U.S. patent application number 09/876727 was filed with the patent office on 2002-12-12 for method for detecting single nucleotide polymorphisms (snps) and point mutations.
Invention is credited to Wong, Jeffrey Tze Fel, Xue, Hong.
Application Number | 20020187477 09/876727 |
Document ID | / |
Family ID | 25368439 |
Filed Date | 2002-12-12 |
United States Patent
Application |
20020187477 |
Kind Code |
A1 |
Xue, Hong ; et al. |
December 12, 2002 |
Method for detecting single nucleotide polymorphisms (SNPs) and
point mutations
Abstract
A method of genotyping single nucleotide polymorphisms (SNP) and
point mutations in nucleic acid based on chain extension by
polymerase. This invention is based on the fact that the nucleoside
immediately 5' adjacent to any SNP/point mutation site is known,
and the neighboring sequence immediately 3' adjacent to the site is
also known. A primer complimentary to the sequence directly
adjacent the 3' side of the SNP in a target polynucleotide is used
for chain elongation. The polymerase reaction mixture contains one
chain terminating nucleotide having the base complimentary to
nucleotide directly adjacent to the 5' side of the SNP of the
target polynucleotide. An additional dNTP may be added to produce a
primer with the maximum of two base extension. The resultant
elongation/termination reaction product are analysed for
incorporation of the chain terminator nucleotide or for chain
length extension of the primer.
Inventors: |
Xue, Hong; (Hong Kong,
HK) ; Wong, Jeffrey Tze Fel; (Hong Kong, HK) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
25368439 |
Appl. No.: |
09/876727 |
Filed: |
June 6, 2001 |
Current U.S.
Class: |
435/6.14 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6858 20130101;
C12Q 1/6858 20130101; C12Q 2537/157 20130101; C12Q 2535/125
20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
1) a method of analysing the base identity of a target nucleotide
in a target polynucleotide molecule, said target polynucleotide
molecule having a 3' portion, a 5' portion and said target
nucleotide therebetween, said method comprising: (a) reacting said
target polynucleotide molecule with a primer oligonucleotide, said
primer oligonucleotide having a sequence complimentary to a section
of said 3' portion directly adjacent to said target nucleotide; (b)
providing a chain termination nucleotide having a base that is
complimentary to the base of the nucleotide directly adjacent to
the 5' side of said target nucleotide; (c) elongating said primer
across said target nucleotide under appropriate polymerisation
condition using a chain extending agent, said chain terminating
nucleotide having functional residues to allow said extending agent
to incorporate said chain terminating nucleotide onto the 3' end of
said primer, said chain terminating nucleotide further terminating
said elongation reaction by blocking further extension of said
primer after said chain terminating nucleotide is incorporated; (d)
analysing said elongation/termination reaction for primer
oligonucleotides containing incorporated chain terminator
nucleotides.
2) A method according to claim (1), wherein step (d) involves
analysing said elongation/termination reaction for oligonucleotides
that have zero, one or two bases longer than said primer.
3) A method according to claim (2) wherein said chain terminating
nucleotide is a 2',3'-dideoxyribonucleoside 5'-triphosphate and
said extending agent is DNA polymerase.
4) A method according to claim (2) wherein said target
polynucleotide molecule is amplified nucleic acid.
5) A method according to claim (1) wherein a second reaction
mixture is provided, said second reaction mixture containing all
the reagents as those mentioned in claim (1), said second reaction
mixture further containing an extender nucleotide with a second
base having an identity different from said base of said chain
terminating nucleotide, said extender nucleotide capable of
supporting further chain elongation after incorporation into said
oligonucleotide, said method further including the steps of
elongating said primer oligonucleotide in said second reaction
mixture using said extending agent under appropriate reaction
conditions, and analysing said second reaction mixture for primer
oligonucleotides containing incorporated chain termination
nucleotide.
6) A method according to claim (1) wherein one, two or three
additional reaction mixtures are provided, each said additional
reaction mixture containing all the reagents as those mentioned in
claim (1), each said additional reaction mixture further containing
an extender nucleotide with a unique base having an identity
different from said base of said chain terminating nucleotide, each
said extender nucleotide capable of supporting further chain
elongation after incorporation into said primer oligonucleotide,
said method further including the step of elongating said primer
oligonucleotide in said additional mixture using said extending
agent under suitable reaction conditions, and analysing said
additional reaction mixture for oligonucleotides containing
incorporated chain terminator nucleotide.
7) A method according to claim (5) wherein said primer
oligonucleotides are analysed for zero, one or two bases extension
after said elongation/termination reaction.
8) A method according to claim (6) wherein said primer
oligonucleotides are analysed for zero, one or two bases extension
after said elongation/termination reaction.
9) A method of detecting single nucleotide polymorphism comprising:
(a) obtaining double-strand nucleic acid from a source for which
said single nucleotide polymorphism analysis method is to be
performed; (b) amplifying a portion of said nucleic acid containing
said single nucleotide polymorphism by polymerase chain reaction
using a first primer and a second primer; (c) separating said first
and second primers from said amplification product; (d) mixing said
amplification product with a reaction mixture, said amplification
product having a 3' portion, a 5' portion and a target nucleotide
therebetween, said target nucleotide having said single nucleotide
polymorphism; (e) providing in said reaction mixture a third primer
oligonucleotide molecule having a sequence complimentary to a
section of said 3' portion directly adjacent to said target
nucleotide; (f) providing in said reaction mixture a polymerase
enzyme capable of extending the 3' end of said third primer; (g)
providing in said reaction mixture a chain terminating nucleotide,
said chain terminating nucleotide having functional residues to
allow said polymerase to polymerise said chain terminating
nucleotide onto the 3' end of that primer while blocking further
extension of said primer after said chain terminating nucleotide is
incorporated; said chain terminating nucleotide further having a
base that is complimentary to the base of the nucleotide directly
adjacent the 5' side of said target nucleotide; (h) reacting said
polymerase with said third primer and said amplification product in
said reaction mixture under appropriate reaction conditions; (i)
analyzing said reaction mixture for oligonucleotides that have
zero, one or two bases longer than said third primer.
10) A method according to claim (9) wherein a second reaction
mixture is provided, said second reaction mixture containing all
the reagents in said second reaction mixture, said second reaction
mixture further containing an extender nucleotide containing a
second base having an identity different from said base of said
chain terminating nucleotide, said extender nucleotide capable of
supporting further chain elongation after incorporation into said
third primer by said polymerase, said method further including the
steps of reacting said polymerase in said second reaction mixture,
and analysing said second reaction mixture for oligonucleotides
that have zero, one or two bases longer than said primer.
11) An oligonucleotide for detecting single nucleotide polymorphism
located at a target site on a target polynucleotide, said
oligonucleotide having a sequence complimentary to a sequence
directly adjacent to the 3' side of said target site.
12) An oligonucleotide according to claim (12) wherein the length
of said oligonucleotide is between 15-55 bases.
13) The method according to claim (1) wherein the 5' end of said
primer oligonucleotide is attached to a solid surface.
14) A method according to claim (9) wherein said third primer
oligonucleotide molecule is attached to a solid surface.
Description
FIELD OF INVENTION
[0001] The present invention relates to the analysis of nucleic
acid sequences. In particular, the present invention relates to the
detection of genetic polymorphisms.
BACKGROUND OF INVENTION
[0002] Single nucleotide polymorphism (SNP) and point mutations are
the most abundant type of genetic variations. These variation sites
are present at high density in genomes, making them powerful tools
for mapping and diagnosing disease-related alleles.
[0003] Many methods have been described for the detection of these
genetic polymorphisms. For example, U.S. Pat. No. 6,110,709
describes a method for detecting the presence or absence of an SNP
in a nucleic acid molecule by first amplifying the nucleic acid of
interest, followed by restrictions analysis and mobilizing the
amplified product to a binding element on a solid support. Patent
Publication WO9302212 describes another method for amplification
and sequencing of nucleic acid in which dideoxy nucleotides are
used to create amplified products of varying lengths. The varying
length products are then separated and visualized by gel
electrophoresis. Patent Publication WO20853 further describes a
method of detecting single base changes using tightly controlled
gel electrophoretic conditions to scan for conformational changes
in the nucleic acid caused by sequence changes.
[0004] In order to screen a large number of different samples,
there is a need to devise a new method with improved efficiency. It
is therefore an object of the present invention to provide a novel
method for scoring single nucleotide polymorphism.
SUMMARY OF INVENTION
[0005] Accordingly, the present invention is directed to a method
of genotyping SNPs (including point mutations) in nucleic acids,
which is based on the simple principle of chain/primer extension.
This invention is based on the fact that the nucleoside immediately
5' adjacent to any SNP/point mutation site is known, and the
neighbouring sequence immediately 3' adjacent to the site is also
known. If primers employed axe complementary to sequences in the
target polynucleotide where the next succeeding 5'-nucleotide of
the target polynucleotide is a potential SNP/point mutation,
polymerase reaction mixture containing one ddNTP complementary to
the 5' nucleotide adjacent the SNP of the target polynucleotide in
combination with dNTPs should produce nucleic acid chains with a
maximum of two-base extensions.
[0006] In one aspect to the present invention, a known SNP at a
defined location of a known nucleic acid may be scored. The nucleic
acid to be analyzed is referred to as the target polynucleotide
molecule and contains a 3' portion, a 5' portion and a target
nucleotide therebetween. This target nucleotide is the position in
which the single nucleotide polymorphism is known to be located.
The next step involves providing for hybridization a primer
oligonucleotide molecule with a sequence complimentary to a section
of the 3' portion of the target polynucleotide molecule directly
adjacent to the target nucleotide. The oligonucleotide and the
target polynucleotide are added to a reaction mixture that is
further provided with a chain extending agent capable of extending
the 3' end of the primer molecule such as a polymerase enzyme. The
reaction mixture is also provided with chain terminating nucleotide
having a base that is complimentary to the base of the nucleotide
directly adjacent to the 5' side of the target nucleotide. The
above reagents are then reacted under appropriate conditions for
primer extension in the presence or absence of an extender
nucleotide, and the numbers of bases that are extended from the
primer are scored using various techniques known in the art.
[0007] Chain terminating nucleotides refer to nucleotides that have
functional residues to allow the chain extending agent to
polymerise and extend one end of the primer while blocking further
extension of the primer after the chain terminating nucleotide has
been incorporated. Extender nucleotide refers to any nucleotides
that can be used by the chain extending agent for continual chain
elongation without causing chain termination. For example, if DNA
polymerase is used as the chain extending agent, then
2'-dideoxyribonucleoside 5'-triphosphate (dNTP) may be used as an
extender nucleotide, while 2',3'-dideoxyribonucleoside
5'-triphosphate (ddNTP) may be used as the chain terminating
nucleotide. If RNA polymerase is used as the chain extending agent,
then 3'-dioxyribonucleoside 5'-triphosphate can be used as the
chain terminating nucleotide while ribonucleoside 5'-triphosphate
(NTP) can be used as the extender nucleotide.
[0008] In one embodiment, the polynucleotide that is to be analysed
for SNP is first isolated and amplified using techniques such as
conventional polymerase chain reaction (PCR) using a pair of first
and second PCR primers. The first and second primers are designed
to amplify the region containing the SNP of interest (i.e. the
target nucleotide). The amplified products (referred to as the
amplified target polynucleotide) are then separated from the first
and second primers. The purified amplified target polynucleotide is
then reacted with a third primer. The third primer is designed to
be complimentary to the 3' portion of the amplified target
polynucleotide directly adjacent to the target nucleotide. The
elongation/termination reaction is then initiated by adding a chain
terminating nucleotide to an appropriate reaction mixture. The
appropriate chain terminating nucleotide contains a base that is
complimentary to the base directly adjacent to the 5' side of the
target nucleotide. An extender nucleotide may also be added to the
reaction mixture to potentially allow the third primer to be
extended across the target nucleotide. The extended third primer,
which would have a maximum of only 2 nucleotides extended, can be
analysed using standard analysis techniques such as electrophoresis
or mass spectroscopy. Other techniques including fluorescence
spectroscopy, capillary electrophoresis (CE), high performance
liquid chromatography (HPLC) can be used for detection.
[0009] Another embodiment of the invention is directed to detection
of SNPs/point mutations in a solid-phase mode including DNA chip.
In this case, oligomers of DNA, RNA, or PNA (peptide nucleic acid)
in modified or unmodified forms with known sequences 5'-upstream to
SNP sites of interest can be coated onto a solid surface, such as
that of glass, metal, plastic, nylon, beads or any other suitable
matrices. Molecules under investigation in the form of, e.g.
purified PCR product, can then be hybridized to the oligomers, or
primers, immobilized on a solid surface. In the presence of a
polymerase reaction mixture containing an unlabelled dNTP and a
labelled ddNTP complimentary to the base 5' to the SNP, the PCR
product will serve as polymeration template, according to which the
immobilized primers will be extended for two residues if the dNTP
present is complimentary to the SNP site on the template sequence.
The primer will however be extended for only one nucleoside if dNTP
is absent and the ddNTP present is in base pairing with the SNP
site.
BREIF DESCRIPTION OF DRAWINGS
[0010] FIG. 1 shows a schematic drawing of one example of how to
use the patent invention.
[0011] FIG. 2A show the result of chain length analysis of the
reaction shown in FIG. 1.
[0012] FIG. 2B is the result of detection of incorporated label
onto the primer in the reaction shown in FIG. 1 using labelled
chain terminator nucleotide and without chain analysis.
[0013] FIGS. 3A and 3D show another example of how to practise the
present invention.
[0014] FIG. 4A shows the result of chain length analysis of the
example shown in FIGS. 3A and 3B.
[0015] FIG. 4B is the result of detection of incorporated label
onto the primer in the reaction shown in FIGS. 3A and 3B using
labelled chain terminator nucleotide and without chain length
analysis.
DETAILS OF INVENTION
[0016] The present invention is applicable to any technology
platforms that use polymerase-based reaction in conjunction with
the use of ddNTP followed by chain length/mass or label analysis
with or without prior separation of free ddNTP from incorporated
ddNTP. The technique may be applied to genetic material of any
organism, including prokaryotic and enkayotic organism.
[0017] In one embodiment, a one-step elongation/termination
reaction followed by chain length analysis provides complete
information on the SNP of interest. In this embodiment, a primer is
provided having a sequence complimentary to the section of the
target polynucleotide that is directly adjacent the 3' side of the
target nucleotide. The target nucleotide refers to the sequence
location in which the SNP to be screened is known to be located.
One ddNTP which is complementary to the nucleotide 5' adjacent to
the SNP/point mutation is also provided in the reaction mixture.
The ddNTP may be in a labelled or unlabelled form, depending on the
method used for product analysis in the subsequent step. There
should be also present in the reaction mixture one dNTP. In the
case where the potential SNP/point mutation site and the 3'
adjacent site in a target DNA accommodate bases of the same
identity, the reaction could proceed without dNTP.
[0018] The identity of the base at the potential SNP/point mutation
site of the target DNA can be determined by checking for chain
extension after the elongation/termination reaction. Fully
informative results can be obtained if different reaction mixtures
are used, each containing a different dNTP. A dideoxy
polymerization that gives no chain extension suggests that the base
of the target nucleotide is not complementary to any dNTP present
in the reaction mature, while one base extension suggests the base
is complementary to the ddNTP present. The production of
oligonucleotide chains with two-base extension indicates that the
target nucleotide contains a base that is complementary to the
specific dNTP added to the reaction. For example, if in the
presence of ddCTP and dATP, but not any other nucleotides, a
two-base extension occurred in the elongation/termination reaction,
the sequences of the target polynucleotide at the potential
SNP/point mutation site and 5' adjacent site should read as T and G
in DNA or U and G in RNA respectively.
[0019] In one specific preferred embodiment, a set of three
reaction and a control are carried out in parallel. The four
reactions mixes are different from one another in the content of
dNTP. For example, the first has dATP, the second dTTP, the third
dGTP, and the fourth, or the control, has no dNTP, while all four
reactions contain ddCTP. The occurrence of incorporation of ddCTP
in labelled and unlabelled form in any of the reactions indicates
that a base complementary to the dNTP present in the reaction
occupies the position of potential SNP/point mutation site in the
target DNA. If chain extension occurred even in the absence of any
dNTP, as in the case of the control reaction, the base of the
target nucleotide is complimentary to that of the ddCTP in the
reaction (i.e. the target nucleotide contains in base G). In this
case, the chain/primer will extent for only one base but not
two.
[0020] In the case where two or three dNTPs are present in the
reaction mixture, the base at the targeted SNP/point mutation site
can be ambiguously identified to be complementary to one of the
two, or three dNTPs. This kind of experimental design can be
meaningful in some special cases.
[0021] In another embodiment, the entire procedure or some of the
reactions may be performed in solid phase. A typical procedure for
the implementation of a solid-phase mode will include essential the
following steps: template amplification, quenching/purification,
probe binding, probe extension, quenching/purification and
detection, where each step can be varied and simplified.
[0022] The principles described above are illustrated by way of the
following examples:
EXAMPLE ONE
[0023] Referring first to FIG. 1, the target polynucleotide
molecule is a human genomic DNA sequence and contains an SNP with
the base A (for ease of description, the location of the SNP on the
target polynucleotide is also referred to as the target
nucleotide). As a further example, it is known that among the human
population, the bases C, G or T also occur at this site at certain
frequencies. The sequences flanking this target nucleotide are
known. The user in this example is given an unknown sample, so he
does not know that the identity of the base of the target
nucleotide is an A, and the following description describes how the
method according to the present invention may be applied to
identify the base at this specific location for this unknown
sample.
[0024] The first step in the process is the purification of the
human genomic DNA and the separation therefrom of other
contaminating material such as cell debris. After DNA purification,
a primer, as indicated in FIG. 1, is provided for use in the
detection method according to the present invention. The primer
contains a sequence that is complimentary to the section of the
target DNA that is directly adjacent to the 3' portion of the SNP.
In this particular example, only the two bases flanking each side
of the SNP are indicated for ease of description. The other bases
are indicated as Ys in the target DNA and the complimentary bases
are indicated as Xs in the primer. In this example, 2 bases TT are
present directly adjacent the 3' side of the SNP. For ease of
description, a 5-mer is used as an example for the primer, and
contains the sequence AAXXX as shown in FIG. 2.
[0025] During the elongation/termination reaction, an appropriate
polymerase enzyme is added to the reaction mix under suitable
reaction condition for the extension of the primer such as the
presence of appropriate substrates and cofactors. For DNA
polymerase, ATP and Mg.sup.++ are provided in the reaction mix. The
components of the reaction mixtures are shown in the top row of
FIG. 2. For convenience, co-factors and reaction conditions, are
not specified. It is understood that the appropriate reaction
conditions have to be provided for chain extension to occur. These
are known in the art and may be obtained from standard laboratory
manuals such as Sambrook et al. in Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratories, CSH, NY, 1989. Since the
nucleotide directly adjacent to the 5' side of the SNP is known to
the user (in this case a G), the dideoxy nucleotide that is
selected is ddCTP. In order to determine the base at the target
location i.e. the SNP, three reaction mixes are used as shown in
row 1 of FIG. 2A. Each of these reaction mixes contains ddCTP, the
primer and the target DNA. As shown in row 1 of FIG. 2A, reaction
mixtures (a), (b) and (c) contain the nucleotides dATP, dTTP and
dGTP respectively, i.e. each reaction mix contains a nucleotide
with a unique base. The last column of FIG. 2A shows the bases that
can possibly be found at the SNP and two flanking bases on either
side of the SNP site. The base at the SNP site is highlighted
within the rectangle.
[0026] As mentioned above, the base at the SNP site for this
particular example is actually an A. Therefore, as shown in row 2,
the results is that reaction mix (b) will contain a product having
a two-base extension of the primer, while no base extension is
found in reaction mixtures (a) and (c).
[0027] To fully illustrate the power of the present invention, the
expected results of chain length analysis for each reaction mixture
if other bases are found at the target nucleotide site are shown in
rows 3-5 of FIG. 2A. In each row, the number of bases expected to
be extended and the actual sequences of the extended primer are
shown. As shown in row 3, a 7-mer would be found in reaction
mixture (a) if the target nucleotide has a base T; no base
extension is expected for tubes (b) and (c). If the target
nucleotide contains a base C, two-base extension (7-mer) would be
expected in reaction mix (c) while no reaction (i.e. only 5-mer)
would be expected in reaction mixtures (a) and (b). Finally, if the
target nucleotide contains a base G, one base is expected to be
added to the primer (i.e. 6-mer) in each of the three tubes as
shown in row 5 of FIG. 4.
[0028] From this example, it can be clearly seen that using a
parallel set of three elongation/termination reactions, the
sequence of a target nucleotide in a single nucleotide polymorphism
can be readily identified. The technique used for the chain length
or label analysis of the extended primer may be any technique that
is available in the art, including electrophoresis or mass
spectroscopy. The length of the primer is dependent on many
factors, including the base composition (which affects the melting
temperature T.sub.m value) of the sequence, reaction temperature
and hybridisation stringency required, and may be any length as
determined by the user. For detection, the dideoxy nucleotide may
be unlabelled or labelled either with a radioactive isotope, with a
fluorescence molecule or with an enzyme that can be used for
colour-based analysis. For a radioactive isotope system,
autoradiography may be performed after gel separation of the primer
and its extended products. If capillary electrophoresis or mass
spectroscopy is used, chain length may be determined without
labelling of the dideoxy nucleotide. Alternatively, fluorescence
labelling may be used.
[0029] If label analysis alone is performed without chain length
analysis, the result would simply be a yes or no answer to the
question of whether there was chain elongation of the primer. FIG.
2B shows such analysis. It is clear that using the present
invention, fully informative results may be obtained even without
chain length analysis.
EXAMPLE TWO
[0030] Example two is similar to example one except that an
amplification reaction is used before the elongation/termination
reaction for the SNP determination. In this example, a very small
quantity of target double stranded genomic DNA is isolated from a
human and polymerase chain reaction (PCR) amplification step is
first used to amplify the purified DNA sample. In this case, two
primers axe first used for the PCR reaction after genomic DNA
purification, referred to as primer 1 and primer 2. As shown in
FIG. 3A, primers 1 and 2 are complimentary to sequence 1 and
sequence 2C within the target DNA and the antisense strand
respectively.
[0031] Sequence 1 is 3' downstream of the target DNA. Sequence 2C
is located on the complimentary nucleic acid strand. Sequence 2 is
complimentary to sequence 2C and identical to primer 2, and is
located 5' upstream of the SNP. For ease of description, the
relative positions of the primers are described in relation to only
one strand (the target DNA) of the double-stranded nucleic acid
(i.e. in relation to sequence 1 and sequence 2). It is understood
that if the PCR amplification reaction is required to amplify the
signal, the primers are complimentary to opposing strands of the
double-stranded nucleic acid.
[0032] After PCR amplification reaction under appropriate
conditions known to the person of the ordinary skilled in the art,
the amplification product is purified from the unreacted primers 1
and 2 as shown in step 2 of FIG. 3A, using conventional methods
such as size exclusion chromatography
[0033] During the elongation/termination reaction (shown in step 3
of FIG. 3B), the amplification product also referred to as the
amplified target DNA containing the SNP of interest is hybridised
with primer 3. Primer 3 is complimentary to the 3' sequence
(referred to as sequence 3) directly adjacent the SNP target
nucleotide. For ease of description, only two bases (TT) directly
adjacent to the 3' side of the SNP is shown in sequence 3. The
other bases are only indicated as Xs. The Ys shown in primer 3 are
bases that are complimentary to the corresponding Xs. In this
example, the bases directly adjacent to the 5' side of the target
SNP location is a C. Therefore, the dideoxy nucleotide that should
be used for chain termination is ddGTP. Also in this example, the
base at the target nucleotide location is an A. The reaction
mixtures that are provided to give completely informative results
include a set of three parallel reactions. These three reaction
mixtures are shown in FIG. 4A. The elongation/termination reactions
are similar to those described in example one. FIG. 3B shows the
elongation/termination reaction that would occur in the presence of
ddGTP and dTTP. The normal dTTP would be added to the 3' end of
primer 3 followed by the addition of ddGTP. Upon the addition of
this dideoxy nucleotide, chain termination would occur and 2-base
extension occurs in this reaction mixture. In this example, the
dideoxyl nucleotide ddCTP is radioactively labelled with .sup.32P.
The incorporation of the radioactive nucleotide would light up the
extended primer and can be detected after separation by gel
electrophoresis or autoradiography.
[0034] To further illustrate how the present invention can be used
to analyse sequences of different bases, FIG. 4A shows the expected
results when three reaction mixes containing nucleotides having
bases differently from that of the dideoxy nucleotide are used for
the reaction. This analysis is similar to that shown in FIG. 3.
Briefly, the base of the SNP and the flanking region around is
shown in the right most column. The expected oligomers to be found
within the three separate tubes are indicated in the space below
each reaction mixture. Briefly, a two-base extension in the mixture
(b) indicates that the sequence of the target nucleotide is an A
while a two-base extension in the (c) reaction mixture indicate
that the SNP nucleotide is a G. If one base extension is found in
all three reaction mixtures, then the sequence nucleotide is a C.
If two bases extension is found in the (a) reaction mixture, then
the sequence of the target nucleotide is a T.
[0035] Alternatively, the incorporation of labled ddGTP into
polynucleotide can be used as an indication that extension has
occurred in a particular reaction, without the need for chain
length analysis. Expected results are shown in FIG. 4B and are
similar to those found in FIG. 3B.
EXAMPLE THREE
[0036] Example three is similar to Example two except that the 5'
end of primer 3 is immobilized onto a solid surface. After PCR
amplification similar to the one described in Example two, the
reaction mixture is quenched with SAP (shrimp alkaline phosphatase)
and Exol, or purified by gel electrophoresis to remove dNTPs and
amplification primers. The amplified template is then denatured and
annealed with the immobilized primer 3.
[0037] The elongation and termination reaction is initiated by
adding the appropriate dideoxynucleotide to an extension mixture.
In this case, the base directly adjacent to the 5' side of the SNP
is a C and ddGTP is provided in the elongation/termination
reaction. In addition, dATP is added to allow the polymerase to
extend the primer across the target nucleotide if the target
nucleotide is a T. In order for the reaction to be fully
informative even if four nucleotides are possibly found in the
target nucleotide sequence, three consecutive extension reactions
may be performed using different dideoxynucleotides, with a
quenching step after each extension reaction.
[0038] As an illustration, the first extension reaction contains
all the reagents shown in column A of FIG. 4A. In this case, a
two-base extension will occur only if the target nucleotide is a T.
A one-base extension will occur if the target nucleotide is a G. A
detector sensitive to labelled molecules e.g. a fluorescence array
scanner or reader can then be used to detect the chain extension
product.
[0039] If no chain extension is detected, a second extension
reaction may be performed. Before the second extension reaction is
performed, the solid surface may be purged or rinsed to eliminate
free ddGTP and dATP. This is followed by the second extension
reaction in which dTTP and additional ddGTP are added to the
reaction mixture. This allows the extension of the immobilized
primer 3 if the base of the target nucleotide is an A. The reaction
is again followed by detection for chain extension.
[0040] If no chain extension is detection in the second reaction, a
further third extension reaction may be performed. This is again
preceded by purging or removal of any free nucleotides. This third
extension reaction is performed using dCTP and additional ddGTP.
Results would be similar to those found in FIGS. 4A and 4B, except
that the three reactions are performed consecutively rather than in
parallel. In this way, only one DNA chip is needed to give a
completely informative result.
[0041] It is clear from the description above that many reaction
combinations may be designed based on the present invention. Thus,
while the present invention is specifically described with
reference to the afore-mentioned examples, it should be understood
that these examples are for illustration only and should not be
taken as limitation on the invention. It is contemplated that many
changes and modifications may be made by one of ordinary skilled in
the art without departing from the spirit and the scope of the
invention described. The primers used in the examples are extremely
short for ease of illustration. As discussed above, the length of
the primers may vary according to the user's needs.
[0042] For example, although the second example describes PCR
reaction as an amplification step, followed by nucleic acid
purification to separate the primers from the amplified products,
it should be understood that should priers 1 and 2 be designed in
such a way as to be distinguishable from primer 3, then it becomes
unnecessary to have the purification step. For example, if primers
1 and 2 are of a length that is substantially longer than primer 3
and dideoxy chain termination occurs directly after polymerization
of primers 1 and 2, then even with a 2 base-extension of primer 3
in the subsequent dideoxy polymerization step, a technique that is
capable of distinguishing between the three different primers and
their dideoxy extension products would be able to produce
informative data without the purification step after DNA
amplification. In general, if size detection is used to distinguish
the dideoxy extension products, then a primer 3 of less than 50
bases would be preferred, as most mass spectroscopic method work
well only with DNA fragments not much longer than 50 base pairs. If
capillary electrophoresis is used as the separation method for
analysing the length of the extension products, then a primer of
less than 100 bases in length may be used. Thus the optimal length
of primer 3 depends on the method of detection used and can be
determined by the end user. As a non-limiting example, a primer of
about 20 bases in length may be used for the chain
elongation/termination reaction.
[0043] The primers used in examples one and two are very short
simply for the sake of ease of illustration. It is clear that
primers of different lengths may be designed and that the length
and location of the PCR primers are dependent on the sequences of
the target nucleotide and the hybridization conditions used. In the
example shown in FIG. 3A, there are only 7 bases shown between
sequence 1 and a SNP, and 6 bases between sequence 2 and the SNP
for ease of illustration. It should be understood that the position
of sequence 2 may be any distance 5' upstream of the SNP as long as
the base directly adjacent to the 5' side of the SNP is also
amplified, and the amplified product has sufficient length for
primer 3 to anneal and for the polymerase to transcribe therefrom
ding the subsequent elongation/termination reaction. Preferably,
there should be a minimum of 100 bases between sequence 2 and the
SNP, and more preferably more than 100 bases therebetween.
[0044] The PCR reaction can be symmetrical, meaning the two
amplification primers are at roughly equal molar concentration, or
it can be asymmetrical, in which one of the primers is at about 100
molar excess and this primer can be modified at its 5' end for the
seek of immobilization in the late steps.
[0045] The position of sequence 1 should be sufficiently 3' distal
from the SNP to accommodate a third primer therebetween during the
subsequent elongation/termination reaction, Primer 3 may be, for
example, 15-55 bases long.
[0046] Also, as shown in example one, if sufficient DNA can be
obtained from the source material, it is possible to do the
elongation/termination reaction without prior DNA amplification.
Furthermore, if a sufficiently sensitive technique is used to
detect the primers after dideoxy chain extension, it is
contemplated that the reaction can be scored without prior
amplification even with the isolation of a small amount of DNA. In
addition, DNA purification is not an essential step, if specific
amplification can be performed using highly specific primers and
PCR conditions.
[0047] DNA and the use of dNTP and ddNTP are used in the
aforementioned examples. It is clear that the polymerase may be DNA
polymerase or RNA polymerase, or any other nuleic acid extender
that may be available to one in the art. Different nucleic acid
extenders may prefer different nucleotides for chain extension. It
is understood that the appropriate nucleotide co-factors and
reaction conditions for use for chain extension would be provided,
and that the appropriate chain terminating nucleotide would be
provided for chain termination. Thus, the term extender nucleotide
is used in the claims to refer to nucleotides that are used for
chain extension including 2'-deoxyribonucleoside 5'-triphospate
(dNTP), and ribonucleoside 5'-triphosphate (NTP) to distinguish
them from the chain terminating nucleotide.
[0048] It is also clear that under certain specific conditions, a
single reaction mixture may provide informative results, such as a
case in which the target nucleotide and its immediate 5' neighbour
both contain the same base. For example, both are Ts. In this case,
the dideoxy nucleotide ddATP would give either a one-base or
two-base extension product. Furthermore, not all four bases are
necessarily found in every SNP polymorphism. Thus, if a particular
polymorphism consists of only two bases, then it is possible that a
reduced number of parallel reaction mixes is required to provide a
completely informative result.
[0049] For instance, if only the frequency of one specific base of
an SNP site of the target nucleotide is required to be scored,
e.g., the base G with a 5' adjacent C, a single reaction contra
dCTP and a ddGTP should in principle be sufficient to provide the
required information. However, parallel reactions containing dATP,
dTTP, and ddGTP together with the ddGTP could be included for
confirmation purposes. A control reaction with no dNTP could also
be included. Alternatively, one of the reaction mixes may be
omitted from the elongation/termination reaction to save costs
while retaining ability to give fully informative results. For
example, reaction mixture (c) in example 2 may be omitted, such
that no chain extension in either mixes (a) or (b) implies that the
target nucleotide is a G and the sequence around the SNP is CGGTT
(FIG. 4A). In other instances, the answer that is required may be a
yes or no answer, in which a particular base in a polymorphic site
is indicative of an important disease. In this case, reduced number
of parallel reaction mixes may again be possibly used to reduce the
cost of the screening.
[0050] In the case of an immobilized probe, an array of, for
example, 96 or 384 probes may be immobilized onto the solid surface
at individually defined locations. Bach extension reaction (in
Example 3 above, the elongation/termination reaction is cared out
in three consecutive extension reactions) may be carried out in a
single cycle or, preferably, carried out in multiple thermal cycles
to increase sensitivity. For example, 25 thermal cycles may be
carried out such that all the immobilized probes are properly
extended.
[0051] Post extension treatment by shrimp alkaline phosphatase
(SAP) or calf intestinal phosphatase (CIP) will remove
unincorporated ddNTPs after each thermal cycle. Post-extension
treatment with phosphatase is needed in cases, e.g. where
incorporation of fluorescence labelled ddNTP is monitored by
capillary electrophoresis, since left untreated, the unincorporated
fluorescent ddNTP's will comigrate with the fragment(s) of
interest. Removal of the 5' phosphoryl groups by phosphatase
treatment alters the migration of the incorporated fluorescent
ddNTP's and thus prohibits interference. This treatment is to be
done before the detection of extension products, and is not needed
for every thermal cycle.
[0052] The extension probe may be immobilized after the extension
reaction instead of prior to the reaction, if proper capture
methods are employed. If a single strand probe is immobilized
before extension reaction, there should be no need for purification
of amplified template. In case that the extended probe is to be
immobilized after extension reaction, isolation of the extended
probe from unincorporated ddNTP and un-extended probe can be
readily achieved without additional post-extension treatment.
[0053] It is also within the scope of the present invention to
provide a SNP scoring method in which multiple probes are
immobilized onto a single chip such that multiple SNPs representing
different polymorphisms may be scored simultaneously for genetic
material from the same individual or isolate. This may be performed
using repetitive extension reactions, an example of which was
described in Example Three above.
[0054] From the description above, it is clear that the present
invention has many advantages:
[0055] Use of only one ddNTP, but not four as in single-base primer
extension methods, reduces the cost on labeled ddNTPs, which can be
significant in large-scale and fluorescence labeled tests. This
feature also makes applicable methods accommodating only one
labeling, such as most radioactive or enzyme-linked color
labels.
[0056] Detection of the incorporation of only one kind of chain
terminating nucleotide is required, which makes the methods more
generally applicable than single-base extension methods, which
typically requires four labeled ddNTPs. Two-base extension methods
can be used on technology platforms less suitable for simultaneous
multiple signal detections. The detection can be carried out on a
solid surface such as a 96-well plastic plate or a chip. It can
also be performed in solution phase, for example using high
performance liquid chromatography or capillary electrophoresis. A
reaction mixture according to the present invention refers to
reaction in any of these environments.
[0057] Relative to the nucleic acid products of single-base primer
extension methods, products with two-base extension are more
readily to be detected by length/mass-based or
length/mass-sensitive methods.
[0058] Methods derived from this invention have most potential
being developed into widely distributable genotyping kits as a
result of its simplicity in detection as well as reduced costs in
labelling with substances such as fluorescence dye or radioactive
material.
[0059] Two-base extension methods can also be developed to suit the
needs of determination of SNP allele frequencies and association
studies in pooled DNAs, which is more sensitive to quantitative
experimental errors. Since only one ddNTP, therefore no more than
one label is used in a two-base extension method, quantification of
incorporated ddNTP should subject to less experimental variations
resulted from, e.g., differences among fluorescent dyes in
physical-chemical properties affecting quantitative detections of
labeled nucleic acid products.
[0060] Since the reactions different in dNTP content are carried
out separately, e.g., in separate tubes, two-base extension methods
are less demanding on separation techniques. Only separation of one
kind of incorporated nucleotide from unincorporated ddNTP, but not
four as in case of single-base extension methods, may be
required.
[0061] In case that the methods such as fluorescence anisotropy, or
mass spectroscopy, capable of detecting in a heterogeneous mode are
employed, there should be no separation needed prior to the
one-step detection method.
* * * * *