METHODS FOR THE ELIMINATION OF DNA SEQUENCING ARTIFACTS

PROCEDES D'ELIMINATION D'ARTEFACTS DE SEQUENCAGE D'ADN

English Abstract

The present invention relates to improvements in methods of DNA sequencing. In particular, the invention relates to the elimination
of stops or pauses in chain termination methods of DNA sequencing by the addition of nitrogen-containing organic compounds such as
betaine, trimethylamine N-oxide and dimethylglycine. The invention also provides for DNA sequencing kits containing these compounds.
The invention also provides for improvements in other laboratory procedures using DNA polymerases, such as polymerase chain reaction
(PCR).

French Abstract

L'invention concerne des procédés améliorés de séquençage d'ADN. Elle concerne, en particulier, l'élimination d'arrêts ou de pauses dans des procédés de séquençage d'ADN par terminaison de la chaîne réalisés par addition de composés organiques contenant de l'azote, tels que la bétaïne, le N-oxyde de triméthylamine et la diméthylglycine. Elle concerne également des trousses de séquençage d'ADN contenant lesdits composés. Elle concerne enfin des améliorations apportées à d'autres procédés de laboratoire selon lesquels on utilise des ADN polymérases, tels que la PCR.

Claims

27
WHAT IS CLAIMED IS:
1. A method of decreasing the incidence of DNA
polymerase stops occurring in a reaction mixture containing a
DNA polymerase comprising adding to the reaction mixture an
amount of a compound of the formula:
<IMG>
wherein:
R1, R 2 and R3 may be the same or different and are
independently selected from the group consisting of hydrogen,
methyl, ethyl, and propyl, with the proviso that no more than
two of R1, R2 and R3 are hydrogen; and
X is a moiety selected from the group consisting of:
(a) =O, and
<IMG>
wherein:
R4 is selected from the group consisting of methyl
and hydrogen; or R4 forms a pyrrolidine ring with
R1;
R5 is selected from the group consisting of -CO2H
and -SO3H; and
n is an integer of from 0 to 2;
with the proviso that, when R1 and R4 form a pyrrolidine
ring, no more than one of R2 and R3 is hydrogen, and
wherein the compound is added in an amount effective to
decrease the incidence of DNA polymerase stops.

28
2. The method of claim 1, wherein R1, R2 and R3 are
the same or different and selected from the group consisting
of methyl, ethyl and hydrogen with the proviso that no more
than two of R1, R2 and R3 are hydrogen and, when R1 and R4 form
a pyrrolidine ring, no more than one of R2 and R3 is hydrogen.
3. The method of claim 1 or 2, wherein X is
-CH2CO2H.
4. The method of claim 1, 2 or 3, wherein R1, R2
and R3 are methyl.
5. The method of claim 1, 2 or 3, wherein R1, R2
are methyl and R3 is hydrogen.
6. The method of claim 1, 2 or 3, wherein R1 is
methyl and R2 and R3 are hydrogen.
7. The method of claim 1 or 2, wherein X is =O.
8. The method of claim 7, wherein R1, R2 and R3 are
methyl.
9. The method of claim 1 or 2, wherein R1 and R4
form a pyrrolidine ring, R2 and R3 are methyl, n is 0, and R5
is -C02H.
10. The method of claim 1 or 2, wherein R1, R2 and
R3 are methyl arid X is -CH2SO3H.
11. The method of claim 1, wherein the compound is
trimethylglycine.

29
12. The method of any one of claims 1 to 11,
wherein said reaction mixture is a reaction mixture for a
chain termination method of DNA sequencing.
13. The method of any one of claims 1 to 11,
wherein said chain termination method of DNA sequencing is a
dideoxy DNA sequencing method.
14. The method of any one of claims 1 to 11,
wherein said reaction mixture is a PCR reaction mixture.
15. The method of any one of claims 1 to 14,
wherein said reaction mixture comprises unpurified DNA.
16. The method of claim 15, wherein said unpurified
DNA is a crude cell lysate.
17. The method of any one of claims 1 to 16,
wherein the reaction mixture comprises an infectious DNA.
18. The method of any one of claims 1 to 17,
wherein the reaction mixture comprises a DNA molecule having
trinucleotide repeats where the trinucleotides are GC rich.
19. A method of decreasing the incidence of DNA
polymerase stops in a chain termination DNA sequencing method
comprising the steps of:
(a) combining in an aqueous solution, a DNA
molecule; a DNA. polymerase capable of producing a nucleic acid
complementary to a portion of said DNA molecule by using the
DNA molecule as a template; a mixture of deoxyribonucleoside
triphosphates; a chain elongation inhibitor; and an amount of
a compound of the formula:

30
<IMG>
wherein:
R1, R2 and R3 may be the same or different and are
independently selected from the group consisting of hydrogen,
methyl, ethyl, and propyl, with the proviso that no more than
two of R1, R2 and R3 are hydrogen; and
X is a moiety selected from the group consisting of:
( a ) =O, and
<IMG>
wherein:
R4 is selected from the group consisting of methyl
and hydrogen; or R4 forms a pyrrolidine ring with
R1;
R5 is selected from the group consisting of -CO2H
and -SO3H; and
n is an integer of from 0 to 2;
with the proviso that, when R1 and R4 form a pyrrolidine
ring, no more than one of R1 and R3 is hydrogen, and
wherein the compound is added in an amount effective to
decrease the incidence of DNA polymerase stops, to form a
reaction mixture; and
(b) incubating the reaction mixture to permit the
DNA polymerase to form nucleic acid fragments of varying
length by using the DNA molecule as a template, wherein said
nucleic acid fragments are complementary to said DNA molecule.

31
20. The method of claim 19, wherein said chain
elongation inhibitors are 2',3'-dideoxyribonucleoside
triphosphates.
21. The method of claim 19 or 20, wherein R1, R2
and R3 are the same or different and selected from the group
consisting of methyl, ethyl and hydrogen with the proviso that
no more than two of R1, R2 and R3 are hydrogen and, when R1 and
R4 form a pyrrolidine ring, no more than one of R2 and R3 is
hydrogen.
22. The method of claim 19, 20 or 21, wherein X is
-CH2CO2-H.
23. The method of any one of claims 19 to 22,
wherein R1, R2 and R3 are methyl.
24. The method of any one of claims 19 to 22,
wherein R1 and R2 are methyl and R3 is hydrogen.
25. The method of any one of claims 19 to 22,
wherein R1 is methyl and R2 and R3 are hydrogen.
26. The method of claim 19, 20 or 21, wherein X is
=O.
27. The method of claim 26, wherein R1, R2 and R3
are methyl.
28. The method of claim 19, 20 or 21, wherein R1
and R4 form a pyrrolidine ring, R1 and R3 are methyl, n is 0,
and R5 is -CO2H.

32
29. The method of claim 19, 20 or 21, wherein R1,
R2 and R3 are methyl and X is -CH2SO3H.
30. The method of claim 19, 20 or 21, wherein the
compound is trimethylglycine.
31. The method of any one of claims 19 to 30,
wherein said DNA molecule is unpurified DNA.
32. The method of claim 31, wherein said unpurified
DNA is a crude cell lysate.
33. The method of any one of claims 19 to 32,
wherein said DNA molecule is an infectious DNA.
34. The method of any one of claims 19 to 33,
wherein said DNA molecule has trinucleotide repeats that are
GC rich.
35. The method of any one of claims 19 to 34,
wherein step (a) further comprises the addition of a primer
complementary to a second portion of said DNA molecule, said
second portion of the DNA molecule located downstream to said
first portion of the DNA molecule, wherein said DNA polymerase
extends the 3' end of said primer to produce a nucleic acid
complementary to said first portion of the DNA molecule.
36. A kit for sequencing DNA by a chain termination
method comprising a container which contains one or more
deoxyribonucleoside triphosphates, a container which contains
a chain elongation inhibitor, and a compound of the formula:

33
<IMG>
wherein:
R1, R2 and R3 may be the same or different and are
independently selected from the group consisting of hydrogen,
methyl, ethyl, and propyl, with the proviso that no more than
two of R1, R2 and R3 are hydrogen; and
X is a. moiety selected from the group consisting of:
(a) =O, and
<IMG>
wherein:
R4 is selected from the group consisting of methyl
and hydrogen; or R4 forms a pyrrolidine ring with
R1;
R5 is selected from the group consisting of -CO2H
and -SO3H; and
n is an integer of from 0 to 2;
with the proviso that, when R1 and R 4 form a pyrrolidine
ring, no more than one of R2 and R3 is hydrogen.
37. The kit of claim 36, wherein R1, R 2 and R3 are
the same or different and selected from the group consisting
of methyl, ethyl and hydrogen with the proviso that no more
than two of R1, R2 and R3 are hydrogen and, when R1 and R4 form
a pyrrolidine ring, no more than one of R2 and R3 is hydrogen.
38. The kit of claim 36 or 37, wherein X is
-CH2CO2H.

34
39. The kit of claim 36, 37 or 38, wherein R1 and
R2 are methyl and R3 is hydrogen.
40. The kit of claim 36, 37 or 38, wherein R1 is
methyl and R2 and R3 are hydrogen.
41. The kit of claim 36, 37 or 38, wherein R1, R2
and R3 are methyl.
42. The kit of claim 36 or 37, wherein X is =O.
43. The kit of claim 42, wherein R1, R2 and R3 are
methyl.
44. The kit of claim 36 or 37, wherein R1 and R4
form a pyrrolidine ring, R2 and R3 are methyl, n is 0, and R5
is -CO2H.
45. The kit of claim 36 or 37, wherein R1, R2 and R3
are methyl and X is -CH2-SO3H.
46. The kit of claim 36, wherein the compound is
trimethylglycine.
47. The kit of any one of claims 36 to 46, further
comprising a DNA polymerase enzyme.
48. The kit of any one of claims 36 to 47, wherein
the DNA is an infectious DNA.
49. The kit of any one of claims 36 to 48, wherein
the DNA contains trinucleotide repeats that are GC rich.

35
50. A method for amplifying a target nucleotide
sequence containing trinucleotide repeats in a reaction
mixture using a Taq polymerase, the method comprising adding
trimethylglycine to the reaction mixture, wherein fewer
amplification products which do not correspond to the target
nucleotide sequence are produced than would be produced in the
absence of the trimethylglycine.
51. The method of claim 50, wherein the nucleotide
sequence being amplified is indicative of a disease state.
52. The method of claim 50 or 51, wherein the
target nucleotide sequence is a DNA.
53. A kit for amplifying a target nucleotide
sequence containing trinucleotide repeats, comprising in
separate containers:
(a) components for a Taq polymerase chain reaction;
and
(b) trimethylglycine.
54. The kit of claim 53, wherein the nucleotide
sequence being amplified is indicative of a disease state.
55. The kit of claim 53 or 54, wherein the target
nucleotide sequence is a DNA.
56. A kit for inhibiting stops during DNA
polymerization comprising:
(a) a DNA polymerase; and
(b) a compound of the formula:

36
<IMG>
wherein:
R1, R2 and R3 may be the same or different and are
independently selected from the group consisting of hydrogen,
methyl, ethyl, and propyl, with the proviso that no more than
two of R1, R2 and R3 are hydrogen; and
X is a moiety selected from the group consisting of:
( a ) =O, and
<IMG>
wherein:
R4 is selected from the group consisting of methyl
and hydrogen; or R4 forms a pyrrolidine ring with
R1;
R5 is selected from the group consisting of -CO2H
and -SO3H; and
n is an integer of from 0 to 2;
with the proviso that, when R1 and R4 form a pyrrolidine
ring, no more than one of R2 and R3 is hydrogen.
57. The kit of claim 56, wherein the compound is
trimethylglycine.
58. The kit of claim 56 or 57, wherein the kit
comprises Taq polymerase.

37
59. The kit of claim 56, 57 or 58, wherein the kit
further comprises components specific for polymerase chain
reaction.
60. A method to reduce premature chain termination
in the replication of a DNA molecule from a template DNA
molecule, the method comprising adding to a reaction mixture
of the DNA template and a DNA polymerase a chain termination
reducing compound selected from the group consisting of
trimethylglycine (betaine) and trimethylamine N-oxide (TMANO)
so as to reduce premature chain termination which would
otherwise occur in the absence of the chain termination
reducing compound.
61. A method to reduce premature chain termination
in the replication of a DNA molecule from a template DNA
molecule, the method comprising adding to a reaction mixture
of the DNA template and a DNA polymerase trimethylglycine
(betaine) so as to reduce premature chain termination which
would otherwise occur in the absence of the trimethylglycine
(betaine).
62. A method for replicating a template nucleotide
sequence containing trinucleotide repeats inhibitory to chain
elongation in a reaction mixture using a Taq polymerase, the
method comprising adding trimethylglycine to a reaction
mixture wherein fewer different replication products which do
not correspond to the template nucleotide sequence are
produced than would be produced in the absence of
trimethylglycine.
63. A kit for replicating a target nucleotide
sequence comprising:

38
(a) a DNA polymerase; and
(b) trimethylglycine.
64. A kit for replicating a target nucleotide
sequence containing trinucleotide repeats inhibitory to chain
elongation, comprising:
(a) components for a Taq polymerase DNA replication
procedure; and
(b) trimethylglycine.
65. A method for sequencing a target nucleotide
sequence containing trinucleotide repeats in a reaction
mixture, the method comprising adding trimethylglycine to the
reaction mixture wherein fewer sequencing products which do
not correspond to the target nucleotide sequence are produced
than would be produced in the absence of trimethylglycine.
66. The method of claim 65, wherein the target
nucleotide sequence is DNA.
67. The method of claim 65 or 66, wherein the
sequencing is enzymatic sequencing.

Description

CA 02181266 2006-09-29
1
METHODS FOR THE ELIlvIINATION
OF DNA SEQUENCING ARTIFACTS
This invention was made with United States
Government support awarded by the National Institutes of
Health (NIH). The United States Government has certain rights
to this invention.
BACKGROUND OF THE INVENTION
The present invention relates primarily to
improvements in methods of DNA sequencing. In particular, the
invention relates primarily to the elimination of stops or
pauses in chain termination methods of DNA sequencing by the
addition of nitrogen-containing organic compounds such as
betaine, trimethylamine N-oxide and dimethylglycine. The
invention also provides for DNA sequencing kits containing
these compounds. The invention also provides for improvements
in other laboratory procedures using DNA polymerases, such as
polymerase chain reaction (PCR).
Efficient DNA sequencing technology is very
important to the development of the biotechnology industry as
well as for basic biological research. Improvements in both
efficiency and accuracy of DNA sequencing are needed to keep
pace with the demands for DNA sequence information. The Human
Genome Project, for example, has set a goal for dramatically
increasing the efficiency, cost-effectiveness and throughput
of DNA sequencing techniques. (See Collins, F., and Galas, D.
(1993) Science 262:43-46.)
Most DNA sequencing today is carried out by a chain
termination method of DNA sequencing. The most popular chain
termination methods are variants of the dideoxynucleotide-
mediated chain termination method of Sanger (see Sanger et al.
(1977) Proc. Nat. Acad. Sci., USA 74:5463-5467). Thousands of
laboratories employ this technique including those doing
automated sequencing for the Human Genome Project. Commercial

CA 02181266 2006-09-29
2
kits containing the reagents needed for this method of DNA
sequencing are available and are widely used.
Although commonly used, the Sanger (dideoxy)
sequencing technique has problems and limitations. One of the
major problems with this method is the incidence of DNA
polymerase stops or pauses which interfere with the
determination of the DNA sequence. Stops are predominantly
problematic in regions of the DNA that are GC-rich or in
regions that are especially far from the primer. In addition,
stops occur more frequently in impure DNA preparations.
Because of this, DNA purification is generally required before
DNA can be sequenced by the dideoxy method.
Various methods have been proposed to eliminate
stops in dideoxy sequencing. For example, researchers have
tried varying the reaction temperature, using a variety of DNA
polymerases, stabilizing the DNA polymerase, and extending the
prematurely terminated DNA molecules with terminal
deoxynucleotidyl transferase (see T.W. Fawcette and S.G.
Bartlett (1990) BioTechniques 9:46-48; D. Pisa-Williamson and
C.W. Fuller (1992) United States Biochemical Corp. Comments
19:29-36; J. Sambrook, E.F. Fritsch and T. Maniatis, ed.
(1989) Molecular Cloning: A Laboratory Manual, second edition,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; and F.M.
Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman,
J.A. Smith and K. Struhl, ed. (1989) Current Protocols in
Molecular Biology, Greene Publishing Associates and Wiley-
Interscientific, John Wiley and Sons, New York.) However,
none of these methods has been reliable. There is a
continuing need to eliminate the problem of stops in.DNA
sequencing and thereby improve the efficiency and cost-
effectiveness of this important process.

CA 02181266 2006-09-29
2a
SUMMARY OF THE INVENTION
Various embodiments of this invention provide a
method of decreasing the incidence of DNA polymerase stops
occurring in a reaction mixture containing a DNA polymerase
comprising adding to the reaction mixture an amount of a
compound of formula (I) as described herein, wherein the
compound is added in an amount effective to decrease the
incidence of DNA polymerase stops.
Other embodiments of this invention provide a method
of decreasing the incidence of DNA polymerase stops in a chain
termination DNA sequencing method comprising the steps of:
(a) combining in an aqueous solution, a DNA molecule; a DNA
polymerase capable of producing a nucleic acid complementary
to a portion of said DNA molecule by using the DNA molecule as
a template; a mixture of deoxyribonucleoside triphosphates; a
chain elongation inhibitor; and an amount of a compound of
formula (I) as described herein, wherein the compound is added
in an amount effective to decrease the incidence of DNA
polymerase stops, to form a reaction mixture; and (b)
incubating the reaction mixture to permit the DNA polymerase
to form nucleic acid fragments of varying length by using the
DNA molecule as a template, wherein said nucleic acid
fragments are complementary to said DNA molecule. This method
may further comprise in step (a) the addition of a primer
complementary to a second portion of said DNA molecule, said
second portion of the DNA molecule located downstream to said
first portion of the DNA molecule, wherein said DNA polymerase
extends the 3' end of said primer to produce a nucleic acid
complementary to said first portion of the DNA molecule.
Various embodiments of this invention provide a kit
for sequencing DNA by a chain termination method comprising a
container which contains one or more deoxyribonucleoside
triphosphates, a container which contains a chain elongation

CA 02181266 2006-09-29
2b
inhibitor, and a compound of the formula (I) as described
herein.
Other embodiments of this invention provide a method
for amplifying a target nucleotide sequence containing
trinucleotide repeats in a reaction mixture using a Taq
polymerase, the method comprising adding trimethylglycine to
the reaction mixture, wherein fewer amplification products
which do not correspond to the target nucleotide sequence are
produced than would be produced in the absence of the
trimethylglycine.
Other embodiments of this invention provide a kit
for amplifying a target nucleotide sequence containing
trinucleotide repeats, comprising in separate containers:
(a) components for a Taq polymerase chain reaction; and
(b)trimethylglycine.
Other embodiments of this invention provide a kit
for inhibiting stops during DNA polymerization comprising:
(a) a DNA polymerase; and (b) a compound of the formula (I),
as described herein.
Other embodiments of this invention provide a method
to reduce premature chain termination in the replication of a
DNA molecule from a template DNA molecule, the method
comprising adding to a reaction mixture of the DNA template
and a DNA polymerase a chain termination reducing compound
selected from the group consisting of trimethylglycine
(betaine) and trimethylamine N-oxide (TMANO) so as to reduce
premature chain termination which would otherwise occur in the
absence of the chain termination reducing compound.
Other embodiments of this invention provide a method
to reduce premature chain termination in the replication of a
DNA molecule from a template DNA molecule, the method
comprising adding to a reaction mixture of the DNA template
and a DNA polymerase trimethylglycine (betaine) so as to

CA 02181266 2006-09-29
2c
reduce premature chain termination which would otherwise occur
in the absence of the trimethylglycine (betaine).
Other embodiments of this invention provide a method
for replicating a template nucleotide sequence containing
trinucleotide repeats inhibitory to chain elongation in a
reaction mixture using a Taq polymerase, the method comprising
adding trirnethylglycine to a reaction mixture wherein fewer
different replication products which do not correspond to the
template nucleotide sequence are produced than would be
produced in the absence of trimethylglycine.
Other embodiments of this invention provide a kit
for replicating a target nucleotide sequence comprising: (a)
a DNA polymerase; and (b) trimethylglycine.
Other embodiments of this invention provide a kit
for replicating a target nucleotide sequence containing
trinucleotide repeats inhibitory to chain elongation,
comprising: (a) components for a Taq polymerase DNA
replication procedure; and (b) trimethylglycine.
Other embodiments of this invention provide a method
for sequencing a target nucleotide sequence containing
trinucleotide repeats in a reaction mixture, the method
comprising addirig trimethylglycine to the reaction mixture
wherein fewer sequencing products which do not correspond to
the target nucleotide sequence are produced than would be
produced in the absence of trimethylglycine.
This invention provides for methods of decreasing
the incidence of DNA polymerase stops that occur in a reaction
mixture containing a DNA polymerase. The incidence of DNA
polymerase stops is decreased by the addition of nitrogen-
containing orgarlic molecules, which are described herein, to

WO 95/20682 2181266 PCT/US95/01200
3
the reaction mixture. These compositions of the invention are
added to the reaction mixture in an amount that is effective
to decrease the incidence of DNA polymerase stops. The
reaction mixture can be used in a chain termination method of
DNA sequencing. Preferably the chain termination method of
DNA sequencing is a dideoxynucleotide DNA sequencing method.
The reaction mixture can also be a PCR reaction mixture. The
DNA which is sequenced or amplified can be purified or
unpurified. Unpurified DNA can be present, for example, as a
crude cell lysate.
The method of decreasing the incidence of DNA
polymerase stops can be combining, in an aqueous solution, a
DNA molecule, a DNA polymerase or DNA polymerases, a mixture
of deoxyribonucleoside triphosphates, a chain elongation
inhibitor, and one or more of the compositions of the
invention to form a reaction mixture. The DNA polymerase is
capable of producing a nucleic acid complementary to a portion
of the DNA molecule by using the DNA molecule as a template.
The reaction mixture is incubated to allow the DNA polymerase
to form nucleic acid fragments of varying length by using the
DNA molecule as a template. The nucleic acid fragments that
are formed are complementary to the DNA molecule that is being
sequenced.
In some variations of the above method, a nucleic
acid primer is also added to the reaction mixture. This
primer is complementary to a second portion of one strand of
the DNA molecule that is located downstream from the first
portion of that strand. The DNA polymerase that is used is
capable of extending the 3' end of the primer to produce a
nucleic acid that is complementary to the first portion of the
DNA molecule.
The invention provides improved methods for
sequencing a DNA molecule by the chain termination method,
wherein the DNA molecule is combined with a DNA polymerase
capable of producing a nucleic acid complementary to a portion
of the DNA molecule by using the DNA molecule as a template;
with a mixture of deoxyribonucleoside triphosphates; and with
chain elongation inhibitor, to form a reaction mixture. This

A 66
WO 95/20682 PCT/US95/01200
4
reaction mixture is incubated to permit the DNA polymerase to
form nucleic acid fragments of varying length by using the DNA
molecule as a template, wherein the nucleic acid fragments are
complementary to the DNA molecule. The improved part of this
method is the addition to the reaction mixture of an amount of
one or more of the nitrogen-containing organic compounds
described herein.
The invention also provides for kits for DNA
sequencing by the chain termination method. These kits have
instructional material, a container which contains one or more
deoxyribonucleoside triphosphates, a container which contains
a chain elongation inhibitor, and an amount of the
compositions of the invention. The kits may also contain
other components. Preferably, the kits are for DNA sequencing
by the dideoxy DNA sequencing method.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure lA. DNA polymerase stops in dideoxy DNA
sequencing. Dideoxy DNA sequencing of double-stranded DNA was
carried out as described in example 1 herein, except that
water was substituted for betaine (results were identical with
or without water). The reaction mixture was separated on a
polyacrylamide/urea DNA sequencing gel as described in example
1. The positions of DNA polymerase stops are shown by arrows.
Figure 1B. Elimination of DNA polymerase stops in
dideoxy DNA sequencing by betaine. Dideoxy DNA sequencing was
carried out in the presence of betaine as described in example
1. The reaction mixture was separated on a
polyacrylamide/urea DNA sequencing gel as described in example
1. The arrows show the positions corresponding to the DNA
polymerase stops of figure 1A.
DEFINITIONS
The term "nucleic acids", as used herein, refers to
either DNA or RNA. It includes plasmids, infectious polymers
of DNA and/or RNA, nonfunctional DNA or RNA, chromosomal DNA
or RNA and DNA or RNA synthesized in vitro (such as by the
polymerase chain reaction). "Nucleic acid sequence" or

WO 95/20682 218~ 266 PCT/US95/01200
"polynucleotide sequence" refers to a single- or double-
stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end.
The term "DNA molecule" as used herein refers to DNA
5 molecules in any form, including naturally occurring,
recombinant, or synthetic DNA molecules. The term includes
plasmids, bacterial and viral DNA as well as chromosomal DNA.
The term encompasses DNA fragments produced by cell lysis or
subsequent manipulation of DNA molecules. Unless specified
otherwise, the left hand end of single-stranded DNA sequences
is the 5' end.
The term "upstream" when used in respect to a single
stranded DNA molecule, refers to those DNA sequences located
5' to a reference region of the DNA molecule. For example,
those sequences located upstream from a defined portion of a
DNA molecule are those located 5' to the defined portion.
Similarly, the term "downstream", when used with respect to
single stranded DNA molecule, refers to those DNA sequences
located 3' to a reference region of the DNA molecule.
The term "complementary" as used herein refers to a
relationship between two nucleic acid sequences. One nucleic
acid sequence is complementary to a second nucleic acid
sequence if it is capable of forming a duplex with the second
nucleic acid, wherein each residue of the duplex forms a
guanosine-cytidine (G-C) or adenosine-thymidine (A-T) basepair
or an equivalent basepair. Equivalent basepairs can include
nucleoside or nucleotide analogues other than guanosine,
cytidine, adenosine, or thymidine, which are capable of being
incorporated into a nucleic acid by a DNA or RNA polymerase on
a DNA template. A complementary DNA sequence can be predicted
from a known sequence by the normal basepairing rules of the
DNA double helix (see Watson J.D., et al. (1987) Molecular
Biology of the Gene, Fourth Edition, Benjamin Cummings
Publishing Company, Menlo Park, California, pp. 65-93).
Complementary nucleic acids may be of different sizes. For
example, a smaller nucleic acid may be complementary to a
portion of a larger nucleic acid.

WO 95/20682 ~~ ~ C PCT/US95101200 _
(~ ks V 6
The term "DNA template" or "template" as used
herein, refers to a DNA molecule or portion of a DNA molecule
that is used by a DNA or RNA polymerase to determine the
sequence of a newly synthesized nucleic acid. (See Watson,
J.D., et al., supra for a detailed description of the action
of DNA and RNA polymerases on template DNA molecules).
The term "replicating", as used herein in reference
to a DNA polymerase, refers to the process of using a DNA
molecule as a template to produce a nucleic acid complementary
to a portion of the DNA molecule.
The terms "purified DNA" or "purified DNA molecule,"
as used herein, refers to DNA that is not contaminated by
other biological macromolecules, such as RNA or proteins, or
by cellular metabolites. Purified DNA contains less than 5%
contamination (by weight) from protein, other cellular nucleic
acids and cellular metabolites. The terms "unpurified DNA" or
"unpurified DNA molecules" refer to preparations of DNA that
have greater than 5% contamination from other cellular nucleic
acids, cellular proteins and cellular metabolites. Unpurified
DNA may be obtained by using a single purification step, such
as precipitation with ethanol combined with either LiCl or
polyethylene glycol. The term "crude cell lysate preparation"
or "crude cell lysate" or "crude lysate" refers to an
unpurified DNA preparation where cells or viral particles have
been lysed but where there has been no further purification of
the DNA.
Some compounds of the invention may be present with
a positive or negative charge or with both a positive and
negative charges, depending on the pH of the solution. It is
understood that these various forms of these compounds are
included in the present invention.
The term betaine, as used herein, refers to N,N,N-
trimethylglycine.
All numerical ranges in this specification are
intended to be inclusive of their upper and lower limits.

WO 95/20682 2181266 PCT/US95/01200
7
DETAILED DESCRIPTION
A. Introduction
There are a number of different methods of
determining the sequence of DNA molecules. Most methods in
common use today are variations on one of two general methods.
One general scheme uses chemical reagents which react with
specific bases to allow other chemicals to cleave the
phosphodiester backbone at those points. The second general
method uses a polymerase enzyme to produce DNA fragments
complementary to the DNA molecule to be sequenced. The
inclusion of low amounts of chain elongation inhibitors in the
reaction mixture causes different sized DNA fragments to be
generated. The DNA fragments generated by either chemical
methods or chain termination methods can be separated and
analyzed by a variety of methods. For example, they can be
separated on a sequencing gel based on size. By examining the
bands present at any position on the gel, the sequence of the
DNA molecule can be determined at the corresponding position.
For example, the top of the sequence noted in Figure 1A
contains three consecutive G nucleotides, since bands are
present at that position in only the G lane.
Chain termination methods of DNA sequencing are much
more widely used than chemical cleavage methods.
Dideoxynucleoside triphosphates are the most common chain
terminators used in DNA sequencing. For examples of specific
protocols for dideoxy DNA sequencing, see Sambrook, et al.,
supra.
In spite of its wide use, there are technical
problems associated with the dideoxy sequencing method. One
of the major problems is the incidence of stops or pauses of
the DNA polymerase which interfere with the determination of
the DNA sequence. When the DNA polymerase stops at a
particular site, bands appear in all four lanes of the
sequencing gel, and the nucleotide residue at that position
cannot be determined.
DNA polymerase stops occur for several reasons.
Stops are observed more frequently as the enzyme extends
farther from the 3' end of the primer. This phenomenon limits

WO 95/20682 PCT/US95/01200
~k 8
the length of DNA fragment that can sequenced at one time.
Reducing the incidence of stops allows longer pieces of DNA to
be sequenced and therefore makes DNA sequencing more
efficient.
DNA polymerase stops also occur more frequently when
contaminants are present in the DNA preparation. Because of
this phenomenon, DNA preparations must be purified prior to
sequencing. The improved DNA sequencing method of the
invention decreases the incidence of stops and therefore
allows less pure preparations of DNA to be reliably sequenced.
This, in turn, increases the efficiency and reduces the cost
of DNA sequencing.
Lastly, even under optimal conditions, DNA
polymerase stops are often seen in GC-rich regions of the DNA.
This makes DNA sequence information in certain areas of a DNA
molecule difficult to determine. This problem is, of course,
compounded when one is trying to determine a DNA sequence in a
GC-rich region far from the 3' end of the primer or in a DNA
preparation that has not been optimally purified. Thus, the
improved method of the invention increases the accuracy and
efficiency of DNA sequencing while reducing its cost.
The reduced incidence of DNA polymerase stops caused
by the compounds of the invention also has utility in
procedures other than DNA sequencing. Numerous common
laboratory techniques, such as PCR (polymerase chain
reaction), in vitro mutagenesis, nick translation, reverse
transcription and blunt ending utilize DNA polymerases. The
addition of appropriate concentrations of the compounds of the
invention can potentially increase the efficiency or speed of
these procedures.
B. Compounds that reduce the incidence of DNA nolymerase
stops
The compounds used in the present invention are
nitrogen-containing organic molecules that are capable of
eliminating or reducing the incidence of stops occurring in
chain-termination methods of DNA sequencing and in other

WO 95/20682 2181 266 PCT/US95/01200
9
laboratory procedures using DNA polymerases, such as PCR.
These compounds are represented by the formula:
Rl
R2-N-X (I)
R3
wherein:
R1, R2, and R3 may be the same or different and are
independently selected from the group consisting of hydrogen,
methyl, ethyl, and propyl, with the proviso that no more than
two of R1, R2, and R3 are hydrogen; and
X is a moiety selected from the group consisting of:
radicals of the formulas
(a) =0 ; and
R4
(b) -CH- (CH2) n-R5
wherein:
R4 is selected from the group consisting of methyl,
hydrogen and, when combined with R1, forms a
pyrrolidine ring;
R5 is selected from the group consisting of -CO2H
and -SO3H; and
n is an integer of from 0 to 2; and
with the proviso that, when R1 and R4 form a pyrrolidine
ring, no more than one of R2 and R3 is hydrogen, and
wherein the composition is added in an amount effective
to decrease the incidence of stops.
When a pyrrolidine ring is formed by R1 and R4, a
compound of formula II is formed.
R2-aN (CH R5 ( I I)
~ 2~n
In certain preferred embodiments, the methods and
kits of this invention use compounds of formula I wherein R1,
R2 and R3 are the same or different and selected from the

WO 95/20682 PCT/US95/01200
group consisting of methyl, ethyl and hydrogen with the
proviso that no more than two of R1, R2 and R3 are hydrogen
and, when R1 and R4 form a pyrrolidine ring, no more than one
of R2 and R3 are hydrogen.
5 In another group of preferred embodiments, the
methods and kits of this invention use a compound of formula I
wherein X is -CH2CO2H. Further preferred embodiments within
this group use compounds where R1, R2 and R3 are methyl; where
R1, R2 are methyl and R3 is hydrogen; or where R1 is methyl
10 and R2 and R3 are hydrogen.
In further preferred embodiments, the methods and
kits of this invention use a compound of formula I wherein X
is =0 and R1, R2 and R3 are methyl.
In still further preferred embodiments, the methods
and kits of this invention use a compound of formula I wherein
Rl and R4 form a pyrrolidine ring, R2 and R3 are methyl, n is
0, and R5 is -CO2H (stachydrine, formula III).
H. (III)
H C-N~-COOH
1
CH3
In yet another group of preferred embodiments, the
methods and kits of this invention use compounds wherein R1,
R2, and R3 are methyl and X is -CH2-SO3H (sulfobetaine).
In general, the compounds used to eliminate or
reduce the incidence of DNA polymerase stops in DNA sequencing
and in other laboratory procedures using a DNA polymerase are
commercially available. For example, betaine,
dimethylglycine, sarcosine, and trimethylamine N-oxide can all
be obtained from Sigma Chemical Company (St. Louis, Missouri,
USA).
These compounds may also be synthesized by routine
methods known to those of skill in the art. For example,
compounds of formula wherein R4 is H, n is 0 and R5 is -CO2H
can be synthesized by the method of Lloyd, et al. (1992) J.
Pharm. Pharmacol. 44: 507-511. In general, ethyl

/, PCT/US95/01200
WO 95/20682 21 gl 26[/
11
chloroacetate is heated to reflux with the appropriate
tertiary amine in ethanol. When the reaction is complete, the
ethanol is removed from the reaction mixture by evaporation
under reduced pressure. The residue is dissolved in 3-6% w/v
aqueous HC1 and warmed to reflux. Evaporation of the solvent
under reduced pressure provides the desired products.
Typically, these products can be recrystallized from an
acetonitrile/water mixture.
Compounds of formula I wherein R4 is H or CH3, n is
1 and R5 is CO2H can be synthesized by the method of Fiedorek,
F.T., U.S. Patent No. 2,548,428. In brief, betalactones are
reacted with tertiary amines to provide the desired compounds.
Compounds of formula I wherein R4 is H, n is 2, and
R5 is -CO2H can be synthesized by the method of Aksnes, G., et
al. J. Chem. Soc. London 1959:103-107. In brief, 4-
bromobutyric acid (Aldrich Chemical Co., Milwaukee, Wisconsin,
USA) is converted to a methyl ester by treatment with methyl
alcohol and catalytic sulfuric acid. Subsequent treatment of
the methyl ester with excess alcoholic tertiary amine provides
the desired compounds.
Compounds of formula I wherein R4 and Rl are taken
together to form a pyrrolidine ring and where R5 is CO2H are
synthesized by the general method of Karer, et al. (1925)
Helv. Chim. Acta. 8: 364. For example, stachydrine is formed
by the methylation of proline, according to this procedure.
Compounds of formula I wherein X is =0 are
synthesized by oxidation of the corresponding tertiary amines
(see March, J. (1992) Advanced Organic Chemistry, Reactions,
Mechanisms and Structure, Fourth Edition, John Wiley and Sons,
New York, pp. 1200-1201). Typically, the oxidation is carried
out with hydrogen peroxide, but other peracids may also be
used.
Sulfobetaine can be synthesized according to the
procedure of King, J.F., et al. (1985) J. Phosphorus Sulfur
25: 11-20. Other compounds of formula I.wherein R5 is -SO3H
can also be synthesized by modifications of this procedure and
by other methods known to those of skill in the art.

---- -- ----- ---
U~
WO 95/20682 Q
Ut%PCT/US95/01200
12
C. Improved methods for DNA sequencina, polymerase chain
reaction and other laboratory procedures using DNA
polymerases
1. Chain termination methods of DNA sequencing
The present invention encompasses an improvement in
DNA sequencing by the chain termination method. As used
herein, the terms "chain termination method of DNA
sequencing," "chain termination method" or "chain termination
DNA sequencing method" refer to a DNA sequencing method that
uses a DNA polymerase to produce nucleic acid fragments
complementary to a portion of the DNA molecule to be
sequenced. Generally, a primer is used that is complementary
to a portion of the DNA molecule. The primer is extended
along the DNA molecule template by the DNA polymerase. The
principle of the method is that low amounts of specific chain
elongation inhibitors are included in the reaction mixture so
that the DNA polymerase will only occasionally incorporate an
inhibitor and terminate. Generally, four reaction mixtures
are set up, each with a different chain elongation inhibitor,
capable of specifically terminating at a guanosine, cytosine,
adenosine or thymidine residue. The DNA fragments generated
by incubation with the DNA polymerase can be separated and
analyzed to determine the sequence of the DNA molecule.
Typically, the fragments are separated by gel electrophoresis
and detected by autoradiography, although other separation or
detection methods may also be used. (See Sanger, et al.,
supra and Sambrook, et al., supra, for a more detailed
description of the chain termipation method of DNA
sequencing.) This procedure is amenable to automation, and
various specific methods have been created for that purpose,
including single lane sequencing, laser detection and
capillary electrophoresis.
The terms "chain elongation inhibitors" or "chain-
terminating inhibitors", as used herein, refer to compounds
that terminate nucleic acid chain elongation by a DNA
polymerase enzyme. As described above, these compounds may be
useful in chain termination methods of DNA sequencing.

WO 95/20682 Z181Z66 PCT/US95/01200
13
The terms "pause", "stop", "DNA polymerase pause" or
"DNA polymerase stop" as used herein refer to a phenomenon
wherein the DNA polymerase stops or does not function at a
particular nucleotide when the DNA polymerase is incubated
with DNA in a chain terminating DNA sequencing method. This
is a phenomenon in which DNA polymerase molecules fail to
continue elongation despite the presence of the next
nucleotide to be incorporated and the absence of any manifest
reason why elongation should cease (such as the incorporation
of a dideoxynucleotide). When the DNA fragments are displayed
on a DNA sequencing gel, stops appear as bands in all four
lanes of the gel at a position corresponding to the length of
the fragment that the DNA polymerase molecules had difficulty
elongating, so the identity of the nucleotide at that position
cannot be determined (see arrows in Figure 1A for examples).
There are a variety of different chain termination
methods for sequencing DNA. The most commonly used method is
the dideoxynucleotide DNA sequencing method. The terms
"dideoxynucleotide DNA sequencing method" or "dideoxy DNA
sequencing method", as used herein, refer to a chain
termination method of DNA sequencing wherein the chain
elongation inhibitors are 2'3'-dideoxynucleosides or their
derivatives. Typically, 2'3'-dideoxynucleoside triphosphates
are used. A variety of other chain elongation inhibitors may
also be used. For example, arabinonucleoside derivatives or
3' 0-methyl deoxyribonucleotide derivatives may be used in
chain termination methods. (See Sanger, et al., supra and
Axelrod, V.O., et al. (1978) N.A.R. 5:3549-3563.)
A variety of different enzymes can be used to
produce DNA fragments complementary to the DNA molecule to be
sequenced. For example, T7 DNA polymerase, Taq polymerase,
and the Klenow fragment of DNA polymerase I are all used in
DNA sequencing. RNA polymerase and reverse transcriptase have
also been used. DNA polymerases may be genetically or
physically altered to optimize the enzyme for use in DNA
sequencing.
The DNA polymerases require a primer sequence. The
primer sequences may be generated by digestion of the DNA

WO 95/20682 PCT/US95/01200
'~k$k 14
molecule or may be added exogenously as a primer molecule.
The terms "primer molecule", "primer" or "nucleic acid primer"
or "DNA primer", as used herein, refer to a single-stranded
nucleic acid molecule which is complementary to a portion of
the DNA molecule to be sequenced. Primers are allowed to
anneal to a DNA molecule, so that subsequent elongation of the
3' end of the primer by a DNA polymerase produces a nucleic
acid sequence complementary to a portion of the DNA molecule
to be sequenced. Primers are commonly used in chain
termination methods of DNA sequencing. However, in certain
modifications of DNA sequencing by the chain termination
method, primer sequences may be generated by digestion with
appropriate enzymes (see below).
In an alternative embodiment, DNA sequencing vectors
may be used to facilitate DNA sequencing by chain termination
methods. A variety of such vectors may be used, including
M13, Bluescript vectors such as pBS-KS+ and pBS-KS-
(Stratagene, San Diego, California, U.S.A.), pUC vectors such
as pUC18 and pUC19 and pBR322 vectors. The DNA molecule to be
sequenced is inserted into the sequencing vector. Primers can
be constructed that are complementary to a region of the DNA
in the sequencing vector, so that elongation from the 3' end
of the primer will produce DNA fragments complementary to
portions of the DNA molecule to be sequenced. Thus, when
sequencing vectors are used, the DNA molecule to be sequenced
can be present as an insert in the vector. Primers that are
complementary to a region of the DNA sequencing vector can be
used, so that chain elongation occurs from the 3' end of the
primer into the region of the DNA insert, thereby allowing
determination of the DNA sequence of the insert.
The DNA preparation for sequencing may be either
purified or unpurified. Unpurified DNA may be a crude cell
lysate. The compounds of the invention that eliminate or
reduce the incidence of DNA polymerase stops allow for the use
in DNA sequencing of unpurified DNA preparations, including
crude cell lysates.
The DNA preparation may be present in single-
stranded or double-stranded form. For instance, linear

WO 95/20682 PCT/US95/01200
1 15
double-stranded DNA may be generated by PCR. When such DNA is
sequenced, the incidence of DNA polymerase stops is reduced or
eliminated.
The mixture of DNA fragments generated in chain
termination methods of DNA sequencing is most commonly
separated and analyzed by gel electrophoresis. See figure 1A
and 1B herein for an example of a DNA sequencing gel. Other
separation methods useful for separating DNA fragments may
also be used (see Sambrook, et al., supra). For instance,
mass spectrometry can be used to analyze mixtures of DNA
fragments (see Jacobson, et al. (1991) J. Genetic Analysis,
Techniques and Applications 8:223-229.
The DNA fragments may be labeled or unlabeled. For
example, unlabeled DNA fragments may be detected by
ultraviolet spectroscopy in an automated technique or by
silver staining after gel electrophoresis. However, it is
generally desirable to produce labeled DNA fragments to
facilitate their detection. For instance, radiolabeled DNA
fragments may be produced. They may be produced, for example,
by use of a radioactive primer. Alternatively, radioactive
nucleotides may be incorporated during the incubation with a
DNA polymerase. When radiolabeled DNA fragments are produced,
they may be separated by gel electrophoresis and visualized by
autoradiography (see figures 1A and 1B, herein). A wide
variety of non-radioactive labels may also be used including
fluorophores, chemiluminescent agents and small detectable
molecules, such as biotin. For example, in automated
sequencing techniques, fluorescent or colorimetric labels can
be detected by laser spectroscopy using capillary tubes or
sequencing gels.
Non-radioactive labels may be attached by indirect
means. Generally, a ligand molecule (e.g., biotin) is
covalently bound to the molecule. The ligand then binds to an
anti-ligand (e.g., streptavidin) molecule which is either
inherently detectable or covalently bound to a signal system,
such as a detectable enzyme, a fluorescent compound, or a
chemiluminescent compound. A number of ligands and anti-
ligands can be used. Where a ligand has a natural anti-

WO 95/20682 PCT/US95/01200
16
ligand, for example, biotin, thyroxine, and cortisol, it can
be used in conjunction with the labelled, naturally occurring
anti-ligands. Alternatively, any haptenic or antigenic
compound can be used in combination with an antibody.
There are a variety of modifications of the chain
termination method of DNA sequencing. For example, DNA
sequencing may be performed manually, by semi-automated
procedures or by the use of an automated DNA sequencing
instrument. In addition, chain termination DNA sequencing
technology may be combined with other techniques. For
example, in "exometh" methods, a DNA molecule is treated with
exonuclease to generate a series of DNA molecules with single-
stranded 5' extensions of varying length. These DNA molecules
can then be sequenced directly by a chain termination method
of DNA sequencing without the addition of an exogenous primer.
(See Sorge, J.A., et al. (1989) Proc. Nat. Acad. Sci, USA 86:
9208-9212 for a more detailed description of DNA sequencing by
the "exometh" modification.) Such modifications of chain
termination DNA sequencing methods are all encompassed by the
present invention.
The compounds of the invention may be added to the
reaction mixture before or during the incubation of the DNA
with the DNA polymerase to eliminate or reduce the incidence
of stops. The order of addition of the DNA molecule, the DNA
polymerase, the chain elongation inhibitors, the
deoxyribonucleoside triphosphates, the primer, and the
compounds of the invention may also be varied. As
demonstrated in Example 4, the compounds that reduce or
eliminate the incidence of stops may even be added after the
normal incubation time for the DNA polymerase, and the
incubation may be extended to remove DNA polymerase stops.
Polvmerase chain reaction procedures
The present invention also encompasses an
improvement in nucleic acid amplification procedures, such as
PCR, which involve chain elongation by a DNA polymerase.
There are a variety of different PCR techniques
which'utilize DNA polymerase enzymes, such as Taq polymerase.

PCT/US95/01200
WO 95/20682 2181266
17
See PCR Protocols: A Guide to Methods and Applications.
(Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.),
Academic Press, San Diego (1990) for detailed description of
PCR methodology. In a typical PCR protocol, a target nucleic
acid, two oligonucleotide primers (one of which anneals to
each strand), nucleotides, polymerase and appropriate salts
are mixed and the temperature is cycled to allbw the primers
to anneal to the template, the polymerase to elongate the
primer, and the template strand to separate from the newly
synthesized strand. Subsequent rounds of temperature cycling
allow exponential amplification of the region between the
primers. The primers can anneal to both the original template
and the newly synthesized nucleic acid, as long as the
polymerase is able to extend at least as far as the position
to which the other primer anneals. For this reason, the
ability of PCR to amplify a product is primarily limited by
the ability of the polymerase to extend the annealed primer.
The ability of the polymerase to extend the annealed primer is
dependent on the distance between the primers and the
nucleotide composition of the sequence between them. The
addition of an appropriate amount of one or more of the
compounds described herein facilitates this elongation. See
example 8 and example 9, herein, for a demonstration of the
effects of betaine on chain elongation in PCR protocols using
Taq polymerase. Other compounds of the invention in addition
to betaine can be used in a similar manner to improve chain
elongation in a variety of different PCR methods.
The compounds of the invention also have utility in
reducing DNA polymerase stops in laboratory procedures other
than DNA sequencing and polymerase chain reactions. Numerous
common laboratory techniques, such as in vitro mutagenesis,
nick translation, reverse transcription and blunt ending
utilize DNA polymerases. See Sambrook, et al. (1989)
Molecular Cloning: A Laboratory Manual, second edition, Vol.
1-3, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)
for a description of these methods. Thus, the compounds of
the invention are useful in a variety of procedures involving
chain elongation by a DNA polymerase.

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WO 95/20682 PCT/US95/01200
18
3. Testina and optimization of comnounds that reduce or
eliminate the incidence of stops
Compounds capable of eliminating or reducing the
incidence of stops in chain termination methods of DNA
sequencing and in other laboratory procedures using DNA
polymerase are described above. These compounds may be tested
for their relative ability to eliminate or reduce DNA
polymerase stops. For instance, the compounds may be tested
by the procedure described in Example 1, herein. Briefly, DNA
sequencing is carried out by a dideoxy sequencing method in
the presence or absence of a selected concentration of a
compound of the invention. The DNA sequencing gels obtained
in the presence and absence of the compound are then compared
to determine the effectiveness of the compound in eliminating
DNA polymerase stops (see for example, figure lA and 1B).
Effective concentrations for each of the compounds may be
determined by this procedure. Optimal concentrations for a
given compound may vary for the different variations of chain
termination DNA sequencing methods. These concentrations may
be readily determined experimentally by adding different
amounts of a compound and determining the incidence of stops
(see for instance, Example 7, herein).
This invention also encompasses kits for DNA
sequencing by the chain termination method which comprise an
amount of one or more of the compounds described herein that
eliminate or reduce the incidence of DNA polymerase stops.
The kits may further comprise instructional material, a
container which contains one or more deoxyribonucleoside
triphosphates, a container which contains a DNA polymerase,
and a container that contains a chain elongation inhibitor.
The containers in the kit may be combined in various ways.
For example, a chain elongation inhibitor may be combined in
the same container with the mixture of deoxyribonucleoside
triphosphates.
This invention also encompasses kits for DNA
polymerase chain reaction which comprise an amount of one or
more of the compounds described herein that eliminate or
reduce the incidence of DNA polymerase stops. The kits may

CA 02181266 2006-09-29
19
further comprise instructional material, a container which
contains a DNA polymerase, and a container that contains
deoxyribonucleoside triphosphates.
Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Although any methods and materials similar
or equivalent to those described herein can be used in the
practice or testing of the present invention, the preferred
methods and materials are now described.
Unless mentioned otherwise, the techniques employed or
contemplated herein are standard methodologies well known to
one of ordinary skill in the art. The materials, methods and
examples are illustrative only and not limiting.
EXAMPLES
Example 1 Elimination of stops in a dideoxy method of DNA
seguencinv by betaine
Betaine eliminates stops that are routinely observed
in a standard dideoxy sequencing procedure.
Betaine monohydrate was obtained from Sigma and
stored as a 5.5M stock solution at -20 C. Modified T7
polymerase (Sequenase 2.OT"" (Amersham, Arlington Heights,
Illinois, U.S.A.)) and nucleotide mixes were purchased from
USB. 35S-dATP was purchased from Amersham.
DNA sequencing generally followed the protocol of
Del Sal et al. (1989) BioTechniques 7:514-519. Sequencing was
performed by mixing 1 picomole (pmole) of supercoiled double
stranded plasmid DNA (purified by running over a QIAGEN
tip-100 column according to the manufacturer's specifications)
with 2-4 pmole of various oligonucleotide primers (generally
purified by polyacrylamide gel electrophoresis, though this
proved unnecessary for primers under 30 nucleotides) and
adjusting the volume to 10 l containing 0.1 N NaOH. The tube
was incubated for ten minutes at 68 C, then it was moved to
room temperature and 4 141 of TDMN (200 mM NaC1, 50 mM DTT, 120
mM nCl, 80 rnM i4gCl2, 280 mM TES) were added. After a further

CA 02181266 2006-09-29
ten minute incubation at room temperature, 2 l of GTP
labeling mix (7.5 M dCTP, 7.5 M dGTP, 7.5 M dTTP) and 5 Ci
of 35S-dATP were added, followed by 2 l of Sequenase (diluted
1:8 in cold 10 mM Tris.Cl pH 8.0, 1 mM EDTA). This mix was
5 incubated for five minutes at room temperature, then 3.5 l
were aliquoted to each of four tubes preheated to 37 C, each
containing 3.5 l of 5.5 M betaine and 2.5 l of one of the
termination mixes (80 M each dNTP, 8 M one ddNTP, 50 mM
NaCl), for a final concentration of approximately 2M betaine.
10 This was incubated for five minutes at 37 C, then stopped with
4 l of stop solution (80% deionized formamide, 1X TBE, 0.05%
xylene cyanol, 0.05% bromophenol blue.) After 2.5 minutes at
95 C, the tubes were briefly chilled on ice. 4.5 l were
loaded on a 6% polyacrylamide (19:1 acrylamide:
15 bisacrylamide)/7M urea/1X TBE gel, which was run for various
periods at 35W. The gels were fixed with a 12% methanol/12%
acetic acid solution, transferred to WhatmanTM paper, and dried.
They were generally exposed overnight on Kodak XAR 5T"' film.
The addition of betaine at a concentration of 2M
20 eliminated virtually all stops when DNA sequencing was
performed using a modified form of the method of Del Sal et
al., supra, as described above. Sequencing without betaine
leads to occasional stops, which tend to occur in GC-rich
regions and in regions particularly far from the primer (Fig.
1A). An analysis of seventeen such stops indicates that they
tend to occur after the middle position of sequences similar
to pyrimidine-guanine-cytosine, with eleven of seventeen stops
examined matching this consensus and the other six differing
at only one position. There was no obvious correlation with
potential secondary structure of the single stranded template,
as determined by the program MFOLD, part of the GCG package.
In the presence of betaine (Fig. 1B), all of these stops
disappear, allowing the correct sequence to be determined.

WO 95/20682 2181 Z66 PCT/US95/01200
21
Example 2 Readable DNA sequences farther from a primer
are obtained in the presence of betaine
The incidence of stops in DNA sequencing by the
dideoxy method increases in regions of the DNA located farther
from the primer. This phenomenon limits the useful DNA
sequence that can be obtained from one primer. However, the
inclusion of betaine in the dideoxy sequencing reaction
mixture decreases the incidence of stops in DNA regions far
downstream from the primer. This allows useful sequence
information to be obtained in these regions of the DNA without
doing an additional sequencing reaction with another primer.
DNA sequencing was performed in the presence and
absence of 2M betaine as described in example 1. The
resulting sequencing reactions were run on a DNA sequencing
gel as described in example 1, except that the gel was run for
12 hours to examine longer DNA fragments. These longer DNA
fragments clearly showed the sequence of DNA regions farther
from the primer than those routinely obtained in dideoxy DNA
sequencing procedures. Little useful sequence information
could be determined from these gels in the absence of betaine.
However, in the presence of betaine, DNA sequence could be
read up to at least 520 nucleotides from the 3' end of the
primer, with sequences beyond that becoming less readable
because of the resolving power of the gel.
Example 3 DNA seauence determination from unpurified DNA
preparations in the presence of betaine
DNA was prepared generally following a standard
alkaline lysis technique (See Silhavy et al. (1984)
Experiments With Gene Fusions, Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York 147-148), followed by an ethanol
precipitation. No organic extraction, RNAse treatment or
other further purification technique was used. XL1-blue cells
(Stratagene, San Diego, California) containing the pDSM3
plasmid were used as a source of the DNA. The DNA was then
sequenced as described for example 1. As expected, there were
a large number of stops in the absence of betaine. However,

WO 95/20682 PCT/US95/01200
(a~~~_G 22
the stops present while sequencing this DNA disappeared when
betaine was added to the termination reaction.
Example 4 Elimination of stops during DNA sequencing by
chasing with betaine
Betaine is capable of eliminating stops when added
after the normal termination reaction. DNA sequencing was
carried out as described in example 1, except that no betaine
was added with the termination mixture. After the 5 minute
incubation at 37 C, either 3.5 l of 5.5 betaine solution or
3.5 l of water was added. The incubation was then continued
for an additional 5 minutes and was stopped with stop solution
as in example 1. Addition of betaine at this point greatly
reduced the stops observed in the absence of betaine. Thus,
the bands representing stops on the sequencing gels appear to
represent halted DNA polymerase complexes. Betaine, added
after the normal incubation with the termination mixes, is
capable of allowing the halted DNA polymerase complexes to
resume elongation. This demonstrates that betaine is not
simply acting by stabilizing the DNA polymerase, since its
addition at this point would have no effect if that were the
case.
Examble 5 Elimination or reduction of the incidence of
stops in modified dideoxy DNA sequencing
procedures
The DNA sequencing procedure of example 1 was
altered to demonstrate that betaine is able to eliminate stops
that occur in a variety of dideoxy DNA sequencing protocols.
The effect of substituting a single-stranded DNA substrate or
using different DNA polymerases in a dideoxy DNA sequencing
procedure was examined.
aj_ Seauencing of single-stranded DNA
Single stranded DNA was prepared from strain
XL1-blue containing plasmid pDSM6, according to routine
procedures. When single stranded DNA was sequenced by the
procedure described in example 1, fewer stops were observed in

WO 95/20682 Z18IZ66 PCT/US95/01200
23
the absence of betaine than in double-stranded specimens.
However, these stops were also eliminated by betaine.
bl Seauencina using Klenow DNA polymerase enzyme
DNA sequencing was carried out essentially by the
procedure described in example 1, except that the Klenow
fragment of E. coli DNA polymerase was used. 7.5 units of the
Klenow enzyme was used and the extension mix was modified to
include 20 Ci of 35S-dATP and 0.1 l of DTT in addition to
the GTP extension mix. Stops were observed in different
places with the use of the Klenow enzyme but were generally
eliminated by betaine, just as described above for the
procedure using Sequenase.
cl Sequencing using Taa molymerase enzyme
DNA sequencing was carried out according to the
Promega "fmol"' DNA Sequencing System" protocol (Promega
Corporation, Madison, Wisconsin, USA). The protocol is
described below.
(1) End label primer.
(2) Mix 4-40 fmol DNA template, 5 l 5x fmol
sequencing buffer (250 mM Tris.Cl pH 9.0, 10 mM MgC12), 1.5
pmole labeled primer, betaine and water to a final volume of
16 l containing an appropriate concentration of betaine
(e.g., 2M).
(3) Add 1 l sequencing Grade Taq DNA Polymerase (5
U/ l)
(4) Add 4 l of the above to each of the dNTP/ddNTP
mixes according to the following table:

WO 95/20682 PCT/US95/01200
21$ E+
24
A C G T
dATP 20 M 20 M 20 M 20 M
dCTP 20 M 20 M 20 M 20 M
7 Deaza dGTP 20 M 20 M 20 M 20 M
dTTP 20 M 20 M 20 M 20 M
ddATP 350 M - - -
ddCTP - 200 M - -
ddGTP - - 30 M -
ddTTP - - - 600 M
(5) Overlay with one drop of mineral oil.
(6) Place tubes in thermal cycler preheated to
95 C.
(7) Start appropriate cycling program (varies with
primer, one example is 95 C for 2 minutes, 30 cycles of (95 C
for 30 sec., 42 C/30 sec., 70 /60 sec.)).
(8) Add 3 l stop solution (10 mM NaOH, 95%
formamide, 0.05% bromophenol blue, 0.05% xylene cyanol).
(9) Heat at 95 C for 2h minutes, ice quench, and
load on a sequencing gel.
Sequencing gels were run and the presence of DNA
polymerase stops was determined as described in Example 1,
herein. The incidence of stops observed in the reaction
mixture was reduced in the presence of betaine.
Example 6 Elimination of stops during DNA sequencing by
Dimethylglycine, sarcosine (monomethylglvcine)
and trimethylamine N-oxide (TMANO)
Three additional compounds were demonstrated to
decrease the incidence of stops in a dideoxy DNA sequencing
procedure. Dimethylglycine, sarcosine (monomethylglycine) and
trimethylamine N-oxide (TMANO) (all from Sigma Chemical
Company) were evaluated for their ability to eliminate stops
in a dideoxy DNA sequencing method, as described in Example 1.
DNA sequencing was otherwise performed as described in example
1. TMANO proved to eliminate stops better than betaine, while
dimethylglycine was somewhat less effective. Sarcosine had a
measurable effect and is less preferred than the other
compounds. Two other N-substituted charged compounds,

CA 02181266 2006-09-29
tetraethylammonium chloride (TEAC1) and tetraethylammonium
acetate (TEAAc) were also tested. TEAC1 inhibits the DNA
polymerase. TEAAc did not inhibit the DNA polymerase as much
as TEAC1, but also did not reduce the number of stops observed
5 in the sequencing gel.
Example 7 Concentration ranges of betaine. TMANO,
sarcosine. and dimethvlglycine effective in
reducing stoRs in a dideoxy method of DNA
10 synthesis
The concentration ranges of TMANO, sarcosine,
dimethylglycine and betaine were determined according to the
procedure described in Example 1.
The results are summarized in the following table.
15 Compound Measurable Effect Maximal Effect
betaine 0.5 M 2 M
TMANO 0.25 M 0.5 M
Dimethylglycine 0.5 M 1.5 M
Sarcosine 0.5 M 1.5 M
Example 8 Increasing the maximum size of PCR
amplification products
Betaine was shown to extend the maximum distance
between primers for chain elongation in PCR. PCR was
performed on T7 DNA as described in B. Krummel, Ph.D. Thesis,
University of California, Berkeley, 1990. Approximately 50
pmole of each primer were combined with 2.5 Units of AmpliTaqTM
Polymerase (Perkin-Elmer, Norwalk, Connecticut, USA) and 0.5
g of phage T7 DNA in a 200 l reaction containing final
concentrations of 200 M dNTP, 50 mM KC1, 10 mM Tris-Cl pH8.3,
2.5 mM MgC12 and 0.01% gelatin. Following a 1' hot start at
94 C, 25 cycles of PCR were performed with cycle times of 90"
at 94 C, 1' at 37 C, and 20' at 72 C. The products were
precipitated, run on a 1% agarose gel and visualized with UV
following ethidium staining.
The addition of betaine at a concentration of 2M
allowed two primers which were separated by approximately 4000

CA 02181266 2006-09-29
26
base pairs to amplify the intervening DNA region. This DNA
region was not appreciably amplified in the absence of
betaine.
Example 9 PCR amplification of reaions containing
problematic DNA sequences
Betaine was shown to allow PCR chain elongation
through GC-rich DNA regions which have impeded chain
elongation in the absence of betaine. PCR was performed on a
plasmid containing multiple repeats of the sequence TGC, which
we have shown to impede the progress of DNA polymerases (see
Example 1, herein). Although the primers were only 500 base
pairs apart, a distance which can normally be easily amplified
by PCR, the standard reaction conditions failed to produce any
amplified product. When betaine was added to a concentration
of 2M, the intervening region was amplified.
It is understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be
suggested to persons skilled in the art and are to be included
within the spirit and preview of this application and scope of
the appended claims.