type ddNTP or
dNTP and, optionally, the other three unlabeled ddNTPs or dNTPs.
Segmented primers can be thus be exposed only to the labeled ddNTP
or dNTP complementary to the known mutant nucleotide or,
alternatively, to all three non-wild-type labeled ddNTPs or dNTPs.
In the K-ras example provided above, if the first nucleotide of
K-ras codon 12 is interrogated for a known C.fwdarw.G mutation,
first probe A and second probe A are added to only one reaction
mixture, the reaction mixture containing labeled ddCTP (or dCTP).
Optionally, methods of the invention may be practiced as described
above using labeled deoxynucleotides.
[0132] However, since several mutations have been identified at
codon 12 of the K-ras gene, the probes are exposed to all
non-wild-type labeled ddNTPs or dNTPs. Thus, second probe A and
first probes A and B are added to the three reaction mixtures
containing labeled ddATP (or dATP), ddTTP (or dTTP), or ddCTP (or
dCTP). Second probe C and first probe C are added to the three
reaction mixtures containing one of labeled ddATP (or dATP), ddCTP
(or dCTP), and ddGTP (or dGTP). Again, the extension of a first
probe with a labeled terminal nucleotide indicates the presence of
a mutation in codon 12 of the K-ras gene in the biological sample
tested.
[0133] In a preferred embodiment, several cycles of extension
reactions are conducted in order to amplify the assay signal.
Extension reactions are conducted in the presence of an excess of
first and second probes, labeled dNTPs or ddNTPs, and heat-stable
polymerase. Once an extension reaction is completed, the first and
second probes bound to target nucleic acids are dissociated by
heating the reaction mixture above the melting temperature of the
hybrids. The reaction mixture is then cooled below the melting
temperature of the hybrids and first and second probes permitted to
associate with target nucleic acids for another extension reaction.
In a preferred embodiment, 10 to 50 cycles of extension reactions
are conducted. In a most preferred embodiment, 30 cycles of
extension reactions are conducted.
Example 4
Methods for Identification of Genetic Alterations
[0134] In one embodiment, the labeled primers or probes are
immobilized as described herein, and are directly subjected to
sequencing, using a chemical method standard in the art (e.g.,
Maxam-Gilbert sequencing, Maxam and Gilbert, 1977, Proc. Natl.
Acad. Sci., USA, 74:560).
[0135] In other embodiments, the immobilized labeled primers or
first probes are removed from the solid support and sequencing of
labeled first probes is performed in aqueous solution. In one
embodiment, the sequence of the labeled first probes is determined
by sequence-specific reverse hybridization by exposing the labeled
first probes to oligonucleotides corresponding to each of the
multiple sequences being interrogated in the assay. Hybridization
analysis can be accomplished by several methods known in the art,
such as dot blots. See, Ausubel et al., Short Protocols in
Molecular Biology, 3rd ed. (John Wiley & Sons, Inc., 1995). In
a preferred embodiment, the oligonucleotides are immobilized to a
solid support at defined locations (i.e., known positions). This
immobilized array is sometimes referred to as a "DNA chip." The
solid support can be a plate or chip of glass, silicon, or other
material. The solid support can also be coated (e.g., with gold or
silver) to facilitate attachment of the oligonucleotides to the
surface of the solid support. Any of a variety of methods known in
the art may be used to immobilize oligonucleotides to a solid
support. A commonly used method consists of the non-covalent
coating of the solid support with avidin or streptavidin and the
immobilization of biotinylated oligonucleotide probes. The
oligonucleotides can also be attached directly to the solid
supports by epoxide/amine coupling chemistry. See Eggers et al.
Advances in DNA Sequencing Technology, SPIE conference proceedings
(1993).
[0136] In another embodiment, the sequence of the labeled first
probe is read by the hybridization and assembly of positively
hybridizing probes through overlapping portions. Drmanac et al.,
U.S. Pat. No. 5,202,231, incorporated herein by reference.
[0137] In yet another embodiment, first probes extended by a
labeled dNTP are identified by enzymatic DNA sequencing (Sanger et
al., 1977, Proc. Natl. Acad. Sci., USA, 74:5463). In this case,
oligonucleotides are synthesized that contain DNA sequences
complementary to the first probes and additional pre-determined
co-linear sequences that act as sequence "tags." When incubated
under Sanger sequencing conditions, the immobilized first probes
hybridize to their complementary sequences and act as primers for
the sequencing reaction. Determination of the resulting primed
sequence "tag" then identifies the first probe(s) present in the
reaction.
[0138] In a further embodiment, first probes extended by a labeled
dNTP are amplified prior to the sequence identification. Labeled
first probes are incubated with complementary oligonucleotides that
contain a sequencing primer sequence with or without an additional
"tag". Initial hybridization of a first probe to its complementary
oligonucleotide allows the first probe to serve as the initial
primer in a single extension reaction. The extension product is
then used directly as template in a cycle sequencing reaction.
Cycle sequencing of the extension products results in amplification
of the sequencing products. In designing the complementary
oligonucleotides, the sequencing primer is oriented so that
sequencing proceeds through the first probe itself, or,
alternatively, through the "tag" sequence. In the latter case, the
determination of the "tag" sequence will identify the colinear
first probe sequence. The amplified products are sequenced by a
chemical method standard in the art or identified by
sequence-specific reverse hybridization methods, as described
above.
[0139] In practicing the present invention, it is not necessary to
determine the entire sequence of the first probe or of the
complementary tagged oligonucleotide. It is contemplated that 1, 2,
or 3 sequencing reactions (instead of the four needed to obtain a
complete sequence) will be effective in producing characteristic
patterns (similar to "bar codes") to allow the immediate
identification of the individual first probes. This approach is
applicable to manual sequencing methods using radioactively labeled
first probes, which produce analog or digitized autoradiograms, as
well as to automated sequencing methods using non-radioactive
reporter molecules, which produce digitized patterns. In either
case, comparisons to an established data base can be performed
electronically. Thus, by reducing the number of required sequencing
reactions, the methods of the present invention facilitate the
economical analysis of multiple samples.
[0140] The present invention accommodates the simultaneous
screening of a large number of potential first probes in a single
reaction. In practice, the actual number of segmented primers that
are pooled for simultaneous hybridization is determined according
to the diagnostic need. For example, in cystic fibrosis (CF), one
particular mutation (.DELTA.508) accounts for more than 70% of CF
cases. This, a preliminary screening with a .DELTA.508-specific
segmented primers according to the present methods, followed by
single base extension of the contiguous primers, and detection of
the extended first probes, will identify and eliminate A508
alleles. In a second ("phase two") screening, a large number of
segmented primers encoding other, less frequent, CF alleles is
performed, followed by single base extension of the contiguous
primers, and detection of the extended first probes as described
above.
[0141] In other clinical situations, however, a single mutation
that appears with as high a frequency as the .DELTA.508 mutation in
CF does not exist. Therefore, pools of segmented primers are
determined only by the number of independent assays that would be
needed in a phase two analysis on a pool positive sample.
[0142] In addition, in current clinical practice, different
clinical syndromes, e.g. cystic fibrosis, thalassemia, and
Gaucher's disease, are screened independently of each other. The
present invention, by contrast, accommodates the simultaneous
screening of large numbers of nucleic acids from different patients
with a large number of first probes that are complementary to
mutations in more than one potential disease-causing gene.
[0143] In the same manner, when clinical indicators suggest
infection by a foreign agent or microorganism, the present
invention provides for simultaneous screening for a large number of
potential foreign nucleic acids. Furthermore, particular strains,
variants, mutants, and the like of one or more microorganisms can
also be distinguished by employing appropriate first probes in the
first screening.
[0144] The methods of the present invention also make it possible
to define potentially novel mutant alleles carried in the nucleic
acid of a patient or an invading microorganism, by the use of
randomly permuted segmented primers in phase one or phase two
screening. In this embodiment, single base extension of contiguous
primers and detection and isolation of extended first probes,
followed by sequencing, reveals the precise mutant sequence.
[0145] The foregoing exemplifies practice of the invention in the
context of multiple mutation detection using segmented primers. As
disclosed herein, numerous additional aspects and advantages of the
invention are apparent upon consideration of the disclosure and the
specific exemplification. Accordingly, the invention is limited
only by the scope of the appended claims.
Sequence CWU 1
1
9 1 26 DNA Artificial Sequence Sequencing reaction probe/primer 1
accagctcca actaccacaa gtttat 26 2 15 DNA Artificial Sequence
Sequencing reaction probe/primer 2 ataaacttgt ggtag 15 3 8 DNA
Artificial Sequence Sequencing reaction probe/primer 3 ttggagct 8 4
8 DNA Artificial Sequence Sequencing reaction probe/primer 4
tggagctg 8 5 17 DNA Artificial Sequence Sequencing reaction
probe/primer 5 ataaacttgt ggtagtt 17 6 8 DNA Artificial Sequence
Sequencing reaction probe/primer 6 ggagctgg 8 7 8 DNA Artificial
Sequence Sequencing reaction probe/primer 7 ataaactt 8 8 8 DNA
Artificial Sequence Sequencing reaction probe/primer 8 tggtagtt 8 9
23 DNA Artificial Sequence Sequencing reaction probe/primer 9
ataaacttgt ggtagttgga gct 23
* * * * *