U.S. patent application number 10/920793 was filed with the patent office on 2005-01-27 for nucleic acid analysis using non-templated nucleotide addition.
This patent application is currently assigned to Applera Corporation. Invention is credited to Bishop, Danielle, Wei, Dong.
Application Number | 20050019817 10/920793 |
Document ID | / |
Family ID | 23073791 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050019817 |
Kind Code |
A1 |
Wei, Dong ; et al. |
January 27, 2005 |
Nucleic acid analysis using non-templated nucleotide addition
Abstract
One embodiment of the invention is a method of producing an
oligonucleotide extended by a single nucleotide base. An
oligonucleotide and an extension terminating nucleotide are mixed
with an enzyme having terminal transferase activity. The reaction
produces an oligonucleotide extended by a single base. The extended
oligonucleotide may be used as a size standard for single base
extension reactions. Another embodiment of the invention is a
method of producing a mixture of oligonucleotides extended by
different single bases. An oligonucleotide, a first extension
terminating nucleotide, and a second extension terminating
nucleotide are mixed with an enzyme having terminal transferase
activity. The first and second extension terminating nucleotides
comprise different nucleotide bases and are labeled with different
labels. The identity of the different extension terminating
nucleotides (and hence the extended oligonucleotides) may be
ascertained by reference to the specific label incorporated.
Inventors: |
Wei, Dong; (Philadelphia,
PA) ; Bishop, Danielle; (Daly City, CA) |
Correspondence
Address: |
MILA KASAN, PATENT DEPT.
APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Assignee: |
Applera Corporation
Foster City
CA
|
Family ID: |
23073791 |
Appl. No.: |
10/920793 |
Filed: |
August 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10920793 |
Aug 17, 2004 |
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10109104 |
Mar 27, 2002 |
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6777189 |
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60280603 |
Mar 30, 2001 |
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Current U.S.
Class: |
435/6.12 ;
435/91.2; 536/25.32 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/68 20130101; C12P 19/34 20130101; C12Q 2525/186 20130101;
C12Q 2525/186 20130101; C12Q 2533/101 20130101; C12Q 2521/131
20130101; C12Q 2545/113 20130101; C12Q 2525/186 20130101; C12Q
1/6869 20130101; C12Q 2521/131 20130101; C12Q 1/6869 20130101; C12Q
1/68 20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 536/025.32 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 019/34 |
Claims
What is claimed is:
1. A method of producing an oligonucleotide extended by a single
nucleotide base, said method comprising, mixing an oligonucleotide
with an extension terminating nucleotide and a terminal
transferase.
2. The method of claim 1, wherein a label is attached to the
extension terminating nucleotide.
3. The method of 2, wherein the label is a fluorescent dye.
4. The method of claim 1, wherein a label is attached to the
oligonucleotide.
5. The method of claim 4, wherein the label is a fluorescent
dye.
6. A method of producing a mixture of oligonucleotides extended by
a single nucleotide base, wherein the mixture comprises
oligonucleotides extended by different single bases, said method
comprising, mixing an oligonucleotide with a first extension
terminating nucleotide, a second extension terminating nucleotide,
and a terminal transferase, wherein the first and second extension
terminating nucleotides comprise different labels and different
nucleotide bases.
7. The method according to claim 6, wherein the labels are
fluorescent dyes.
8. A kit for producing oligonucleotides extended by a single
nucleotide base, said kit comprising, an extension terminating
nucleotide, and an enzyme having terminal transferase activity.
9. The kit according to claim 8, wherein the extension terminating
nucleotides is labeled.
10. The kit according to claim 9, wherein the label is a
fluorescent label.
11. The kit according to claim 8, said kit comprising a first
extension terminating nucleotide and a second extension terminating
nucleotide, wherein the first and second extension terminating
nucleotides comprise different labels and different nucleotide
bases.
12. The kit according to claim 11, said kit comprising a four
different extension terminating nucleotides, wherein each of the
four extension terminating nucleotides has a different nucleotide
base and a different label.
13. The method of claim 12, wherein the labels are fluorescent
dyes.
14. A method of identifying the reaction product of a single
nucleotide base extension reaction on a detection instrument, said
method comprising, forming a single base oligonucleotide extension
product standard by mixing an oligonucleotide with an extension
terminating nucleotide and a terminal transferase, and resolving
the single base oligonucleotide extension product standard on a
detection instrument, whereby a signal indicative of the single
base oligonucleotide extension product standard is created.
15. The method of claim 14, comprising, forming a single base
extension reaction product standard by mixing a second
oligonucleotide with an extension terminating nucleotide and an
enzyme having DNA polymerase activity, resolving the single base
extension product on the detection instrument, whereby a signal
indicative of the single base extension product is created,
comparing the signal indicative of the single base oligonucleotide
extension product with the signal indicative of the single base
extension product.
16. A method of producing an oligonucleotide extended by a single
nucleotide base, said method comprising, mixing an oligonucleotide
with an extension terminating nucleotide and an enzyme having
terminal transferase activity, wherein the mixture does not contain
a template that permits the oligonucleotide to function as a
primer.
17. A method of producing a mixture of oligonucleotides extended by
a single nucleotide base, wherein the mixture comprises
oligonucleotides extended by different single bases, said method
comprising, mixing an oligonucleotide with a first extension
terminating nucleotide, a second extension terminating nucleotide,
and an enzyme having terminal transferase activity and an enzyme
having terminal transferase activity, wherein the first and second
extension terminating nucleotides comprise different labels and
different nucleotide bases, wherein the mixture does not contain a
template that permits the oligonucleotide to function as a
primer.
18. A method of identifying the reaction product of a single
nucleotide base extension reaction on a detection instrument, said
method comprising, forming a single base oligonucleotide extension
product standard by mixing an oligonucleotide with an extension
terminating nucleotide and an enzyme having terminal transferase
activity, wherein the mixture does not contain a template that
permits the oligonucleotide to function as a primer, and resolving
the single base oligonucleotide extension product standard on a
detection instrument, whereby a signal indicative of the single
base oligonucleotide extension product standard is created.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
10/109,104 filed Mar. 27, 2002 which claims the benefit under 35
USC .sctn. 119(e) of provisional application Ser. No. 60/280,603,
filed Mar. 30, 2001, the entire contents of each are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The invention is in the field of molecular biology.
BACKGROUND
[0003] The determination of nucleic acid base sequences is
important for both research and diagnostics. Many techniques for
determining nucleic acid base sequences have been developed over
the years, e.g., controlled chemical degradation (Maxim and
Gilbert, Proceedings of the National Academy of Sciences USA 74:
560-564 (1977), 2'3' dideoxy chain termination method (Sanger et
al. Proceedings of the National Academy of Sciences USA 74:
5463-5467 (1977). A variation of the technique of chain termination
sequencing is known as single base extension or "mini-sequencing"
is performed when nucleic acid base sequence information is
required for only a single base site adjacent to the 3' terminus
oligonucleotide primer. The technique of single base extension is
described in U.S. Pat. No. 5,856,092 and Syvanen et al. Genomics 8,
684-692(1990). A problem with single base extension techniques is
the difficulty associated with identifying the single base
extension product, particularly in an electrophoresis . Variations
in the signal produced by the single base extension product, e.g.,
as detected by an electrophoresis apparatus, may be the result of
variations in signal produced by differences between
oligonucleotide primers. The problems associated with identifying
single base extension products become particularly troublesome in
multiplexed single base extension reactions. The inventors have
provided methods, compositions, kits and software for ameliorating
these problems associated with identifying single base extension
reaction products.
SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION
[0004] One embodiment of the invention is a method of producing an
oligonucleotide extended by a single nucleotide base. An
oligonucleotide and an extension terminating nucleotide are mixed
with an enzyme having terminal transferase activity. The reaction
produces an oligonucleotide extended by a single base. The extended
oligonucleotide may be used as a size standard for single base
extension reactions.
[0005] Another embodiment of the invention is a method ofproducing
a mixture of oligonucleotides extended by different single bases.
An oligonucleotide, a first extension terminating nucleotide, and a
second extension terminating nucleotide are mixed with an enzyme
having terminal transferase activity. The first and second
extension terminating nucleotides comprise different nucleotide
bases and are labeled with different labels. The identity of the
different extension terminating nucleotides (and hence the extended
oligonucleotides) may be ascertained by reference to the specific
label incorporated.
[0006] Another embodiment of the invention is a method of
identifying the reaction products of single nucleotide base
extension reactions on a detection instrument, e.g., an automated
fluorescence detecting electrophoresis system, such as an Applied
Biosystems PRISM.RTM. 377, PRISM.RTM. 3700 or PRISM.RTM. 3100. An
oligonucleotide extension product is produced by mixing an
oligonucleotide with an extension terminating nucleotide and an
enzyme having terminal transferase activity, e.g., a terminal
transferase. The single base oligonucleotide extension product may
be used as a standard for comparison with the reaction products of
single base extension reactions produced using a DNA polymerase,
e.g., a mini-sequencing reaction product. Single base
oligonucleotide extension products produced by the enzyme having
terminal transferase activity may be resolved on a detection
instrument, e.g. an electrophoresis apparatus, so as to produce a
signal indicative of the single base extension product standard.
The signal may be used as a standard for comparison with signals
produced by the reaction products of template-dependent single base
extension reaction products.
[0007] Other embodiments of the invention are kits for performing
one or more methods of the invention. Embodiments of the subject
kits include kits that comprise a terminal transferase and one or
more extension terminating nucleotides. The extension terminating
nucleotides may be labeled with the detectable moieties, such as
fluorescent dyes.
[0008] Definitions
[0009] The term "terminal transferase" as used herein refers to an
enzyme having terminal transferase activity, but not having
significant DNA polymerase activity. The term "significant" as used
in reference to DNA polymerase activity means DNA polymerase
activity sufficient to perform a polynucleotide extension reaction
that is template dependent at level sufficient to produce
detectable amounts of template-dependent oligonucleotide extension
product from an oligonucleotide primed template. Examples of
terminal transferases include E. coli terminal transferase, calf
thymus terminal transferase, and the like. Terminal transferases
are commercially available from many companies such as Aphonix,
Finnzymes, MBI Fermentas, New England Biolabs, Promega, Panvera,
Sigma Biochemicals, and Roche Molecular Biochemicals.
[0010] The term "terminal transferase activity" as used herein
refers to the enzymatic catalysis as of a reaction in which
nucleotide triphosphates (including extension terminating
nucleotides) are covalently attached to the 3' terminus of an
oligonucleotide primer in a template independent manner. Thus, by
mixing an enzyme having terminal transferase activity within
oligonucleotide having a free 3'-OH (or functional equivalent to)
and with a nucleotide triphosphate, one or more nucleotides are
added to the 3' prime terminus of the oligonucleotide, irrespective
of the presence or absence of a template complementary to the
oligonucleotide.
[0011] The term "oligonucleotide" as used herein, unless clearly
indicated otherwise by context, broadly refers to a polymer of
natural or synthetic nucleobases, or a combination of both. The
backbone of the capture polynucleotide can be composed entirely of
"native" phosphodiester linkages, or it may contain one or modified
linkages, such as one or more phosphorothioate, phosphoramidite or
other modified linkages. As a specific example, a polynucleotide
may be a peptide nucleic acid (PNA), which contains amide
interlinkages.
[0012] Additional examples of modified bases and backbones that can
be used in conjunction with the invention, as well as methods for
their synthesis can be found, for example, in U.S. Pat. No.
6,001,983; Uhlman & Peyman, 1990, Chemical Review
90(4):544-584; Goodchild, 1990, Bioconjugate Chem. 1(3): 165-186;
Egholm et al., 1992, J. Am. Chem. Soc. 114:1895-1897; Gryaznov et
al., J. Am. Chem. Soc. 116:3143-3144, as well as the references
cited in all of the above. Common modified or synthetic nucleobases
of which polynucleotides may be composed include 3-methlyuracil,
5,6-dihydrouracil, 4-thiouracil, 5-bromouracil, 5-thorouracil,
5-iodouracil, 6-dimethyl amino purine, 6-methyl amino purine,
2-amino purine, 2,6-diamino purine, 6-amino-8-bromo purine,
inosine, 5-methyl cytosine, 7-deazaadenine, and 7-deaza guanosine.
Additional non-limiting examples of modified or synthetic
nucleobases of which the target nucleic acid may be composed can be
found in Fasman, CRC PRACTICAL HANDBOOK OF BIOCHEMISTRY AND
MOLECULAR BIOLOGY, 1985, pp. 385-392; Beilstein's Handbuch der
Organischen Chemie, Springer Verlag, Berlin and Chemical Abstracts,
all of which provide references to publications describing the
structures, properties and preparation of such nucleobases. The
term "oligonucleotide" as used herein includes oligonucleotides
that comprise additional molecules (or atoms) that have beenjoined,
either covalently or non-covalently, to an oligonucleotide. These
additional molecules (or atoms) may be attached to virtually any
site on the oligonucleotide, provided the attachment does not
prevent the oligonucleotide from being used as a substrate for the
enzyme having terminal transferase activity used in a given
embodiment of the subject methods. Examples of such additional
molecules include mobility modifier compounds such as those
described as the subject of U.S. Pat. Nos. 5,514,543, 5,777,096,
5,703,096 and 5,470,705.
[0013] The term "extension terminating nucleotide" as used herein
refers to refers to an enzymatically-incorporable nucleotide or
nucleotide analog in which the sugar moiety does not support
incorporation of subsequent nucleotides or nucleotide analogs.
Typical terminators are those in which the nucleobase is a purine,
a 7-deaza-purine, a pyrimidine, a normal nucleobase or a common
analog thereof and the sugar moiety is a pentose which includes a
3'-substituent that blocks further synthesis, such as a ddNTP.
Substituents that block further synthesis include, but are not
limited to, amino, deoxy, halogen, alkoxy and aryloxy groups.
Exemplary terminators include, but are not limited to, those in
which the sugar-phosphate ester moiety is
3'-(C.sub.1-C.sub.6)alkylribose-5'-triphosphate,
2'-deoxy-3'-(C.sub.1-C.s- ub.6) alkylribose-5'-triphosphate,
2'-deoxy-3'-(C.sub.1-C.sub.6)alkoxyribo- se-5-triphosphate,
2'-deoxy-3'-(C.sub.5-C.sub.14)aryloxyribose-5'-triphosp- hate,
2'-deoxy-3'-haloribose-5'-triphosphate,
2'-deoxy-3'-aminoribose-5'-t- riphosphate,
2',3'-dideoxyribose-5'-triphosphate or
2',3'-didehydroribose-5'-triphosphate.
[0014] The term "detection instrument" as used herein refers to an
analytical instrument capable of analyzing polynucleotides based on
the size (or weight) of the polynucleotide. Examples of such
detection instruments to include, but are not limited to,
electrophoresis instruments (included automated DNA sequencers such
as the Applied Biosystems ABI PRISM 377, ABI PRISM 310, ABI PRISM
3100, and ABI PRISM 3700) and mass spectragraphs, HPLC, and the
like.
[0015] The term "resolved" as used herein with respect to a
detection instrument refers to detection of a specific signal
indicative of a polynucleotide by the instrument. A polynucleotide
that is said to be resolved by the instrument may be, but is not
necessarily, separated or purified, from other polynucleotides in
the mixture.
[0016] The term "DNA polymerase" as used herein refers to an enzyme
capable of catalyzing in a template dependent manner the addition
of nucleotide triphosphates to the 3' terminus of an
oligonucleotide that is hybridized to a complementary template.
[0017] The term "label" as used, refers to a detectable moiety that
may be attached to a nucleotide in such a way as to permit the
addition of the nucleotide (bearing the moiety) to the 3' terminus
of an oligonucleotide in a reaction catalyzed by a DNA polymerase.
Detectable moieties produce a distinctive signal indicative of the
presence of the moiety. Examples of detectable moieties may be
fluorescent dyes, chromophores, chemiluminescent compounds,
purified isotopes, and the like.
[0018] The term "single base extension reaction" (or also
"template-dependent single base extension reaction") refers to a
method of determining the identity of a nucleotide base at a
specific location on a polynucleotide template by extending an
oligonucleotide hybridized to the template or single base at the 3'
position in a reaction catalyzed by a DNA polymerase. The extension
of the oligonucleotide is by only a single base (as opposed to
multiple bases) may be achieved by catalyzing the extension in the
presence of one or more extension terminating nucleotides and in
the absence of extendable nucleotides, e.g., 2' deoxynucleotide
triphosphates. The extension terminating nucleotides maybe
differentially labeled so as to easily identified which bases been
incorporated into the oligonucleotide. Thus, the 3' position is
extended by only a single base in a template dependent manner.
Another method of achieving single base extension is by adding a
single extendable nucleotide so that either extension with a lack
of extension is detected. Descriptions of various methods of single
base extension reactions can be found in U.S. Pat. No. 5,856,092
and Syvanen et al. Genomics 8, 684-692(1990). The oligonucleotide
that has been extended by single base in a single base extension
reaction in a template dependent manner (also referred to as
"template-dependent single base extension reaction products") is
said to be a "single base extension reaction products."
EMBODIMENTS OF THE INVENTION
[0019] Several embodiments of the invention relate to the use of
terminal transferases (and other enzymes having terminal
transferase activity) to produce oligonucleotides extended by a
single nucleotide base. These extended oligonucleotide can be used
as standards for comparison with template-dependent single base
extension reaction products. The extended oligonucleotide produced
by the enzyme having terminal transferase activity may be identical
to the products of template-dependent single base extension
reaction.
[0020] One embodiment of the invention is a method of producing an
oligonucleotide of interest extended by a single nucleotide base.
An oligonucleotide of interest is mixed with one or more extension
terminating nucleotides and an enzyme having terminal transferase
activity. The enzyme having terminal transferase activity may be a
terminal transferase. The extension terminating nucleotides may be
labeled or unlabeled. When using more than one extension
terminating nucleotides, the different chain terminating
nucleotides maybe differentially labeled so as to correspond to
different nucleotide bases on the different chain terminating
nucleotides, thereby providing a method of conveniently identifying
the base on the chain terminating nucleotide that has been
incorporated into the oligonucleotide. After mixing, the reaction
components mixture is permitted to incubate for an amount of time
sufficient to permit the formation of the desired amount of
reaction products. Suitable incubation times may be determined
through routine experimentation and may vary in accordance with
parameters such as the specific enzyme selected, the buffer
employed, the reaction component concentrations, reaction
temperature, and the like.
[0021] Another embodiment invention is a method of producing
oligonucleotides extended by a single base in which the
oligonucleotide is labeled prior to the addition of an extension
terminating nucleotide. A labeled oligonucleotide of interest is
mixed with one or more extension terminating nucleotides and an
enzyme having terminal transferase activity. The enzyme having
terminal transferase activity may be a terminal transferase. The
extension terminating nucleotides may be labeled or unlabeled. When
using more than one extension terminating nucleotides, the
different chain terminating nucleotides maybe differentially
labeled so as to correspond to different nucleotide bases on the
different chain terminating nucleotides, thereby providing a method
of conveniently identifying the base on the chain terminating
nucleotide that has been incorporated into the oligonucleotide.
After mixing, the reaction components mixture is permitted to
incubate for an amount of time sufficient to permit the formation
of the desired amount of reaction products. Suitable incubation
times may be determined through routine experimentation and may
vary in accordance with parameters such as the specific enzyme
selected, the buffer employed, the reaction component
concentrations, reaction temperature, and the like.
[0022] The extended oligonucleotide provided by the methods of the
invention may be used as standards for comparison with reaction
products of template-dependent single base extension reactions.
Meaningful comparisons may obtained by using an enzyme having
terminal transferase activity to produce the same reaction product
produced in a template-dependent single base extension reaction.
The reaction products of the subject methods employing enzymes
having terminal transferase activity may easily be designed to be
identical to the predicted reaction products of template-dependent
single nucleotide base extension reactions. For example, identity
of reaction products may be achieved by using the same
oligonucleotide and the same chain terminating nucleotides in both
reactions. While it is preferable to use standards that are
essentially identical to reaction products of template-dependent
single nucleotide base extension reactions, it is not necessary to
use such identical reaction products.
[0023] One example of a need for standards produced by the methods
of the invention is in multiplexed template dependent single
nucleotide extension reactions that are analyzed when an automated
fluorescent nucleic acid analyzer (e.g., an Applied Biosystems
3100). In a multiplexed template dependent single base extension
reaction, it may be difficult to identify specific reaction
products because of changes in mobility to different labeled
extension terminating nucleotides. The reaction products produced
by the methods of the invention may be used to mitigate this
problem.
[0024] In another embodiment of the invention, multiple different
oligonucleotide are mixed with one or more different labeled
extension terminating nucleotides and an enzyme having terminal
transferase activity. This reaction produces multiple different
reaction products. The different reaction products can be used as
standards for identification of reaction products produced in
template-dependent single base extension reactions that employ the
same oligonucleotide primers as used to produce the standard.
[0025] In addition to employing terminal transferases to catalyze
the formation of oligonucleotide extended by a single nucleotide
base, the subject methods may use other enzymes having terminal
transferase activity. Many DNA polymerases are known to have
terminal transferase activity, in embodiments of the invention
employing DNA polymerases having terminal transferase activity, it
is desirable, but not necessary, to perform the in the
non-templated addition in a reaction mixture that lacks significant
amounts of template (template as defined with respect to the
oligonucleotide primer). By omitting template, template directed
addition.
[0026] Other embodiments of the invention include kits for
producing oligonucleotide that are extended by a single base. The
extended oligonucleotides produced using the subject kits are
produced in accordance with the methods of the invention employing
enzymes having terminal transferase activity. The kits of the
invention comprise and enzyme having terminal transferase and an
extension terminating nucleotide. The enzyme having terminal
transferase activity may a terminal transferase. The kits may
comprise one or more different extension terminating nucleotides.
The different extension terminating nucleotides may be present in
separate solution or maybe present in a single or multiple
solution. The kits may also comprise a single solution comprising
one or more extension terminating nucleotides and enzyme having
terminal transferase activity. The chain extension terminating
nucleotides may be labeled.
[0027] One example of the kit of the invention is a kit comprising
a terminal transferase and four different to chain terminating
nucleotides, wearing each nucleotide comprises a different
nucleotide base (the four canonical bases A, G, C, and T) and
wherein each base is labeled with a different fluorescent dye. The
kits are reagents or sets of reagents that are placed in together
in a single package unit (or functional equivalent thereof), so as
to provide for the convenient practice of the subject methods. Kits
may supply the reagents in premeasured form so as to increased the
ease or reproduceability outperforming the subject methods. Kits
may contain instructions for performing the subject methods.
[0028] The invention, having been described above, may be better
understood by reference to the following examples. The examples are
offered, for among other reasons, to illustrate specific embodiment
of the invention and should not be construed as a limitation on the
invention.
EXAMPLES
dRhodamine Terminal Transferase Assay
[0029] 1. Completely thaw Buffer 1, Buffer 2 and dRhodamine Mix on
ice.
[0030] 2. Vortex and spin briefly.
[0031] 3. Prepare the reaction mix on ice:
[0032] 4 .mu.l dRhodamine Mix
[0033] 1 .mu.l Buffer 1
[0034] 1 .mu.l Buffer 2
[0035] 1 .mu.l primer (2 .mu.M)
[0036] 0.1 .mu.l Terminal Transferase
[0037] 2.9 l water
[0038] Total: 10 .mu.l
[0039] 4. Put the reaction tube in a thermocycler. Run
conditions:
[0040] 37.degree. C. for 15 minutes
[0041] 7.degree. C. for 10 minutes
[0042] 4.degree. C. hold
[0043] 5. Add 1 .mu.l Shrimp alkaline phosphatase; mix
thoroughly.
[0044] 6. Put the reaction tube in a thermocycler.
[0045] Run conditions:
[0046] 37.degree. C. for 60 minutes
[0047] 72.degree. C. for 15 minutes
[0048] 4.degree. C. hold
[0049] 7. Analysis on 3700 DNA Analyzer:
[0050] 1). Dilute 1 .mu.l of the final product in 5 .mu.l of
formamide.
[0051] 2). Mix 1 .mu.l of diluted final product, 0.5 .mu.l of GS120
size standard and 8.5 .mu.l of formamide.
[0052] 3). The sample can then be transferred into one well of the
96 well plate and run on 3700 along with SNaPshot products.
[0053] 8. The final result should show four colored peaks for each
primer, with each peak corresponding to the (N+1 ddNTP), which are
the same as SNaPshot.TM. products.
[0054] Incorporation by Reference
[0055] This application incorporates all publications, patents, and
patent application referenced herein in their entirety.
* * * * *