U.S. patent number RE43,096 [Application Number 10/389,663] was granted by the patent office on 2012-01-10 for tagged extendable primers and extension products.
This patent grant is currently assigned to California Institute of Technology. Invention is credited to Charles R. Connell, Leroy E. Hood, Michael W. Hunkapiller, Timothy Hunkapiller, Lloyd M. Smith.
United States Patent |
RE43,096 |
Smith , et al. |
January 10, 2012 |
Tagged extendable primers and extension products
Abstract
This invention provides a duplex comprising an oligonucleotide
primer and a template, wherein the primer is coupled chemically to
a chromophore or fluorophore so as to allow chain extension by a
polymerase. In one embodiment, the primer is extended by a
polymerase to generate the complement of the template. In a further
embodiment, the extended primer is separated from the template for
use in a number of methods, including sequencing reactions. Methods
of generating these compositions of matter are further
provided.
Inventors: |
Smith; Lloyd M. (Madison,
WI), Hood; Leroy E. (Seattle, WA), Hunkapiller; Michael
W. (San Carlos, CA), Hunkapiller; Timothy (Mercer
Island, WA), Connell; Charles R. (Redwood City, CA) |
Assignee: |
California Institute of
Technology (Pasadena, CA)
|
Family
ID: |
27557712 |
Appl.
No.: |
10/389,663 |
Filed: |
March 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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08361176 |
Dec 21, 1994 |
5821058 |
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07898019 |
Jun 12, 1992 |
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07660160 |
Feb 21, 1991 |
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07106232 |
Oct 7, 1987 |
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06722742 |
Apr 11, 1985 |
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06689013 |
Jan 2, 1985 |
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06570973 |
Jan 16, 1984 |
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Reissue of: |
08484340 |
Jun 7, 1995 |
6200748 |
Mar 13, 2001 |
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Current U.S.
Class: |
435/6.1;
536/26.6; 435/91.51; 536/24.3; 435/91.2; 435/91.1; 536/25.32 |
Current CPC
Class: |
C12Q
1/6816 (20130101); G01N 27/44726 (20130101); G01N
27/44721 (20130101); C12Q 1/6869 (20130101); C12Q
1/6869 (20130101); C12Q 2563/107 (20130101); C12Q
2535/101 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); G01N 27/447 (20060101) |
Field of
Search: |
;435/6,91.2
;536/24.33,23.1,24.3,26.6 |
References Cited
[Referenced By]
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Nov 1986 |
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WO |
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WO 86/07361 |
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Dec 1986 |
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WO |
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California Institute of Technology Motion 3 (Substantive Motion to
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California Institute of Technology Motion 4 (for Judgment Based on
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California Institute of Technology Motion 5 (to Deny Enzo Benefit
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|
Primary Examiner: Mummert; Stephanie K
Attorney, Agent or Firm: Keddie; James S. Francis; Carol L.
Bozicevic, Field & Francis, LLP
Parent Case Text
This application is a continuation of application Ser. No.
08/361,176 filed Dec. 21, 1994, now U.S. Pat. No. 5,821,058 which
is a continuation of application Ser. No. 07/898,019, filed Jun.
12, 1992, now abandoned, which is a continuation of application
Ser. No. 07/660,160, filed Feb. 21, 1991, now abandoned, which is a
continuation of application Ser. No. 07/106,232, filed Oct. 7,
1987, now abandoned, which is a CIP of application Ser. No.
06/722,742, filed Apr. 11, 1985, now abandoned, which is CIP of
application Ser. No. 06/689,013,filed Jan. 2, 1985, now abandoned,
which is a CIP of application Ser. No. 06/570,973, filed Jan. 16,
1984, now abandoned.
Claims
What is claimed is:
.[.1. A duplex comprising an oligonucleotide primer and a template,
wherein the primer is covalently coupled to a chromophore or
fluorophore so as to allow chain extension by a polymerase..].
.[.2. A duplex comprising an extended oligonucleotide primer and a
template, produced by providing a duplex according to claim 1 and
extending the oligonucleotide primer with a polymerase..].
.[.3. A single-stranded labeled polynucleotide produced by
separating the extended oligonucleotide primer from the duplex of
claim 2..].
.[.4. A set of duplexes comprising two or more of the duplexes of
claim 1..].
.[.5. A set of duplexes comprising two or more of the duplexes of
claim 2..].
.[.6. A set of polynucleotides comprising two or more
single-stranded labeled polynucleotides of claim 3..].
.[.7. A set of reagents comprising oligonucleotide primers
covalently coupled to one or more chromophores or fluorophores so
as to allow chain extension by a polymerase, and a
polymerase..].
.[.8. A single-stranded labeled polynucleotide comprising a first
portion and a second portion, wherein the first portion comprises
an oligonucleotide primer covalently coupled to a chromophore or
fluorophore; and wherein the second portion is produced by
extension of the first portion along a complementary
template..].
.[.9. The polynucleotide of claim 8, wherein the chromophore or
fluorophore is covalently coupled to the first portion through an
amine linkage..].
.[.10. The polynucleotide of claim 8, wherein the chromophore or
fluorophore is covalently coupled to the first portion at its 5'
end..].
.[.11. The duplex of claim 1, prepared by a method comprising
hybridizing an oligonucleotide primer to a template, wherein the
primer is covalently coupled to a chromophore or fluorophore so as
to allow chain extension by a polymerase..].
.[.12. The duplex of claim 11, wherein the chromophore or
fluorophore is covalently coupled to the primer through an amine
linkage..].
.[.13. The duplex of claim 11, wherein the chromophore or
fluorophore is covalently coupled to the primer at its 5'
end..].
.[.14. A single-stranded labeled polynucleotide produced by the
method comprising the steps of extending the oligonucleotide primer
of the duplex of claim 1 by a polymerase to produce a labeled
polynucleotide and separating the labeled polynucleotide from the
template..].
.[.15. The polynucleotide of claim 14, wherein the chromophore or
fluorophore is covalently coupled to the oligonucleotide through an
amine linkage..].
.[.16. The polynucleotide of claim 14, wherein the chromophore or
fluorophore is covalently coupled to the oligonucleotide at its 5'
end..].
.[.17. A chain termination DNA sequencing method comprising
extending the primer of the duplex of claim 1 by a polymerase to
produce a labeled polynucleotide, and separating the labeled
polynucleotide from the template..].
.[.18. A chain termination DNA sequencing method comprising
extending the primers of the set of duplexes of claim 4 by a
polymerase to produce a set of labeled polynucleotides..].
.[.19. The chain termination DNA sequencing method of claim 18,
wherein the set of duplexes comprises four DNA sequencing
reactions, wherein each labeled polynucleotide is distinguishable
by spectral characteristics of the chromophore or fluorophore
covalently coupled thereto..].
.[.20. The oligonucleotide primer of claim 1, wherein the primer is
DNA..].
.[.21. The oligonucleotide primer of claim 1 wherein the
chromophore or fluorophore is detectable by exposure to a
high-intensity monochromatic light source..].
.[.22. The duplex of either of claim 1 or 2, wherein the
chromophore or fluorophore is detectable by exposure to a
laser..].
.[.23. The set of duplexes of either of claim 4 or 5, wherein the
primers are DNA..].
.[.24. The set of duplexes of either of claim 4 or 5, wherein the
chromophore or fluorophore is detectable by exposure to a
high-intensity monochromatic light source..].
.[.25. The set of duplexes of either of claim 4 or 5, wherein the
chromophore or fluorophore is detectable by exposure to a
laser..].
.[.26. The set of reagents of claim 7, wherein the primers are
DNA..].
.[.27. The set of reagents of claim 7, wherein the chromophore or
fluorophore is detectable by exposure to a high-intensity
monochromatic light source..].
.[.28. The set of reagents of claim 7, wherein the chromophore or
fluorophore is detectable by exposure to a laser..].
.[.29. The polynucleotide of any of claims 14 to 16, wherein the
primer is DNA..].
.[.30. The polynucleotide of any of claims 14 to 16, wherein the
chromophore or fluorophore is detectable by exposure to a
high-intensity monochromatic light source..].
.[.31. The polynucleotide of any of claims 14 to 16, wherein the
chromophore or fluorophore is detectable by exposure tc a
laser..].
.[.32. The duplex of any of claims 11 to 13, wherein the primer is
DNA..].
.[.33. The duplex of any of claims 11 to 13, wherein the
chromophore or fluorophore is detectable by exposure to a
high-intensity monochromatic light source..].
.[.34. The duplex of any of claims 11 to 13, wherein the
chromophore or fluorophore is detectable by exposure to a
laser..].
.[.35. The duplex of either of claim 1 or 2, wherein the
chromophore or fluorophore is covalently coupled to the primer
through an amine linkage..].
.[.36. The set of duplexes of either of claim 4 or 5, wherein the
chromophore or fluorophore is covalently coupled to the primer
through an amine linkage..].
.[.37. The set of reagents of claim 7, wherein the chromophore or
fluorophore is covalently coupled to the primer through an amine
linkage..].
.[.38. The duplex of either of claim 1 or 2, wherein the
chromophore or fluorophore is covalently coupled to the primer at
its 5' end..].
.[.39. The set of duplexes of either of claim 4 or 5, wherein the
chromophore or fluorophore is covalently coupled to the primer at
its 5' end..].
.[.40. The set of reagents of claim 7, wherein the chromophore or
fluorophore is covalently coupled to the primer at its 5'
end..].
.[.41. The polynucleotide of claim 3, wherein the chromophore or
fluorophore is covalently coupled to the primer through an amine
linkage..].
.[.42. The polynucleotide of claim 3, wherein the chromophore or
fluorophore is covalently coupled to the primer at its 5'
end..].
.[.43. The polynucleotide of claim 3, wherein the chromophore or
fluorophore is detectable by exposure to a laser..].
.[.44. The set of polynucleotides of claim 6, wherein the primers
are DNA..].
.[.45. The set of polynucleotides of claim 6, wherein the
chromophore or fluorophore is detectable by exposure to a
high-intensity monochromatic light source..].
.[.46. The set of polynucleotides of claim 6, wherein the
chromophore or fluorophore is detectable by exposure to a
laser..].
.[.47. The set of polynucleotides of claim 6, wherein the
chromophore or fluorophore is covalently coupled to the primer
through an amine linkage..].
.[.48. The set of polynucleotides of claim 6, wherein the
chromophore or fluorophore is covalently coupled to the primer at
its 5' end..].
.[.49. A duplex comprising an oligonucleotide primer and a
template, wherein the primer hybridizes to a specific region of the
template and wherein the primer is covalently coupled to a
chromophore or fluorophore so as to allow chain extension by a
polymerase..].
.[.50. A plurality of identical oligonucleotide primers of defined
length and base sequences wherein each primer is covalently coupled
to a fluorophore or chromophore so as to allow chain extension by a
polymerase..].
.[.51. The plurality of claim 50 wherein said primers have a free
3' hydroxyl group..].
.[.52. The plurality of claim 51 wherein the chromophore or
fluorophore is covalently coupled to the primer at its 5'
end..].
.[.53. The plurality of claim 50 wherein said primers are coupled
to said fluorophore or chromophore by an amine linkage..].
.[.54. A composition comprising the plurality of claim 50..].
.[.55. The composition of claim 54 further comprising a
buffer..].
.[.56. A set of reagents comprising the plurality of claim 50 and a
polymerase..].
.[.57. A set of reagents comprising two or more pluralities of
oligonucleotide primers of claim SO wherein each plurality has a
different emission spectra..].
.[.58. A plurality of single-stranded labeled polynucleotides
produced by the method comprising the steps of hybridizing the
plurality of oligonucleotide primers of claim 50 to a template
thereby forming a plurality of duplexes; extending the primers of
said duplexes by a polymerase thereby forming labeled
polynucleotides; and separating said labeled polynucleotides from
said duplexes..].
.[.59. A set of single stranded labeled polynucleotides comprising
two or more pluralities of polynucleotides of claim 58, wherein
each plurality has a different emission spectra..].
.[.60. The plurality of claim 50 wherein the chromophore or
fluorophore is detectable by exposure to a high-intensity
monochromatic light source..].
.[.61. The plurality of claim 50 wherein the chromophore or
fluorophore is detectable by exposure to a laser..].
.Iadd.62. A method of nucleic acid sequence analysis, comprising
extending an oligonucleotide along a complementary strand of DNA of
a duplex by a polymerase to produce a labeled extension product,
wherein the duplex comprises the oligonucleotide specifically
hybridized to the complementary strand of DNA, and wherein the
oligonucleotide is covalently coupled to a fluorophore so as to
allow chain extension by the polymerase..Iaddend.
.Iadd.63. The method of claim 62, further comprising separating
said labeled extension product from said duplex..Iaddend.
.Iadd.64. A DNA sequencing method, comprising extending
oligonucleotides of a set of duplexes along hybridized
complementary strands of DNA by a polymerase to produce a set of
labeled extension products, wherein the set of labeled extension
products comprises two or more extension products, wherein an
extension product comprises an extended oligonucleotide
specifically hybridized to a complementary strand of DNA, thereby
producing four sets of labeled extension products, wherein the
extension products of each set are distinguishably labeled with a
different type of fluorophore from the extension products of the
other sets..Iaddend.
.Iadd.65. The method of claim 64 or claim 62, wherein the
fluorophore is covalently coupled to the oligonucleotide through an
amine linkage..Iaddend.
.Iadd.66. A mixture comprising a polymerase and a duplex, wherein
the duplex comprises an oligonucleotide specifically hybridized to
a complementary strand of DNA, wherein the oligonucleotide is
covalently coupled to a fluorophore so as to allow chain extension
by the polymerase..Iaddend.
.Iadd.67. A composition comprising four sets of oligonucleotides,
wherein oligonucleotides of each of the four sets are
distinguishably labeled with a different type of fluorophore from
the oligonucleotides of the other three sets..Iaddend.
.Iadd.68. The method of claim 64, wherein the extension products
comprise a terminal nucleotide having any one of four different
types of terminal base components, wherein substantially all
molecules of the same set of labeled extension products have the
same type of terminal base component, and substantially all
molecules of different sets of labeled extension products have
different types of terminal base components..Iaddend.
.Iadd.69. The composition of claim 67, wherein the oligonucleotides
comprise a terminal nucleotide having any one of four different
types of terminal base components, wherein substantially all
oligonucleotide molecules of the same set have the same type of
terminal base component, and substantially all oligonucleotide
molecules of different sets have different types of terminal base
components..Iaddend.
.Iadd.70. The method of claim 62, wherein substantially all
molecules of the labeled extension product individually comprise a
single fluorescent nucleotide..Iaddend.
.Iadd.71. The method of claim 64, wherein substantially all
molecules of the labeled extension products individually comprise a
single fluorescent nucleotide..Iaddend.
.Iadd.72. The mixture of claim 66, wherein substantially all
oligonucleotide molecules individually comprise a single
fluorescent nucleotide..Iaddend.
.Iadd.73. The composition of claim 67, wherein substantially all
oligonucleotide molecules of each set individually comprise a
single fluorescent nucleotide..Iaddend.
.Iadd.74. The method of claim 62, wherein substantially all
molecules of the labeled extension product are individually coupled
to a fluorophore by a single covalent linkage..Iaddend.
.Iadd.75. The method of claim 64, wherein substantially all
molecules of the labeled extension products are individually
coupled to a fluorophore by a single covalent linkage..Iaddend.
.Iadd.76. The mixture of claim 66, wherein substantially all
oligonucleotide molecules are individually coupled to a fluorophore
by a single covalent linkage..Iaddend.
.Iadd.77. The composition of claim 67, wherein substantially all
oligonucleotide molecules of each set are individually coupled to a
fluorophore by a single covalent linkage..Iaddend.
.Iadd.78. The method of claim 68, wherein substantially all
molecules of the labeled extension products individually comprise a
single fluorescent nucleotide..Iaddend.
.Iadd.79. The composition of claim 69, wherein substantially all
oligonucleotide molecules of each set individually comprise a
single fluorescent nucleotide..Iaddend.
.Iadd.80. The method of claim 74, wherein substantially all
molecules of the labeled extension product individually are
terminally labeled with a fluorophore..Iaddend.
.Iadd.81. The method of claim 75, wherein substantially all
molecules of the labeled extension products individually are
terminally labeled with a fluorophore..Iaddend.
.Iadd.82. The mixture of claim 76, wherein substantially all
oligonucleotide molecules individually are terminally labeled with
a fluorophore..Iaddend.
.Iadd.83. The composition of claim 77, wherein substantially all
oligonucleotide molecules of each set individually are terminally
labeled with a fluorophore..Iaddend.
.Iadd.84. The method of claim 68, wherein substantially all
molecules of the labeled extension products individually are
terminally labeled with a fluorophore..Iaddend.
.Iadd.85. The composition of claim 69, wherein substantially all
oligonucleotide molecules of each set individually are terminally
labeled with a fluorophore..Iaddend.
.Iadd.86. The method of claim 70, wherein substantially all
molecules of the labeled extension product individually are
terminally labeled with a fluorophore..Iaddend.
.Iadd.87. The method of claim 71, wherein substantially all
molecules of the labeled extension products individually are
terminally labeled with a fluorophore..Iaddend.
.Iadd.88. The mixture of claim 72, wherein substantially all
oligonucleotide molecules individually are terminally labeled with
a fluorophore..Iaddend.
.Iadd.89. The composition of claim 73, wherein substantially all
oligonucleotide molecules of each set individually are terminally
labeled with a fluorophore..Iaddend.
.Iadd.90. The method of claim 78, wherein substantially all
molecules of the labeled extension products individually are
terminally labeled with a fluorophore..Iaddend.
.Iadd.91. The composition of claim 79, wherein substantially all
oligonucleotide molecules of each set individually are terminally
labeled with a fluorophore..Iaddend.
.Iadd.92. The method of claim 74, wherein substantially all
molecules of the labeled extension product individually comprise a
5' terminal fluorescent nucleotide..Iaddend.
.Iadd.93. The method of claim 75, wherein substantially all
molecules of the labeled extension products individually comprise a
5' terminal fluorescent nucleotide..Iaddend.
.Iadd.94. The mixture of claim 76, wherein substantially all
oligonucleotide molecules individually comprise a 5' terminal
fluorescent nucleotide..Iaddend.
.Iadd.95. The composition of claim 77, wherein substantially all
oligonucleotide molecules of each set individually comprise a 5'
terminal fluorescent nucleotide..Iaddend.
.Iadd.96. The method of claim 84, wherein substantially all
molecules of the labeled extension products individually comprise a
5' terminal fluorescent nucleotide..Iaddend.
.Iadd.97. The composition of claim 85, wherein substantially all
oligonucleotide molecules of each set individually comprise a 5'
terminal fluorescent nucleotide..Iaddend.
.Iadd.98. The method of claim 86, wherein substantially all
molecules of the labeled extension product individually comprise a
5' terminal fluorescent nucleotide..Iaddend.
.Iadd.99. The method of claim 87, wherein substantially all
molecules of the labeled extension products individually comprise a
5' terminal fluorescent nucleotide..Iaddend.
.Iadd.100. The mixture of claim 88, wherein substantially all
oligonucleotide molecules individually comprise a 5' terminal
fluorescent nucleotide..Iaddend.
.Iadd.101. The composition of claim 89, wherein substantially all
oligonucleotide molecules of each set individually comprise a 5'
terminal fluorescent nucleotide..Iaddend.
.Iadd.102. The method of claim 90, wherein substantially all
molecules of the labeled extension products individually comprise a
5' terminal fluorescent nucleotide..Iaddend.
.Iadd.103. The composition of claim 91, wherein substantially all
oligonucleotide molecules of each set individually comprise a 5'
terminal fluorescent nucleotide..Iaddend.
.Iadd.104. The composition of claim 69, wherein substantially all
oligonucleotide molecules of each set individually comprise a 3'
terminal fluorescent nucleotide..Iaddend.
.Iadd.105. The composition of claim 73, wherein substantially all
oligonucleotide molecules of each set individually comprise a 3'
terminal fluorescent nucleotide..Iaddend.
.Iadd.106. The composition of claim 79, wherein substantially all
oligonucleotide molecules of each set individually comprise a 3'
terminal fluorescent nucleotide..Iaddend.
.Iadd.107. The method of claim 68, wherein substantially all
molecules of the labeled extension products individually comprise a
3' terminal nucleotide that is complementary to a corresponding
nucleotide on the complementary strand of DNA..Iaddend.
.Iadd.108. The composition of claim 69, wherein substantially all
oligonucleotide molecules of each set individually (i) are
specifically hybridized to a complementary strand of DNA, and (ii)
comprise a 3' terminal nucleotide that is complementary to a
corresponding nucleotide on the complementary strand of
DNA..Iaddend.
.Iadd.109. The method of claim 71, wherein substantially all
molecules of the labeled extension products individually comprise a
3' terminal nucleotide that is complementary to a corresponding
nucleotide on the complementary strand of DNA..Iaddend.
.Iadd.110. The composition of claim 73, wherein substantially all
oligonucleotide molecules of each set individually (i) are
specifically hybridized to a complementary strand of DNA, and (ii)
comprise a 3' terminal nucleotide that is complementary to a
corresponding nucleotide on the complementary strand of
DNA..Iaddend.
.Iadd.111. The method of claim 75, wherein substantially all
molecules of the labeled extension products individually comprise a
3' terminal nucleotide that is complementary to a corresponding
nucleotide on the complementary strand of DNA..Iaddend.
.Iadd.112. The composition of claim 77, wherein substantially all
oligonucleotide molecules of each set individually (i) are
specifically hybridized to a complementary strand of DNA, and (ii)
comprise a 3' terminal nucleotide that is complementary to a
corresponding nucleotide on the complementary strand of
DNA..Iaddend.
.Iadd.113. The composition of claim 79, wherein substantially all
oligonucleotide molecules of each set individually comprise a 3'
terminal nucleotide that is complementary to a corresponding
nucleotide in a complementary strand of DNA..Iaddend.
.Iadd.114. The method of claim 81, wherein substantially all
molecules of the labeled extension products individually comprise a
3' terminal nucleotide that is complementary to a corresponding
nucleotide on the complementary strand of DNA..Iaddend.
.Iadd.115. The composition of claim 83, wherein substantially all
oligonucleotide molecules of each set individually (i) are
specifically hybridized to a complementary strand of DNA, and (ii)
comprise a 3' terminal nucleotide that is complementary to a
corresponding nucleotide on the complementary strand of
DNA..Iaddend.
.Iadd.116. The method of claim 68, wherein substantially all
molecules of the labeled extension products individually comprise a
3' terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.117. The composition of claim 69, wherein substantially all
oligonucleotide molecules of each set individually comprise a 3'
terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.118. The method of claim 70, wherein substantially all
molecules of the labeled extension product individually comprise a
3' terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.119. The method of claim 71, wherein substantially all
molecules of the labeled extension products individually comprise a
3' terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.120. The composition of claim 73, wherein substantially all
oligonucleotide molecules of each set individually comprise a 3'
terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.121. The method of claim 74, wherein substantially all
molecules of the labeled extension product individually comprise a
3' terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.122. The method of claim 75, wherein substantially all
molecules of the labeled extension products individually comprise a
3' terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.123. The composition of claim 77, wherein substantially all
oligonucleotide molecules of each set individually comprise a 3'
terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.124. The method of claim 78, wherein substantially all
molecules of the labeled extension products individually comprise a
3' terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.125. The composition of claim 79, wherein substantially all
oligonucleotide molecules of each set individually comprise a 3'
terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.126. The method of claim 80, wherein substantially all
molecules of the labeled extension product individually comprise a
3' terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.127. The method of claim 81, wherein substantially all
molecules of the labeled extension products individually comprise a
3' terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.128. The composition of claim 83, wherein substantially all
oligonucleotide molecules of each set individually comprise a 3'
terminal nucleotide that is adapted to terminate polymerase
extension..Iaddend.
.Iadd.129. The composition of claim 69, further comprising a
polymerase or nucleotides adapted to terminate polymerase
extension..Iaddend.
.Iadd.130. The composition of claim 73, further comprising a
polymerase or nucleotides adapted to terminate polymerase
extension..Iaddend.
.Iadd.131. The composition of claim 77, further comprising a
polymerase or nucleotides adapted to terminate polymerase
extension..Iaddend.
.Iadd.132. The composition of claim 79, further comprising a
polymerase or nucleotides adapted to terminate polymerase
extension..Iaddend.
.Iadd.133. The composition of claim 83, further comprising a
polymerase or nucleotides adapted to terminate polymerase
extension..Iaddend.
.Iadd.134. The composition of claim 85, further comprising a
polymerase or nucleotides adapted to terminate polymerase
extension..Iaddend.
.Iadd.135. The composition of claim 89, further comprising a
polymerase or nucleotides adapted to terminate polymerase
extension..Iaddend.
.Iadd.136. The composition of claim 91, further comprising a
polymerase or nucleotides adapted to terminate polymerase
extension..Iaddend.
.Iadd.137. The method of claim 68, wherein the four different types
of terminal base components are adenosine, guanosine, thymidine and
cytosine..Iaddend.
.Iadd.138. The composition of claim 69, wherein the four different
types of terminal base components are adenosine, guanosine,
thymidine and cytosine..Iaddend.
.Iadd.139. The method of claim 81, wherein the oligonucleotides are
fluorescently labeled before being extended..Iaddend.
.Iadd.140. The method of claim 84, wherein the oligonucleotides are
fluorescently labeled before being extended..Iaddend.
.Iadd.141. The method of claim 87, wherein the oligonucleotides are
fluorescently labeled before being extended..Iaddend.
.Iadd.142. The method of claim 90, wherein the oligonucleotides are
fluorescently labeled before being extended..Iaddend.
.Iadd.143. A method of nucleic acid sequence analysis, comprising
producing the composition of claim 69, and detecting the type of
fluorophore on oligonucleotides of the composition..Iaddend.
.Iadd.144. A method of nucleic acid sequence analysis, comprising
producing the composition of claim 73, and detecting the type of
fluorophore on oligonucleotides of the composition..Iaddend.
.Iadd.145. A method of nucleic acid sequence analysis, comprising
producing the composition of claim 77, and detecting the type of
fluorophore on oligonucleotides of the composition..Iaddend.
.Iadd.146. A method of nucleic acid sequence analysis, comprising
producing the composition of claim 83, and detecting the type of
fluorophore on oligonucleotides of the composition..Iaddend.
.Iadd.147. A method of nucleic acid sequence analysis, comprising
producing the composition of claim 85, and detecting the type of
fluorophore on oligonucleotides of the composition..Iaddend.
.Iadd.148. A method of nucleic acid sequence analysis, comprising
producing the composition of claim 104, and detecting the type of
fluorophore on oligonucleotides of the composition..Iaddend.
.Iadd.149. A method of nucleic acid sequence analysis, comprising
producing the composition of claim 105, and detecting the type of
fluorophore on oligonucleotides of the composition..Iaddend.
.Iadd.150. A method of nucleic acid sequence analysis, comprising
producing the composition of claim 108, and detecting the type of
fluorophore on oligonucleotides of the composition..Iaddend.
.Iadd.151. A method of nucleic acid sequence analysis, comprising
producing the composition of claim 117, and detecting the type of
fluorophore on oligonucleotides of the composition..Iaddend.
.Iadd.152. The method of claim 68, wherein the oligonucleotides are
fluorescently labeled before being extended..Iaddend.
.Iadd.153. The method of claim 71, wherein the oligonucleotides are
fluorescently labeled before being extended..Iaddend.
.Iadd.154. The method of claim 75, wherein the oligonucleotides are
fluorescently labeled before being extended..Iaddend.
.Iadd.155. The method of claim 78, wherein the oligonucleotides are
fluorescently labeled before being extended..Iaddend.
.Iadd.156. The method of claim 93, wherein the oligonucleotides are
fluorescently labeled before being extended..Iaddend.
.Iadd.157. The method of claim 107, wherein the oligonucleotides
are fluorescently labeled before being extended..Iaddend.
.Iadd.158. The method of claim 116, wherein the oligonucleotides
are fluorescently labeled before being extended..Iaddend.
Description
BACKGROUND OF THE INVENTION
The development of reliable methods for sequence analysis of DNA
(deoxyribonucleic acid) and RNA (ribonucleic acid) has been one of
the keys to the success of recombinant DNA and genetic engineering.
When used with the other techniques of modern molecular biology,
nucleic acid sequencing allows dissection and analysis of animal,
plant and viral genomes into discrete genes with defined chemical
structure. Since the function of a biological molecule is
determined by its structure, defining the structure of a gene is
crucial to the eventual manipulation of this basic unit of
hereditary information in useful ways. Once genes can be isolated
and characterized, they can be modified to produce desired changes
in their structure that allow the production of gene
products--proteins--with different properties than those possessed
by the original proteins. Microorganisms into which the natural or
synthetic genes are placed can be used as chemical "factories" to
produce large amounts of scarce human proteins such as interferon,
growth hormone, and insulin. Plants can be given the genetic
information to allow them to survive harsh environmental conditions
or produce their own fertilizer.
The development of modem nucleic acid sequencing methods involved
parallel developments in a variety of techniques. One was the
emergence of simple and reliable methods for cloning small to
medium-sized strands of DNA into bacterial plasmids,
bacteriophages, and small animal viruses. This allowed the
production of pure DNA in sufficient quantities to allow its
chemical analysis. Another was the near perfection of gel
electrophoretic methods for high resolution separation of
oligonucleotides on the basis of their size. The key conceptual
development, however, was the introduction of methods of generating
size-nested sets of fragments cloned, purified DNA that contain, in
their collection of lengths, the information necessary to define
the sequence of the nucleotides comprising the parent DNA
molecules.
Two DNA sequencing methods are in widespread use. These are the
method of Sanger, F., Nicken, S. and Coulson, A. R. Proc. Natl.
Acad. Sci. U.S.A. 74, 5463 (1977) and the method of Maxam, A. M.
and Gilbert, W. Methods in Enzymology 65, 499-599 (1980).
The method developed by Sanger is referred to as the dideoxy chain
termination method. In the most commonly used variation of this
method, a DNA segment is cloned into a single-stranded DNA phage
such as M13. These phage DNAs can serve as templates for the primed
synthesis of the complementary strand by the Klenow fragment of DNA
polymerase I. The primer is either a synthetic oligonucleotide or a
restriction fragment isolated from the parental recombinant DNA
that hybridizes specifically to a region of the M13 vector near the
3'' end of the cloned insert. In each of four sequencing reactions,
the primed synthesis is carried out in the presence of enough of
the dideoxy analog of one of the four possible deoxynucleotides so
that the growing chains are randomly terminated by the
incorporation of these "dead-end" nucleotides. The relative
concentration of dideoxy to deoxy forms is adjusted to give a
spread of termination events corresponding to all the possible
chain lengths that can be resolved by gel electrophoresis. The
products from each of the four primed synthesis reactions are then
separated on individuals tracks of polyacrylamide gels by the
electrophoresis. Radioactive tags incorporated in the growing
chains are used to develop an autoradiogram image of the pattern of
the DNA in each electrophoresis track. The sequence of the
deoxynucleotides in the cloned DNA is determined from an
examination of the pattern of bands in the four lanes.
The method developed by Maxam and Gilbert uses chemical treatment
of purified DNA to generate size-nested sets of DNA fragments
analogous to those produced by the Sanger method. Single or
double-stranded DNA, labeled with radioactive phosphate at either
the 3' or 5' end, can be sequenced by this procedure. In four sets
of reactions, cleavage is induced at one or two of the four
nucleotide bases by chemical treatment. Cleavage involves a
three-stage process: modification of the base, removal of the
modified base from its sugar, and strand scission at that sugar.
Reaction conditions are adjusted so that the majority of
end-labeled fragments generated are in the size range (typically 1
to 400 nucleotides) that can be resolved by gel electrophoresis.
The electrophoresis, autoradiography, and pattern analysis are
carried out essentially as is done for the Sanger method. (Although
the chemical fragmentation necessarily generates two pieces of DNA
each time it occurs, only the piece containing the end label is
detected on the autoradiogram.)
Both of these DNA sequencing methods are in widespread use, and
each has several variations.
For each, the length of sequence that can be obtained from a single
set of reactions is limited primarily by the resolution of the
polyacrylamide gels used for electrophoresis. Typically, 200 to 400
bases can be read from a single set of gel tracks. Although
successful, both methods have serious drawbacks, problems
associated primarily with the electrophoresis procedure. One
problem is the requirement of the use of radiolabel as a tag for
the location of the DNA bands in the gels. One has to contend with
the short half-life of phosphorus-32, and hence the instability of
the radiolabeling reagents, and with the problems of radioactive
disposal and handling. More importantly, the nature of
autoradiography (the film image of a radioactive gel band is
broader than the band itself) and the comparison of band positions
between four different gel tracks (which may or may not behave
uniformly in terms of band mobilities) can limit the observed
resolution of bands and hence the length of sequence that can be
read from the gels. In addition, the track-to-track irregularities
make automated scanning of the autoradiograms difficult--the human
eye can presently compensate for these irregularities much better
than computers can. This need for manual "reading" of the
autoradiograms is time-consuming, tedious and error-prone.
Moreover, one cannot read the gel patterns while the
electrophoresis is actually being performed, so as to be able to
terminate the electrophoresis once resolution becomes insufficient
to separate adjoining bands, but must terminate the electrophoresis
at some standardized time and wait for the autoradiogram to be
developed before the sequence reading can begin.
.Iadd.An oligonucleotide is a short polymer consisting of a linear
sequence of four nucleotides in a defined order. The nucleotide
subunits are joined by phosphodiester linkages joining the 3'
hydroxyl moiety of one nucleotide to the 5' hydroxyl moiety of the
next nucleotide. An example of an oligonucleotide is 5'
ApCpGpTpApTpGpGpCp 3'. The letters A, C, G and T refer to the
nature of the purine of pyrimidine base coupled at the 1-position
of deoxyribose. A, adenine; C, cytosine; G, guanine; T, thymidine.
P represents the phosphodiester bond. The structure of a section of
an oligonucleotide is shown below..Iaddend.
##STR00001##
.Iadd.The single stranded oligonucleotides of this invention are
further characterized by being homogenous with respect to the
sequence of the nucleoside subunits and are of uniform molecular
weight..Iaddend.
.Iadd.Synthetic oligonucleotides are powerful tools in modern
molecular biology and recombinant DNA work. There are numerous
applications for these molecules, including a) as probes for the
isolation of specific genes based on the protein sequence of the
gene product, b) to direct the in vitro mutagenesis of a desired
gene, c) as primers for DNA synthesis on a single-stranded
template, d) as steps in the total synthesis of genes, and many
more, reviewed in Wm. R. Bahl et al, Prog. Nucl. Acid Res. Mol.
Biol., 21, 101 (1978)..Iaddend.
.Iadd.A very considerable amount of effort has therefore been
devoted to the development of efficient chemical methods for the
synthesis of such oligonucleotides. A brief review of these methods
as they have developed to the present is found in Crockett, G. C.,
Aldrichimica Acta 16(3), 47 55 (1983). The best methodology
currently available utilizes the phosphoramidite derivatives of the
nucleosides in combination with a solid phase synthetic procedure,
Matteucci et al, J. Am. Chem. Soc., 103, 3185 (1981); and Beaucage
et al, M. H. Tet. Lett., 22 (20), 1858-1862 (1981).
Oligonucleotides of length up to 30 bases may be made on a routine
basis in this matter, and molecules as long as 50 bases have been
made. Machines that employ this technology are now commercially
available..Iaddend.
.Iadd.There are other reports in the literature of the
derivitization of DNA. A modified nucleoside triphosphate has been
developed wherein a biotin group is conjugated to an aliphatic
amino group at the 5 position of uracil, Langer et al, Proc. Nat.
Acad. Sci., U.S.A., 78, 6633-6637 (1981). This nucleotide
derivative is effectively incorporate into double stranded DNA.
Once in DNA it may be bound by anti-biotin antibody which can then
be used for detection by fluorescence or enzymatic methods. The DNA
which has had biotin conjugated nucleosides incorporated therein by
the method of Langer et al is fragmented into smaller single and
double stranded pieces which are heterogeneous with respect to the
sequence of nucleoside subunits and variable in molecular weight.
Draper and Gold, Biochemistry, 19, 1774-1781 (1980), reported the
introduction of aliphatic amino groups by a bisulfite catalyzed
transamination reaction, and their subsequent reaction with the
fluorescent tag. In Draper and Gold the amino group is attached
directly to the pyrimidine base. The amino group so positioned
inhibits hydrogen bonding and for this reason, these materials are
not useful in hybridization and the like. Chu et al, Nucleic Acid
Res. 11(18), 6513-6529 (1983), have reported a method for attaching
an amine to the terminal 5' phosphate of oligonucleotides or
nucleic acids..Iaddend.
.Iadd.There are many reasons to want a method for covalently
attaching other chemical species to synthetic oligonucleotides.
Fluorescent dyes attached to the oligonucleotides permits one to
eliminate radioisotopes from the research, diagnostic and clinical
procedures in which they are used, and improve shelf-life
availability. As described in the assignee's co-pending application
for a DNA sequencing machine (Serial No. the synthesis of
fluorescent-labeled oligonucleotides permits the automation of the
DNA sequencing process..Iaddend.
The invention of the present patent application addresses these and
other problems associated with DNA sequencing procedures and is
believed to represent a significant advance in the art. The
preferred embodiment of the present invention represents a further
and distinct improvement.
SUMMARY OF THE INVENTION
Briefly, this invention comprises a novel process for the
electrophoetic analysis of DNA fragments produced in DNA sequencing
operations wherein chromophores or fluorophores are used to tag the
DNA fragments produced by the sequencing chemistry and permit the
detection and characterization of the fragments as they are
resolved by electrophoresis through a gel. The detection employs an
absorption or fluorescent photometer capable of monitoring the
tagged bands as they are moving through the gel.
This invention further comprises a novel process for the
electrophoretic analysis of DNA fragments produced in DNA
sequencing operations wherein a set of four chromophores are used
to tag the DNA fragments produced by the sequencing chemistry and
permit the detection and characterization of the fragments as they
are resolved by electrophoresis through a gel; the improvement
wherein the four different fragment sets are tagged with the
fluorophores fluorescein, Texas Red, tetramethyl rhodamine, and
7-nitrobenzofurazan.
This invention also includes a novel system for the electrophoretic
analysis of DNA fragments produced in DNA sequencing operations
comprising: a source of chromophore or fluorescent tagged DNA
fragments. a zone for containing an electrophoresis gel, means for
introducing said tagged DNA fragments to said zone; and photometric
means for monitoring or detecting said tagged DNA fragments as they
move through and are separated by said gel.
It is an object of this invention to provide a novel process for
the sequence analysis of DNA.
It is another object of our invention to provide a novel system for
the analysis of DNA fragments.
More particularly, it is an object of this invention to provide an
improved process for the sequence analysis of DNA.
These and other objects and advantages of this invention will be
apparent from the detailed description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
Turning to the drawings:
FIG. 1 is an illustration of one means of end-labeling a DNA
fragment with a fluorescent tag. Pst. I and T4 DNA ligase are
enzymes commonly used in recombinant DNA research.
FIG. 2 is a block diagram of automated DNA sequencer, gel
electrophoretic system.
FIG. 3 is a comparison of the type of data produced by DNA
sequencing of the sequence shown in FIG. 1.
FIG. 4 is a block diagram of a preferred DNA sequencer according to
this invention.
FIG. 5 shows the emission spectra for the four fluorophores used as
tags in the preferred embodiment of this invention.
FIG. 6 is a schematic diagram of a possible optical configuration
in the detector unit. P, lamp source; L1, objective lens; L2,
collimating lens; F1, UV blocking filter; F2, heat blocking filter;
F3, band pass excitation filter; F4, long pass emission filter; DM,
dichroic mirror; C, polyacrylamide gel; PMT, photomultiplier
tube.
FIG. 7 is a schematic diagram of another possible optical
configuration in the detector unit. F1 to F4 are bandpass filters
centered at the emission maximum of the different dyes. P1 to P4
are photomultiplier tubes. The excitation light is of a wavelength
such that it is not transmitted through any of the filters F1 to
F4.
DETAILED DESCRIPTION OF THE INVENTION
In the previous methods of DNA sequencing, including those based on
the Sanger dideoxy chain termination method, a single radioactive
label, phosphorus-32, is used to identify all bands on the gels.
This necessitates that the fragment sets produced in the four
synthesis reactions be run on separate gel tracks and leads to the
problems associated with comparing band mobilities in the different
tracks. This problem is overcome in the present invention by the
use of a set of four chromophores or fluorophores with different
absorption or fluorescent maxima, respectively. Each of these tags
is coupled chemically to the primer used to initiate the synthesis
of the fragment strands. In turn, each tagged primer is then paired
with one of the dideoxynucleotides and used in the primed synthesis
reaction with the Klenow fragment of DNA polymerase.
The primers must have the following characteristics. 1) They must
have a free 3' hydroxyl group to allow chain extension by the
polymerase. 2) They must be complementary to a unique region 3' of
the cloned insert. 3) They must be sufficiently long to hybridize
to form a unique, stable duplex. 4) The chromophore or fluorophore
must not interfere with the hybridization or prevent 3'-end
extension by the polymerase.
Conditions 1, 2 and 3 above are satisfied by several synthetic
oligonucleotide primers which are in general use for Sanger-type
sequencing utilizing M13 vectors.
One such primer is the 15 mer 5' CCC AG TCA CGA CGT T 3' where A,
C, G and T represent the four different nucleoside components of
DNA; A, adenosine; C, cytosine; G, guanosine; T, thymidine.
In the preferred embodiment of the present invention a set of four
fluorophores with different emission spectra, respectively, are
used. These different emission spectra are shown in FIG. 5. Each of
these tags is coupled chemically to the primer used to initiate the
synthesis of the fragment strands. In turn, each tagged primer is
then paired with one of the dideoxynucleotides and used in the
primed synthesis reaction with the Klenow fragment of DNA
polymerase.
The dyes used must have high extinction coefficients and/or
reasonably high quantum yields for fluorescence. They must have
well resolved adsorption maxima and/or emission masima.
Representative of such amino reactive dues are: fluorescein
isothiocyanage (FITC, .lamda..sub.max.sup.Ex=495,
.lamda..sub.max.sup.Em=520 ,
.epsilon..sub.495.apprxeq.8.times.10.sup.4), tetramethyl rhodamine
isothiocyanate (TMRITC, .lamda..sub.max.sup.Ex=550,
.lamda..sub.max.sup.Em=578,
.epsilon..sub.550.apprxeq.4.times.10.sup.4), and substituted
rhodamine isothiocyanate (XRITC, .lamda.=580,
.lamda..sub.max.sup.Em=604,
.epsilon..sub.580.apprxeq.8.times.10.sup.4)
where .lamda. represents the wavelength in nanometers, Ex is
excitation, Em is emission, max is maximum, and .epsilon. is the
molar extinction coefficient. These dyes have been attached to the
M13 primer and the conjugates electrophoresed on a 20%
polyacrylamide gel. The labeled,primers are visible by both their
absorption and their fluorescence in the gel. All four labeled
primers have identical electrophoretic mobilities. The dye
conjugated primers retain their ability to specifically hybridize
to DNA, as demonstrated by their ability to replace the
underivitized oligonucleotide normally used in the sequencing
reactions.
The chemistry for the coupling of the chromophoric or fluorophoric
tags is described in assignee's copending patent applications Ser.
No. 565,010, filed Dec. 20, 1983, now abandoned, and Ser. No.
709,579, filed Mar. 8, 1985, the disclosures of which are expressly
incorporated herein by reference. The strategy used is to introduce
an aliphatic amino group at the 5' terminus as the last addition in
the synthesis of the oligonucleotide primer. This reactive amino
group may then readily be coupled with a wide variety of amino
reactive fluorophores or chromophores. This approach aids
compatibility of the labeled primers with condition 4 above.
End Labeling of DNA for Use With Maxam/Gilbert Method. In the
Maxam/Gilbert method of DNA sequencing, the end of the piece of DNA
whose sequence is to be determined must be labeled. This is
conventionally done enzymatically using radioactive nucleosides. In
order to use the Maxam/Gilbert method in conjunction with the dye
detection scheme described in this invention, the DNA piece must be
labeled with dyes. One manner in which this maybe accomplished is
shown in FIG. 1. Certain restriction endonucleases generate what is
known as a 3' overhang as the product of DNA cleavage. These
enzymes generate a "sticky end," a short stretch of single stranded
DNA at the end of a piece of double stranded DNA. This region will
anneal with a complementary stretch of DNA, which may be covalently
joined to the duplex DNA with the enzyme ligase. In this manner one
of the strands is covalently linked to a detectable moiety. This
moiety may be a dye, an amino group or a protected amino group
(which could be deprotected and reacted with dye subsequent to the
chemical reactions).
Sequencing Reactions. The dideoxy sequencing reactions are
performed in the standard fashion Smith, A. J. H., Methods in
Enzymology 65, 560 580 (1980), except that the scale may be
increased if necessary to provide an adequate signal intensity in
each band for detection. The reactions are done using a different
color primer for each different reaction. No radiolabeled
nucleoside triphosphate need be included in the sequencing
reaction.
The Maxam/Gilbert sequencing reactions are performed in the usual
manner, Gil, S. F. Aldrichimica Acta 16(3), 59 61 (1983), except
that the end label is either one or four colored dyes, or a free or
protected amino group which may be reacted with dye
subsequently.
Detection. There are many different ways in which the tagged
molecules which have been separated by length using polyacrylamide
gel electrophoresis may be detected. Four illustrative modes are
described below. These are i) detection of the fluorescence excited
by light of different wavelengths for the different dyes, ii)
detection of fluorescence excited by light of the same wavelength
for the different dyes, iii) elution of the molecules from the gel
and detection by chemiluminescence, and iv) detection by the
absorption of light by molecules. In modes i) and ii) the
fluorescence detector should fulfill the following requirements. a)
The excitation light beam should not have a height substantially
greater than the height of a band. This is normally in the range of
0.1 to 0.5 mm. The use of such a narrow excitation beam allows the
attainment of maximum resolution of bands. b) The excitation
wavelength can be varied to match the absorption maxima of each of
the different dyes or can be a single narrow, high intensity light
band that excites all four fluorophores and does not overlap with
any of the fluorescence emission. c) The optical configuration
should minimize the flux of scattered and reflected excitation
light to the photodetector 14. The optical filters to block out
scattered and reflected excitation light are varied as the
excitation wavelength is varied. d) The photodetector 14 should
have a fairly low noise level and a good spectral response and
quantum efficiency throughout the range of the emission of the dyes
(500 to 600 nm for the dyes listed above). e) The optical system
for collection of the emitted fluorescence should have a high
numerical aperture. This maximizes the fluorescence signal.
Furthermore, the depth of field of the collection optics should
include the entire width of the column matrix.
Two illustrative fluorescence detection systems are diagrammed in
FIGS. 6 and 7. The system in FIG. 6 is compatible with either
single wavelength excitation or multi wavelength excitation. For
single wavelength excitation, the filter F4 is one of four band
pass filters centered at the peak emission wavelength of each of
the dyes. This filter is switched every few seconds to allow
continual monitoring of each of the four fluorophores. For multi
wavelength excitation, the optical elements F3 (excitation filter),
DM (dichroic mirror), and F4 (barrier filter) are switched
together. In this manner both the excitation light and the observed
emission light are varied. The system in FIG. 7 is a good
arrangement for the case of single wavelength excitation. This
system has the advantage that no moving parts are required, and
fluorescence from all four of the dyes may be simultaneously and
continuously monitored. A third approach (iii above) to detection
is to elute the labeled molecules at the bottom of the gel, combine
them with an agent for excitation of chemiluminescence such as 1,2
dioxetane dione, Gill, S. K. Aldrichimica Acta 16(3), 59 61 (1983);
Mellbin, G. J. Liq. Chrom. 6(9), 1603 1616 (1983), and flow the
mixture directly into a detector which can measure the emitted
light at four separate wavelengths. The background signal in
chemiluminescence is much lower than in fluorescence, resulting in
higher signal to noise ratios and increased sensitivity. Finally,
the measurement may be made by measurements of light absorption (iv
above). In this case, a light beam of variable wavelength is passed
through the gel, and the decrease in the beam intensity due to
absorption of light at the different wavelengths corresponding to
the absorption maximum of the four dyes, it is possible to
determine which dye molecule is in the light path. As disadvantage
of this type of measurement is that absorption measurements are
inherently less sensitive than fluorescence measurements.
The above-described detection system is interfaced to a computer
16. In each time interval examined, the computer 16 receives a
signal proportional to the measured signal intensity at that time
for each of the four colored tags. This information tells which
nucleotide terminates the DNA fragment of the particular length in
the observation window at that time. The temporal sequence of
colored bands gives the DNA sequence. In FIG. 3 is shown the type
of data obtained by conventional methods, as well as the type of
data obtained by the improvements described in this invention.
The following Examples are presented solely to illustrate the
invention. In the Examples, parts and percentages are by weight
unless otherwise indicated.
EXAMPLE I
Gel electrophoresis. Aliquots of the sequencing reactions are
combined and loaded onto a 5% polyacrylamide column 10 shown in
FIG. 2 from the upper reservoir 12. The relative amounts of the
four different reactions in the mixture are empirically adjusted to
give approximately the same fluorescence or absorptive signal
intensity from each of the dye DNA conjugates. This permits
compensation for differences in dye extinction coefficients, dye
fluorescence quantum yields, detector sensitivities and so on. A
high voltage is placed across the column 10 so as to electrophorese
the labeled DNA fragments through the gel. The labeled DNA segments
differing in length by a single nucleotide are separated by
electrophoresis in this gel matrix. At or near the bottom of the
gel column 10, the bands of DNA are resolved from one another and
pass through the detector 14 (more fully described above). The
detector 14 detects the fluorescent or chromophoric bands of DNA in
the gel and determines their color, and therefore to which
nucleotide they correspond. This information yields the DNA
sequence.
EXAMPLE II
FIG. 4 shows a block diagram of a DNA sequenator for use with one
dye at a time. The beam (4880 A) from an argon ion laser 100 is
passed into the polyacrylamide gel tube (sample) 102 by means of a
beamsteerer 104. Fluorescence exited by the beam is collected using
a low f-number lens 106, passed through an appropriate set of
optical filters 108 and 110 to eliminate scattered excitation light
and detected using a photomultiplier tube (PMT) 112. The signal is
readily detected on a strip chart recorder. DNA sequencing
reactions are carried out utlizing a fluorescein labeled
oligonucletide primer. The peaks on the chart correspond to
fragments to fluorescein labeled DNA of varying lengths synthesized
in the sequencing reactions and separated in the gel tube by
electrophoresis. Each peak contains on the order of 10.sup.-15 to
10.sup.-16 moles of fluorescein, which is approximately equal to
the amount of DNA obtained per band in an equivalent sequencing gel
utilizing radioisotope detection. This proves that the fluorescent
tag is not removed or degraded from the oligonucleotide primer in
the sequencing reactions. It also demonstrates that the detection
sensitivity is quite adequate to perform DNA sequence analysis by
this means.
Materials
Fluorescein-5-isothiocyanate (FITC) and Texas Red were obtained
from Molecular Probes, Inc. (Junction City, Oreg.). tetramethyl
rhodamine isothiocyanate (TMRITC) was obtained from Research
Organics, Inc. (Cleveland, Ohio.). 4-fluoro-7-nitro-benzofurazan
(NBD-fluoride) was obtained from Sigma Chemical Co. (St. Louis,
Mo.). Absorption spectra were obtained on a H/P 8491
spectrophotometer. High performance liquid chromatography was
performed on a system composed of two Altex 110A pumps, a dual
chamber gradient mixer, Rheodyne injector, Kratos 757 UV detector,
and an Axxiom 710 controller.
EXAMPLE III
Addition of 5'-aminothymidine phosphoramidites to
oligonucleotides.
The protected 5'-aminothymidine phosphoramidites,
5'-(N-9-fluorenylmethyloxycarbonyl)-5'-amino-5'-deoxy-3'-N,
N-diisopropylaminomethoxyphosphinyl thymidine, is coupled to the
5'-hydroxyl of an oligonucleotide using well established DNA
synthetic procedures. The solvents and reaction conditions used are
identical to those used in oligonucleotide synthesis.
EXAMPLE IV
Dye Conjugation
The basic procedure used for the attachment of fluorescent dye
molecules to the amino oligonucleotides is to combine the amino
oligonucleotide and the dye in aqueous solution buffered to pH 9,
to allow the reaction to stand at room temperature for several
hours, and then to purify the product in two stages. The first
purification step is to remove the bulk of the unreacted or
hydrolyzed dye by gel filtration. The second purification stage is
to separate the dye conjugate from unreacted oligonucleotide by
reverse phase high performance liquid chromatography. Slight
variations upon these conditions are employed for the different
dyes, and the specific procedures and conditions used for four
particular dyes are given below and in Table 1.
TABLE-US-00001 TABLE 1 Reverse Phase HPLC Conditions for
Dye-oligonucleotide Purification Sample Retention time PLP-15.sup.a
18' PLP-15-T-NH.sub.2.sup.b 18' FITC PLP-15.sup.c 27' NBD PLP-15
25' TMRITC PLP-15 32' and 34'.sup.d Texas Red PLP-15 42' Retention
limes shown are for HPLC gradients of 20% solvent B/80% solvent A
to 60% solvent B/40% solvent A in 40 min., where solvent A is 0.1 M
triethylammonium acetate pH 7.0 and solvent B is 50% acelonitrile,
50% 0.1 M triethylammonium acetate pH 7.0. The column was an Axxiom
ODS 5 micron C 18 column #555-102 available from Cole Scientific,
Calabasas, CA. This gradient is not optimized tor purification of
PLP-15 and PLP-15-T-NH.sub.2, but the retention times are included
for comparison with the dye primer conjugates. .sup.aPLP-15 is an
oligonucleotide primer for DNA sequence analysis in the M13
vectors. Its sequence is 5'CCC AGT CAC GAC FTT 3'.
.sup.bPLP-15-T-NH.sub.2 is the oligonucleotide PLP-15 to which a
5'-amino-5'-deoxythymidine base has been added to==at the 5'
terminus. .sup.cThe nomenclature Dye PLP-15 signifies the conjugate
of PLP-15-T-NH.sub.2 and the dye molecule. .sup.dTwo fluorescent
oligonucleotide products were obtained with TMRITC. Both were
equally effective in sequencing. This is presumed to be due to the
two isomers of TMRITC which are present in the commercially
available material.
The following procedure is for use with fluorescein isothiocyanate
or 4-fluoro-7-nitro-benzofurazan. Amino oligonucleotide (0.1 ml of
.about.1 mg/ml oligonucleotide in water) is combined with 1 M
sodium carbonate/bicarbonate buffer pH 9 (50 .mu.l), 10 mg/ml dye
in dimethylformamide (20 .mu.l) and H.sub.2O (80 .mu.l). This
mixture is kept in the dark at room temperature for several hours.
The mixture is applied to a 10 ml column of Sephadex G-25 (medium)
and the colored band of material eluting in the excluded volume is
collected. The column is equilibrated and run in water. In control
reactions with underivatized oligonucleotides, very little if any
dye is associated with the oligonucleotide eluting in the void
volume. The colored material is further purified by reverse phase
high performance liquid chromatography on an Axxiom C.sub.18 column
(#555-102, Cole Scientific, Calabasas, Calif.) in a linear gradient
of acetonitrile:0.1 M triethylammonium acetate, pH 7.0. It is
convenient for this separation to run the column eluant through
both a UV detector (for detecting the DNA absorbance) and a
fluorescence detector (for detecting the dye moiety). The desired
product is a peak on the chromatogram which is both strongly UV
absorbing and strongly fluorescent. The dye oligonucleotide
conjugates elute at higher acetonitrile concentrations than the
oligonucleotides alone, as shown in Table 1. The oligonucleotide is
obtained from the high performance liquid chromatographyin solution
in a mixture of acetonitrile and 0.1 M triethylammonium acetate
buffer. This is removed by lyophilization and the resulting
material is redissolved by vortexing in 10 mM sodium hydroxzide
(for a minimum amount of time) followed by neutralization with a
five fold molar excess (to sodium hydroxide) of Tris buffer, pH
7.5.
The conjugation with Texas Red is identical to that described for
fluorescein isothiocyanate and 4-fluoro-7-nitro-benzofurazan,
except that: a) prior to separation on Sephadex G-25 the reaction
is made 1 M in ammonium acetate and kept at room temperature for 30
minutes, and b) the Sephadex G-25 column is run in 0.1 M ammonium
acetate. This largely eliminates nonspecific binding of the dye
molecule to the oligonucleotide.
The conjugation with tetramethyl rhodamine isothiocyanate cyanate
is identical to that for Texas Red except that the reaction-is
carried out in 10 mM sodium carbonate/bicarbonate buffer, pH 9.0,
and 50% dioxane. This increases solubility of the tetramethyl
rhodamine and a much higher yield of dye oligonucleotide conjugate
is obtained.
In some cases, particularly with the rhodamine-like dyes, a
substantial amount of nonspecific binding of dye was observed, as
manifested by an inappropriately large dye absorption present in
the material eluted from the gel filtration column. In these cases
the material was concentrated and reapplied to a second gel
filtration column prior to high performance liquid chromatography
purification. This generally removed the majority of the
noncovalently associated dye.
EXAMPLE V
Properties of Dye-Oligonucleotide Conjugates
The development of chemistry for the synthesis of dye
oligonucleotide conjugates allows their use as primers in DNA
sequence analysis. Various fluorescent dye primers have been tested
by substituting them for the normal primer in DNA sequence analysis
by the enzymatic method. An autoradiogram of a DNA sequencing gel
in which these dye-conjugated primers were utilized in T reactions
in place of the normal oligonucleotide primer was prepared. This
autoradiogram was obtained by conventional methods employing
.alpha.-.sup.32P-dCTP as a radiolabel. The autoradiogram showed
that the underivitized primer, amino-derivitized primer, and dye
conjugated primers all give the same pattern of bands
(corresponding to the DNA sequence), indicating that the
derivitized primers retain their ability to hybridize specifically
to the complementary strand. Secondly, the bands generated using
the different primers differ in their mobilities, showing that it
is indeed the dye-primers which are responsible for the observed
pattern, and not a contaminant of unreacted or underivitized
oligonucleotide. Thirdly, the intensity of the bands obtained with
the different primers is comparable, indicating that the strength
of hybridization is not significantly perturbed by the presence of
the dye molecules.
The separations are again carried out in an acrylamide gel column.
The beam from an argon ion laser is passed into the polyacrylamide
gel tube (sample) by means of a beamsteerer. Fluorescence exited by
the beam is collected using a low f-number lens, passed through an
appropriate set of optical filters to eliminate scattered
excitation light and detected using a photomultiplier tube (PMT).
The signal is monitored on a strip chart recorder. DNA sequencing
reactions have been carried out utilizing each of the four
different dye coupled oligonucleotide primers. In each case a
series of peaks are observed on the chart paper. The peaks
correspond to fragments of dye labeled DNA of varying lengths
synthesized in the sequencing reactions and separated in the gel
tube by electrophoresis. Each peak contains of the order of
10.sup.-14 to 10.sup.-16 moles of dye, which is approximately equal
to the amount of DNA obtained per band in an equivalent sequencing
gel utilizing radioisotope detection. This proves that the
fluorescent tag is not removed or degraded from the oligonucleotide
primer in the sequencing reactions. It also demonstrates that the
detection sensitivity is quite adequate to perform DNA sequence
analysis by this means, and that adequate resolution of the DNA
fragments is obtained in a tube gel system.
Having fully described the invention it is intended that it be
limited only by the lawful scope of the appended claims.
* * * * *