U.S. patent application number 12/305633 was filed with the patent office on 2010-01-28 for methods for fragmentation and labeling of nucleic acids.
Invention is credited to Pengchin Chen, Nurith Kurn.
Application Number | 20100022403 12/305633 |
Document ID | / |
Family ID | 38895198 |
Filed Date | 2010-01-28 |
United States Patent
Application |
20100022403 |
Kind Code |
A1 |
Kurn; Nurith ; et
al. |
January 28, 2010 |
METHODS FOR FRAGMENTATION AND LABELING OF NUCLEIC ACIDS
Abstract
The invention provides methods, compositions, and kits for
fragmentation and labeling of nucleic acids. More particularly, the
invention relates to methods for fragmentation of nucleic acids to
produce fragments with 3' end hydroxyl groups within a desired size
range. In methods of the invention, nucleic acids are fragmented at
abasic sites to produce fragments with blocked 3' ends. The 3' ends
are unblocked to produce polynucleotide fragments with hydroxyl
groups at their 3' ends. Methods, kits, and compositions for
carrying out fragmentation of a polynucleotide template in a single
reaction mixture to yield fragments with 3'-hydroxyl ends within
the desired size range are disclosed.
Inventors: |
Kurn; Nurith; (Palo Alto,
CA) ; Chen; Pengchin; (San Jose, CA) |
Correspondence
Address: |
WILSON, SONSINI, GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Family ID: |
38895198 |
Appl. No.: |
12/305633 |
Filed: |
July 2, 2007 |
PCT Filed: |
July 2, 2007 |
PCT NO: |
PCT/US07/15409 |
371 Date: |
July 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60817890 |
Jun 30, 2006 |
|
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|
Current U.S.
Class: |
506/9 ; 435/6.12;
435/6.15; 435/91.1; 435/91.2 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12Q 1/6811 20130101; C12Q 1/6806 20130101; C12Q 1/6806 20130101;
C12Q 2525/119 20130101; C12Q 2523/107 20130101; C12Q 2523/107
20130101; C12Q 2525/119 20130101; C12Q 2533/101 20130101; C12Q
2521/319 20130101 |
Class at
Publication: |
506/9 ; 435/91.1;
435/6; 435/91.2 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C12P 19/34 20060101 C12P019/34; C12Q 1/68 20060101
C12Q001/68 |
Claims
1-74. (canceled)
75. A method for generating a polynucleotide fragment, within a
desired size range and comprising a hydroxyl group at the 3' end,
from a polynucleotide comprising an abasic site, said method
comprising: (a) contacting said polynucleotide comprising an abasic
site with a reaction mixture comprising: i. a chemical capable of
fragmenting a phosphodiester backbone of a polynucleotide at an
abasic site, whereby generating a polynucleotide fragment within
the desired size range and comprising a blocked 3' end; and ii. an
enzyme capable of unblocking the blocked 3' end of said fragment,
whereby generating a polynucleotide fragment within the desired
size range and comprising a hydroxyl group at the 3' end.
76. The method according to claim 75, wherein the chemical is a
polyamine.
77. The method according to claim 76, wherein the polyamine is
N,N'-dimethylethylenediamine (DMED).
78. The method according to claim 75, wherein the enzyme capable of
unblocking the blocked 3, end comprises a 3' to 5' exonuclease
activity.
79. The method according to claim 78, wherein the enzyme comprises
non-processive exonuclease activity and does not comprise an
endonuclease activity.
80. The method according to claim 78, wherein the exonuclease
activity is a non-processive exonuclease activity, wherein the
enzyme that comprises an exonuclease activity also comprises an
endonuclease activity, and wherein contacting the polynucleotide
fragment with the enzyme is under conditions in which the
endonuclease activity is minimized or absent.
81. The method according to claim 78, wherein the enzyme comprising
a 3' exonuclease activity is selected from the group consisting of
endonuclease 4, exonuclease T, and apurinic/apyrimidinic
endonuclease (APE 1).
82. The method according to claim 75, further comprising: (b)
extending the polynucleotide fragment from the 3' hydroxyl group
with a polymerase.
83. The method according to claim 82, wherein the extension further
comprises a labeled nucleotide, whereby a polynucleotide fragment
within the desired size range and labeled at the 3' end is
generated.
84. The method of claim 83 wherein the polymerase is template
independent.
85. The method according to claim 84, wherein the template
independent polymerase is terminal deoxynucleotidyl transferase
(TdT).
86. The method according to claim 83, wherein the labeled
nucleotide is selected from the group consisting of a labeled
nucleotide triphosphate (NTP), a labeled deoxynucleotide
triphosphate (dNTP), and a labeled dideoxynucleotide triphosphate
(ddNTP).
87. The method according to claim 83, wherein the labeled
nucleotide is a biotinylated nucleotide.
88. The method according to claim 83, wherein the labeled
nucleotide comprises a fluorophore.
89. The method according to claim 83, wherein a mixture of labeled
and unlabeled nucleotides is used for labeling the polynucleotide
fragment.
90. The method according to claim 75, wherein the reaction mixture
further comprises: iii. an agent capable of cleaving a base portion
of a non-canonical nucleotide in a polynucleotide comprising a
non-canonical nucleotide, whereby generating an abasic site; or iv.
an agent capable of non-enzymatically converting a canonical or
non-canonical nucleotide in a polynucleotide into an abasic
site.
91. The method according to claim 90, wherein the non-canonical
nucleotide is selected from the group consisting of dUTP, dITP, and
5-OH-Me-dCTP.
92. The method according to claim 90, wherein the agent capable of
cleaving a base portion of the non-canonical nucleotide is an
N-glycosylase enzyme.
93. The method according to claim 92, wherein the N-glycosylase is
selected from the group consisting of Uracil N-Glycosylase (UNG),
hypoxanthine-N-Glycosylase, and hydroxy-methyl
cytosine-N-glycosylase.
94. The method according to claim 90, wherein the non-canonical
nucleotide is dUTP and the enzyme capable of cleaving a base
portion of the non-canonical nucleotide is UNG.
95. The method according to claim 90, wherein the non-canonical
nucleotide is dUTP, the enzyme capable of cleaving a base portion
of the non-canonical nucleotide is UNG, and the phosphodiester
backbone is cleaved with DMED.
96. The method according to claim 90, wherein the polynucleotide
comprising a non-canonical nucleotide is synthesized in the
presence of two or more different non-canonical nucleotides,
whereby a polynucleotide comprising two or more different
non-canonical nucleotides is synthesized.
97. The method according to claim 90, wherein the polynucleotide
comprising a non-canonical nucleotide is synthesized in the
presence of all four canonical nucleotides and a non-canonical
nucleotide, wherein the non-canonical nucleotide is provided at a
ratio suitable for generating fragments within the desired size
range.
98. The method according to claim 83 further comprising: (c)
characterizing a polynucleotide template of interest, comprising
analyzing a polynucleotide fragment within the desired size range
and labeled at the 3' end.
99. The method according to claim 98, wherein analyzing the labeled
polynucleotide fragment within the desired size range comprises
determining amount of said products, whereby the amount of the
polynucleotide template present in a sample is quantified.
100. The method according to claim 98, wherein analyzing the
labeled polynucleotide fragment within the desired size range
comprises contacting the labeled polynucleotide fragment with at
least one probe.
101. The method according to claim 100, wherein the at least one
probe is provided as a microarray.
102. The method according to claim 83 further comprising: (c)
determining a gene expression profile in a sample, said method
comprising determining the amount of the polynucleotide fragment
within the desired size range and labeled at the 3' end wherein the
amount is indicative of the amount of a polynucleotide template in
said sample from which the labeled polynucleotide fragment was
generated, whereby a gene expression profile is determined.
103. The method according to claim 102, wherein the polynucleotide
template is RNA or mRNA.
104. The method according to claim 102, wherein the amounts of a
plurality of polynucleotide fragments within the desired size range
derived from a plurality of polynucleotide templates in a sample
are determined.
105. The method according to claim 83 further comprising: (c)
hybridizing a first population of polynucleotide fragments within
the desired size range and labeled at the 3' end, to at least one
probe.
106. The method according to claim 105 further comprising: (d)
comparing hybridization of the first population of polynucleotide
fragments within the desired size range and labeled at the 3' end
to at least one probe with hybridization of a second population of
polynucleotide fragments within the desired size range and labeled
at the 3' end to the at least one probe.
107. The method according to claim 83 further comprising: (c)
detecting presence or absence of a mutation in a template,
comprising analyzing a polynucleotide fragment within the desired
size range and labeled at the 3' end, whereby presence of absence
of a mutation is detected, wherein analyzing comprises comparison
of the polynucleotide fragment within the desired size range and
labeled at the 3' end to a polynucleotide prepared from a reference
polynucleotide.
108. The method according to claim 107, wherein the mutation is
selected from the group consisting of a base substitution, a base
insertion, a base deletion, and a single nucleotide
polymorphism.
109. The method according to claim 75, wherein the polynucleotide
comprising an abasic site is generated by cleaving a base portion
of a methylated nucleotide with an agent capable of cleaving a base
portion of the methylated nucleotide to create an abasic site,
whereby an abasic site is generated.
110. The method according to claim 75, wherein the polynucleotide
comprising an abasic site is generated by cleaving a base portion
of a canonical nucleotide with an agent capable of cleaving a base
portion of the canonical nucleotide to create an abasic site,
whereby an abasic site is generated.
111. The method according to claim 110, wherein the canonical
nucleotide is cytosine and the agent capable of cleaving a base
portion of the canonical nucleotide comprises cytosine deaminase in
conjunction with UNG.
112. The method according to claim 75, wherein the polynucleotide
comprising an abasic site is synthesized from a polynucleotide
template comprising DNA or RNA.
113. The method according to claim 112, wherein the polynucleotide
template is selected from the group consisting of mRNA, cDNA, and
genomic DNA.
114. The method according to claim 75, wherein the polynucleotide
comprising an abasic site is single stranded or double
stranded.
115. The method according to claim 75, wherein the polynucleotide
comprising an abasic site is synthesized by an amplification method
selected from the group consisting of polymerase chain reaction
(PCR), strand displacement amplification (SDA), multiple
displacement amplification (MDA), rolling circle amplification
(RCA), single primer isothermal amplification (SPIA), and
Ribo-SPIA.
116. The method according to claim 75, wherein the polynucleotide
comprising an abasic site is synthesized by a method selected from
the group consisting of reverse transcription, primer extension,
limited primer extension, replication, and nick translation.
117. A method for fragmenting a polynucleotide comprising an abasic
site to generate fragments within a desired size range, said method
comprising: (a) chemically cleaving a phosphodiester backbone of a
polynucleotide comprising an abasic site at the abasic site,
whereby a polynucleotide fragment within the desired size range and
comprising a blocked 3' end is generated; and (b) contacting the
polynucleotide fragment with an enzyme capable of unblocking the
blocked 3' end of said fragment, whereby a polynucleotide fragment
within the desired size range and comprising a 3' end hydroxyl
group is generated; wherein (a) and (b) are performed
simultaneously and performed in the same reaction mixture.
118. A composition or kit comprising: (a) a chemical agent capable
of cleaving a phosphodiester backbone at an abasic site to produce
a polynucleotide fragment with a blocked 3' end within a desired
size range; and (b) an enzyme capable of unblocking a blocked 3'
end to generate a polynucleotide comprising a 3' hydroxyl
group.
119. The composition or kit according to claim 118 wherein the
composition or kit further comprises: (c) an agent capable of
cleaving a base portion of a nucleotide to generate an abasic site
in a polynucleotide.
120. The composition or kit according to claim 118, wherein (a) is
a polyamine, (b) is an enzyme comprising a 3' exonuclease activity,
and (c) is an N-glycosylase.
121. The composition or kit according to claim 120, wherein the
polyamine is DMED, the enzyme comprising a 3' exonuclease activity
is selected from the group consisting of endonuclease 4,
exonuclease T, and APE 1, and the N-glycosylase is UNG.
122. The kit according to claim 119, further comprising: (d) an
agent capable of labeling a 3' hydroxyl group of a
polynucleotide.
123. The composition or kit according to claim 119, further
comprising: (c) a non-canonical nucleotide; and (d) an enzyme
capable of synthesizing a polynucleotide comprising the
non-canonical nucleotide.
124. The composition or kit according to claim 123, wherein the
non-canonical nucleotide is dUTP and the agent capable of cleaving
a base portion of a nucleotide to generate an abasic site in a
polynucleotide is UNG.
125. The composition or kit according to claim 122, further
comprising: (e) a labeled nucleotide.
126. The composition or kit according to claim 125, wherein (a) is
a polyamine, (b) is an enzyme comprising a 3, exonuclease activity;
(c) is an N-glycosylase (d) is a template independent polymerase;
and (e) is a biotinylated nucleotide.
127. The composition or kit according to claim 125, wherein the
labeled nucleotide is selected from the group consisting of a
labeled nucleotide triphosphate (NTP), a labeled deoxynucleotide
triphosphate (dNTP), and a labeled dideoxynucleotide triphosphate
(ddNTP).
128. The composition or kit according to claim 127, wherein the
labeled nucleotide is a biotinylated nucleotide.
129. The composition or kit according to claim 125, wherein the
polyamine is DMED, the enzyme comprising a 3' exonuclease activity
is selected from the group consisting of endonuclease 4,
exonuclease T, and APE 1, the N-glycosylase is UNG, an agent
capable of labeling a 3a hydroxyl group of a polynucleotide is TdT,
and the labeled nucleotide is selected from the group consisting of
biotin 2',3'-dideoxy-UTP and biotin 2',3'-dideoxy-CTP 17.
130. A population of single-stranded polynucleotide fragments of a
desired size range representative of a complete genome or complete
transcriptome of an organism wherein each of said fragments
comprises a hydroxyl group at the 3' end.
131. The population of claim 130 wherein the sizes of the fragments
are about 50 to 200 nucleotides.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/817,890, filed Jun. 30, 2006, which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The invention relates to methods for fragmentation of
nucleic acids, in particular to generate fragments with 3' end
hydroxyl groups, and methods for labeling the fragments.
BACKGROUND
[0003] Nucleic acid fragmentation and labeling has gained
importance in the field of nucleic acid analysis. Fragmented and
labeled nucleic acids are useful for efficient hybridization based
nucleic acid analysis, particularly when hybridizing to immobilized
probes, such as in multiplex detection using microarrays or bead
based hybridization assays. Fragmentation of nucleic acids to
generate fragments having hydroxyl groups at their 3' termini is
useful since a 3' hydroxyl group permits extension with a
template-dependent polymerase or labeling with a
template-independent polymerase, which permits introduction of a
label. The extension of fragmented, hybridized nucleic acid along a
nucleic acid probe or template molecule by target directed nucleic
acid synthesis is useful for both nucleic acid analysis and the
generation of recombinant nucleic acids.
[0004] A method for controlled nucleic acid fragmentation has been
previously described which is based on the incorporation of
non-canonical nucleotides into a polynucleotide strand which is
synthesized in vitro, followed by generation of an abasic site at
the site of incorporation of a non-canonical nucleotide, permitting
subsequent fragmentation of the synthesized polynucleotide and/or
labeling at the abasic site. (U.S. Patent Application No.
2004/0005614; PCT Application No. WO 04/011665). The size
distribution of the fragmented products may be controlled by the
level of incorporation of non-canonical nucleotides. The level of
incorporation, and subsequent nucleic acid fragment size, may be
adjusted to provide a suitable fragment size for the particular
downstream use of the fragmented products.
[0005] A process utilizing dUTP as the non-canonical nucleotide
during DNA synthesis, uracil N-glycosylase ("UNG") as the enzyme
which removes the base portions of the non-canonical nucleotides to
generate abasic sites, and cleavage of abasic sites with a
polyamine, such as N,N'-dimethylethylenediamine, as described in
U.S. Application No. 2004/0005614 and PCT Application No. WO
04/011665, results in the generation of fragmented polynucleotides
with modified ("blocked") 3' ends that are capable of reacting with
an aldehyde-reactive reagent. This process is useful for generation
of fragmented and labeled nucleic acids suitable for analysis, for
example, on a microarray, or fragmented nucleic acid targets
suitable for immobilization.
[0006] The process of controlled fragmentation and labeling of
single stranded nucleic acids is difficult to achieve using other
methods, such as non-specific digestion with an enzyme such as
DNase or chemical nucleotide modification, since these reactions
cannot be carried out to completion without complete, or nearly
complete, degradation of the single stranded nucleic acid to be
fragmented and labeled. Double stranded DNA may be digested with
restriction endonucleases to generate fragments of defined size
distribution. Such enzymes are specific for a defined sequence
content, both with respect to composition and length of a
recognition site, and afford higher or lower frequency of cleavage
depending on the restriction enzyme used. However, restriction
endonucleases are specific for double stranded DNA and this
approach does not apply to fragmentation of single stranded nucleic
acid molecules.
[0007] A desirable feature for fragmented nucleic acids is the
presence of a hydroxyl group at the 3' end, which may serve as a
substrate for polynucleotide synthesis with a polymerase via
template-dependent or template-independent extension, or ligation
with another polynucleotide. Such manipulations may facilitate
introduction of a label. For example, a label may be introduced by
extending from the 3' end with terminal transferase (a
template-independent polymerase) and a labeled nucleotide. The
method described above, in which nucleic acid molecules are cleaved
at an abasic site with a polyamine, results in nucleic acids with
"blocked" 3' ends, precluding use of the fragments as primers for
polynucleotide synthesis or substrates for labeling at a 3'
hydroxyl group.
[0008] New methods for the efficient fragmentation of nucleic acids
to generate fragments comprising 3' end hydroxyl groups, and of
defined size distribution, are desirable.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention provides methods, compositions, and kits for
fragmenting, or fragmenting and labeling, a polynucleotide.
[0010] In one aspect, the invention features the generation and/or
use of a polynucleotide with abasic sites distributed such that
cleavage at the abasic sites generates fragments of a desired size
range.
[0011] In one aspect, the invention provides a method for
fragmenting a polynucleotide, said method comprising: (a)
chemically cleaving a phosphodiester backbone of a polynucleotide
comprising an abasic site at or near the abasic site, whereby a
polynucleotide fragment comprising a blocked 3' end is generated;
and (b) contacting the polynucleotide fragment with an enzyme
capable of unblocking (i.e., which unblocks) the blocked 3' end of
said fragment, whereby a polynucleotide fragment comprising a 3'
end hydroxyl group is generated. In some embodiments, steps (a) and
(b) are performed simultaneously in the same reaction mixture. In
some embodiments, fragments within a desired size range are
generated.
[0012] In some embodiments, the phosphodiester backbone is cleaved
with a polyamine to generate a polynucleotide fragment with a
blocked 3' end. In one embodiment, the polyamine is
N,N'-dimethylethylenediamine (DMED).
[0013] In some embodiments, the enzyme capable of unblocking the
blocked 3' end comprises a 3' to 5' exonuclease activity. In one
embodiment, the exonuclease activity is a non-processive
exonuclease activity. In one embodiment, the enzyme that comprises
an exonuclease activity does not comprise an endonuclease activity.
In one embodiment, the enzyme that comprises an exonuclease
activity also comprises an endonuclease activity, and contacting
the polynucleotide fragment with the enzyme is under conditions in
which the endonuclease activity is minimized or absent. In one
embodiment, the enzyme comprising a 3' exonuclease activity is
selected from the group consisting of endonuclease 4, exonuclease
T, and apurinic/apyrimidinic endonuclease (APE 1).
[0014] In some embodiments, the method further comprises extending
the polynucleotide fragment from the 3' hydroxyl group with a
template independent polymerase and a labeled nucleotide, whereby a
polynucleotide fragment labeled at the 3' end is generated. In one
embodiment, the template independent polymerase is terminal
deoxynucleotidyl transferase (TdT). In one embodiment, the labeled
nucleotide is a biotinylated nucleotide. In various embodiments,
the biotinylated nucleotide is selected from the group consisting
of a biotinylated nucleotide triphosphate (NTP), a biotinylated
deoxynucleotide triphosphate (dNTP), and a biotinylated
dideoxynucleotide triphosphate (ddNTP). In some embodiments, the
biotinylated nucleotide is selected from the group consisting of
biotin 2',3'-dideoxy-UTP and biotin 2',3'-dideoxy-CTP. In one
embodiment, the labeled nucleotide comprises a fluorophore. In one
embodiment, a mixture of labeled and unlabeled nucleotides is used
for labeling the polynucleotide fragment. As used herein, the term
"nucleotide" encompasses nucleotide analogs, which are known in the
art. The term "labeled nucleotide" encompasses labeled nucleotide
analogs.
[0015] In some embodiments, the polynucleotide comprising an abasic
site is generated by: (i) synthesizing a polynucleotide from a
polynucleotide template in the presence of a non-canonical
nucleotide, whereby a polynucleotide comprising the non-canonical
nucleotide is generated; and (ii) cleaving a base portion of the
non-canonical nucleotide from the synthesized polynucleotide with
an enzyme capable of cleaving (i.e., which cleaves) the base
portion of the non-canonical nucleotide, whereby an abasic site is
generated. In some embodiments, the method involves synthesizing
the polynucleotide from the polynucleotide template in the presence
of all four canonical nucleotides and a non-canonical nucleotide,
wherein the non-canonical nucleotide is provided at a ratio
suitable for generating fragments within the desired size range. In
various embodiments, the non-canonical nucleotide is selected from
the group consisting of dUTP, dITP, and 5-OH-Me-dCTP. In one
embodiment, the enzyme capable of cleaving a base portion of the
non-canonical nucleotide is an N-glycosylase. In some embodiments,
the N-glycosylase is selected from the group consisting of Uracil
N-Glycosylase (UNG), hypoxanthine-N-Glycosylase, and hydroxy-methyl
cytosine-N-glycosylase. In one embodiment, the non-canonical
nucleotide is dUTP and the enzyme capable of cleaving a base
portion of the non-canonical nucleotide is UNG. In one embodiment,
the non-canonical nucleotide is dUTP, the enzyme capable of
cleaving a base portion of the non-canonical nucleotide is UNG, and
the phosphodiester backbone is cleaved with DMED. In one
embodiment, the polynucleotide comprising a non-canonical
nucleotide is synthesized using a primer comprising a non-canonical
nucleotide. In one embodiment, the polynucleotide comprising a
non-canonical nucleotide is synthesized in the presence of two or
more different non-canonical nucleotides, whereby a polynucleotide
comprising two or more different non-canonical nucleotides is
synthesized. In one embodiment, polynucleotides comprising a
non-canonical nucleotides are synthesized from two or more
different polynucleotide templates. In some embodiments, the
polynucleotide comprising an abasic site is generated by
non-enzymatically converting a canonical or non-canonical
nucleotide in a polynucleotide into an abasic site. Exemplary
non-enzymatic methods for generating an abasic site include
depurination or depyrimidination of a nucleotide using an acidic
pH, an oxidizing agent, an alkylating agent, and any two or more of
the foregoing.
[0016] In some embodiments, the polynucleotide comprising an abasic
site is generated by cleaving a base portion of a methylated
nucleotide with an agent capable of cleaving (i.e., which cleaves)
a base portion of the methylated nucleotide to create an abasic
site, whereby an abasic site is generated. In some embodiments, the
method includes cleaving a base portion of a methylated nucleotide
in a polynucleotide with an agent capable of cleaving a base
portion of the methylated nucleotide to create an abasic site,
whereby the polynucleotide comprising an abasic site is
generated.
[0017] In some embodiments, the polynucleotide comprising an abasic
site is generated by cleaving a base portion of a canonical
nucleotide with an agent capable of cleaving (i.e., which cleaves)
a base portion of the canonical nucleotide to create an abasic
site, whereby an abasic site is generated. In some embodiments, the
method includes cleaving a base portion of a canonical nucleotide
in a polynucleotide with an agent capable of cleaving a base
portion of the canonical nucleotide to create an abasic site,
whereby the polynucleotide comprising an abasic site is generated.
In one embodiment, the canonical nucleotide is cytosine and the
agent capable of cleaving a base portion of the canonical
nucleotide comprises cytosine deaminase in conjunction with
UNG.
[0018] In some embodiments, the polynucleotide comprising an abasic
site is synthesized from a polynucleotide template comprising DNA
or RNA. In various embodiments, the polynucleotide template is
selected from the group consisting of RNA, mRNA, cDNA, and genomic
DNA. In one embodiment, the polynucleotide comprising an abasic
site is single stranded. In one embodiment, the polynucleotide
comprising an abasic site is double stranded.
[0019] In some embodiments of the methods of the invention, the
polynucleotide to be fragmented (i.e., the polynucleotide
comprising an abasic site) is synthesized using a method
comprising: (a) extending a composite primer in a complex
comprising: (i) a polynucleotide template; and (ii) the composite
primer, said composite primer comprising an RNA portion and a 3'
DNA portion, wherein the polynucleotide template is hybridized to
the composite primer; and (b) cleaving RNA of the annealed
composite primer with an enzyme that cleaves RNA from an RNA/DNA
hybrid such that another composite primer hybridizes to the
template and repeats primer extension and strand displacement,
whereby multiple copies of the complementary sequence of the
polynucleotide template are produced. In one embodiment, the
complex of part (a) comprises: (i) a complex of first and second
primer extension products, wherein the first primer extension
product is produced by extension of a first primer hybridized to a
target RNA with at least one enzyme comprising RNA-dependent DNA
polymerase activity, wherein the first primer is a composite primer
comprising an RNA portion and a 3' DNA portion; wherein RNA in the
complex of first and second primer extension products is cleaved
with at least one enzyme that cleaves RNA from an RNA/DNA hybrid
such that a composite primer hybridizes to the second primer
extension product; and (ii) the composite primer.
[0020] In some embodiments of the methods of the invention, the
polynucleotide to be fragmented (i.e., the polynucleotide
comprising an abasic site) is synthesized by an amplification
method selected from the group consisting of polymerase chain
reaction (PCR), strand displacement amplification (SDA), multiple
displacement amplification (MDA), rolling circle amplification
(RCA), single primer isothermal amplification (SPIA), and
Ribo-SPIA. In some embodiments, the polynucleotide is synthesized
by a method selected from the group consisting of reverse
transcription, primer extension, limited primer extension,
replication, and nick translation. In one embodiment, the
polynucleotide is synthesized using a labeled primer.
[0021] In one embodiment, the invention provides a method for
fragmenting and labeling a polynucleotide, said method comprising:
(a) synthesizing a polynucleotide from a polynucleotide template in
the presence of a non-canonical nucleotide, whereby a
polynucleotide comprising the non-canonical nucleotide is
generated; (b) cleaving a base portion of the non-canonical
nucleotide from the synthesized polynucleotide with an enzyme
capable of cleaving (i.e., which cleaves) the base portion of the
non-canonical nucleotide, whereby an abasic site is generated; (c)
cleaving the phosphodiester backbone of the polynucleotide
comprising the abasic site at or near the abasic site, whereby a
polynucleotide fragment comprising a blocked 3' end is generated;
(d) contacting the polynucleotide fragment with an enzyme capable
of unblocking (i.e., which unblocks) the blocked 3' end of said
fragment, whereby a polynucleotide fragment comprising a 3'
hydroxyl group is generated; and (e) contacting the polynucleotide
fragment comprising a 3' hydroxyl group with an enzyme capable of
extending (i.e., which extends) the polynucleotide fragment from
the 3' end and a labeled nucleotide, whereby a labeled
polynucleotide fragment is generated. In some embodiments, steps
(b), (c), and (d) are performed simultaneously. In some
embodiments, steps (b), (c), and (d) are performed simultaneously
in the same reaction mixture. In some embodiments, steps (b) and
(c) are performed simultaneously. In some embodiments, steps (b)
and (c) are performed simultaneously in the same reaction mixture.
In some embodiments, steps (c) and (d) are performed
simultaneously. In some embodiments, steps (c) and (d) are
performed simultaneously in the same reaction mixture. In some
embodiments, the method involves synthesizing the polynucleotide
from the polynucleotide template in the presence of all four
canonical nucleotides and a non-canonical nucleotide, wherein the
non-canonical nucleotide is provided at a ratio suitable for
generating fragments within the desired size range. In some
embodiments, labeled fragments within a desired size range are
generated.
[0022] In one embodiment, the invention provides a method for
fragmenting a polynucleotide, said method comprising: (a)
incubating a reaction mixture, said reaction mixture comprising:
(i) a polynucleotide template; and (ii) a non-canonical nucleotide;
wherein the incubation is under conditions that permit synthesis of
a polynucleotide comprising the non-canonical nucleotide, whereby a
polynucleotide comprising the non-canonical nucleotide is
generated; (b) incubating a reaction mixture, said reaction mixture
comprising: (i) the polynucleotide comprising the non-canonical
nucleotide; and (ii) an enzyme capable of cleaving (i.e., which
cleaves) a base portion of the non-canonical nucleotide, wherein
the incubation is under conditions that permit cleavage of the base
portion of the non-canonical nucleotide, whereby a polynucleotide
comprising an abasic site is generated; (c) incubating a reaction
mixture, said reaction mixture comprising: (i) the polynucleotide
comprising the abasic site; and (ii) an agent capable of chemically
cleaving (i.e., which cleaves) the phosphodiester backbone of the
polynucleotide comprising the abasic site at or near the abasic
site, wherein the incubation is under conditions that permit
cleavage of the phosphodiester backbone of the polynucleotide at or
near the abasic site, whereby a polynucleotide fragment comprising
a blocked 3' end is generated; and (d) incubating a reaction
mixture, said reaction mixture comprising: (i) the fragment of the
polynucleotide comprising a blocked 3' end; and (ii) an enzyme
capable of unblocking (i.e., which unblocks) the blocked 3' end,
whereby a polynucleotide fragment comprising a 3' hydroxyl group is
generated. In some embodiments, the method further comprises: (e)
incubating a reaction mixture, said reaction mixture comprising:
(i) the polynucleotide fragment comprising a 3' hydroxyl group; and
(ii) an agent capable of extending (i.e., which extends) the
fragment from the 3' hydroxyl group; and (iii) a labeled
nucleotide, wherein the incubation is under conditions that permit
extension of the polynucleotide fragment from the 3' hydroxyl
group, whereby a labeled polynucleotide fragment is generated. In
one embodiment, the agent capable of extending the polynucleotide
fragment from the 3' hydroxyl group is TdT, wherein the
polynucleotide fragment is labeled at the 3' hydroxyl group with a
labeled nucleotide. In some embodiments, steps (b), (c), and (d)
are performed simultaneously in the same reaction mixture. In some
embodiments, the incubation is under conditions that permit
synthesis of the polynucleotide from the polynucleotide template in
the presence of all four canonical nucleotides and a non-canonical
nucleotide, wherein the non-canonical nucleotide is provided at a
ratio suitable for generating fragments within the desired size
range. In some embodiments, fragments within a desired size range
are generated.
[0023] In one embodiment, the invention provides a method for
generating a polynucleotide fragment with a 3' end hydroxyl group,
comprising contacting a polynucleotide fragment with a blocked 3'
end with an enzyme capable of unblocking (i.e., which unblocks) the
blocked 3' end, wherein the polynucleotide fragment with a blocked
3' end is generated by cleaving a polynucleotide fragment
comprising an abasic site at or near the abasic site.
[0024] In another aspect, the invention provides, a method of
characterizing a polynucleotide template of interest, comprising
analyzing a labeled polynucleotide fragment produced by a method as
described herein. In one embodiment, the method comprises (a)
generating a labeled polynucleotide fragment by a method as
described herein; and (b) analyzing the labeled polynucleotide
fragment. In one embodiment, analyzing the labeled polynucleotide
fragment comprises determining amount of said products, whereby the
amount of the polynucleotide template present in a sample is
quantified. In one embodiment, analyzing the labeled polynucleotide
fragment comprises contacting the labeled polynucleotide fragment
with at least one probe. In one embodiment, the at least one probe
is provided as a microarray. In one embodiment, the microarray is a
high density polynucleotide microarray. In one embodiment, the
microarray is a high density oligonucleotide microarray. In some
embodiments, the microarray comprises at least one probe
immobilized on a substrate fabricated from a material selected from
the group consisting of paper, glass, ceramic, plastic,
polypropylene, polystyrene, nylon, polyacrylamide, nitrocellulose,
silicon, and optical fiber. In one embodiment, the at least one
probe is immobilized on the substrate in a two-dimensional
configuration or a three-dimensional configuration comprising pins,
rods, fibers, tapes, threads, beads, particles, microtiter wells,
capillaries, and cylinders.
[0025] In another aspect, the invention provides a method of
determining gene expression profile in a sample, said method
comprising determining the amount of labeled polynucleotide
fragment from at least one polynucleotide fragment produced by a
method as described herein, wherein the amount is indicative of
amount of a polynucleotide template from which the polynucleotide
fragment was generated in a sample, whereby a gene expression
profile is determined. In one embodiment, the method comprises (a)
generating a labeled polynucleotide fragment from at least one
polynucleotide template in the sample using a method as described
herein; and (b) determining amount of labeled polynucleotide
fragment from a polynucleotide template, wherein said amount is
indicative of amount of the polynucleotide template in the sample,
whereby the a gene expression profile in the sample is determined.
In one embodiment, the polynucleotide template is RNA or mRNA. In
one embodiment, the amounts of a plurality of polynucleotide
fragments derived from a plurality of polynucleotide templates in a
sample is determined.
[0026] In another aspect, the invention provides a method of
generating hybridization probes, comprising generating a labeled
polynucleotide fragment using a method as described herein.
[0027] In another aspect, the invention provides a method for
nucleic acid hybridization, comprising hybridizing a labeled
polynucleotide fragment with at least one probe, wherein the
labeled polynucleotide fragment is generated using a method as
described herein. In one embodiment, the method comprises (a)
generating a labeled polynucleotide fragment using a method as
described herein; and (b) hybridizing the labeled polynucleotide
fragment with at least one probe.
[0028] In another aspect, the invention provides a method for
comparative hybridization, comprising comparing hybridization of a
first population of labeled polynucleotide fragments prepared using
a method as described herein to at least one probe with
hybridization of a second population of labeled polynucleotide to
the at least one probe. In one embodiment, the method comprises (a)
preparing a first population of labeled polynucleotides fragments
from a first template polynucleotide sample using a method as
described herein; and (b) comparing hybridization of the first
population to at least one probe with hybridization of a second
population of labeled polynucleotide. In one embodiment, the first
population and second population comprise detectably different
labels. In one embodiment, the second population of labeled
polynucleotides are prepared from a second polynucleotide sample
using a method as described herein. In one embodiment, comparing
comprises determining amount of said products, whereby the amount
of the first and second polynucleotide templates is quantified. In
one embodiment, the first and/or second template polynucleotides
from which the first and/or second populations of labeled
polynucleotides are prepared comprise genomic DNA.
[0029] In another aspect, the invention provides a method for
detecting presence or absence of a mutation in a template,
comprising analyzing a labeled polynucleotide fragment prepared by
a method as described herein, whereby presence of absence of a
mutation is detected. In one embodiment, the method comprises (a)
generating a labeled polynucleotide fragment by a method as
described herein; and (b) analyzing the labeled polynucleotide
fragment, whereby presence or absence of a mutation is detected. In
one embodiment, the labeled polynucleotide fragment is compared to
a reference template. In various embodiments, the mutation is
selected from the group consisting of a base substitution, a base
insertion, a base deletion, and a single nucleotide
polymorphism.
[0030] In another aspect, the invention provides a composition
comprising: (a) an agent capable of cleaving (i.e., which cleaves)
a base portion of a nucleotide to generate an abasic site in a
polynucleotide; (b) an agent capable of cleaving (i.e., which
cleaves) a phosphodiester backbone at or near an abasic site to
produce a polynucleotide fragment with a blocked 3' end; and (c) an
enzyme capable of unblocking (i.e., which unblocks) a blocked 3'
end to generate a polynucleotide comprising a 3' hydroxyl group. In
one embodiment, (a) is an N-glycosylase, (b) is a polyamine, and
(c) is an enzyme comprising a 3' exonuclease activity. In one
embodiment, (a) is UNG, (b) is DMED, and (c) is selected from the
group consisting of endonuclease 4, exonuclease T, and APE 1.
[0031] In another aspect, the invention provides a kit comprising:
(a) an agent capable of cleaving (i.e., which cleaves) a base
portion of a nucleotide to generate an abasic site in a
polynucleotide; (b) an agent capable of cleaving (i.e., which
cleaves) a phosphodiester backbone at or near an abasic site to
produce a polynucleotide fragment with a blocked 3' end; and (c) an
enzyme capable of unblocking (i.e., which unblocks) a blocked 3'
end to generate a polynucleotide comprising a 3' hydroxyl group. In
one embodiment, the kit further comprises: (d) an agent capable of
labeling (i.e., which labels) a 3' hydroxyl group of a
polynucleotide. In one embodiment, (a) is an N-glycosylase, (b) is
a polyamine, (c) is an enzyme comprising a 3' exonuclease activity;
and (d) is a template independent polymerase. In one embodiment,
(a) is UNG, (b) is DMED, (c) is selected from the group consisting
of endonuclease 4, exonuclease T, and APE 1; and (d) is TdT. In
some embodiments, the kit further comprises a non-canonical
nucleotide. In some embodiments, the kit further comprises a
non-canonical nucleotide and an enzyme capable of synthesizing a
polynucleotide comprising the non-canonical nucleotide. In one
embodiment, the non-canonical nucleotide is dUTP and the agent
capable of cleaving a base portion of a nucleotide to generate an
abasic site in a polynucleotide is UNG.
[0032] In some embodiments, the kit further comprises: (e) a
labeled nucleotide. In one embodiment, (a) is an N-glycosylase, (b)
is a polyamine, (c) is an enzyme comprising a 3' exonuclease
activity; (d) is a template independent polymerase; and (e) is a
biotinylated nucleotide. In some embodiments, (e) is selected from
the group consisting of a biotinylated nucleotide triphosphate
(NTP), a biotinylated deoxynucleotide triphosphate (dNTP), and a
biotinylated dideoxynucleotide triphosphate (ddNTP). In one
embodiment, (a) is UNG, (b) is DMED, (c) is selected from the group
consisting of endonuclease 4, exonuclease T, and APE 1; (d) is TdT,
and (e) is selected from the group consisting of biotin
2',3'-dideoxy-UTP and biotin 2',3'-dideoxy-CTP.
[0033] In some embodiments, a kit of the invention comprises, in
addition to the components described above, a template dependent
DNA polymerase; a composite primer, wherein the composite primer
comprises a 5' RNA portion and a 3' DNA portion; and an agent
capable of cleaving RNA from an RNA-DNA hybrid. In some
embodiments, the RNA portion of the composite primer is 5' with
respect to the 3' DNA portion, the 5' RNA portion is adjacent to
the 3' DNA portion, the RNA portion of the composite primer
consists of about 5 to about 50 nucleotides and the DNA portion of
the composite primer consists of 1 to about 20 nucleotides. In one
embodiment, the agent that cleaves RNA from an RNA-DNA hybrid is
RNAse H.
[0034] Kits of the invention generally comprise packaging, and may
comprise instructions for use in a method for polynucleotide
fragmentation, or polynucleotide fragmentation and labeling, as
described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 schematically depicts an embodiment of a nucleic acid
fragmentation and labeling procedure as described herein. A single
stranded nucleic acid comprising non-canonical nucleotide dU (A) is
used to generate a nucleic acid with abasic sites (B).
Non-canonical nucleotides are denoted as "U" in nucleic acid A and
canonical nucleotides are denoted as "N." Abasic sites are denoted
as "X" in nucleic acid B. The nucleic acid is fragmented at the
abasic sites to generate fragments with blocked 3' termini (C). A
3'-5' exonuclease is used to unblock the 3' termini of fragments C,
thereby generating nucleic acid fragments with 3' hydroxyl groups
(D). The 3' hydroxyl groups serve as substrates for end labeling
using terminal transferase and labeled nucleotide triphosphates,
thereby producing labeled nucleic acid fragments (E). The end
labels are denoted as "L" in fragments E.
[0036] FIG. 2 shows data from an experiment comparing the ability
of various 3' exonucleases to unblock blocked 3' ends of
polynucleotide fragments, as described in Example 1.
[0037] FIG. 3 shows data from an experiment demonstrating the
ability of terminal deoxynucleotidyl transferase (TdT) to label 3'
hydroxyl groups of polynucleotide fragments which after blocked 3'
ends were unblocked by treatment with apurinic/apyrimidinic
exonuclease 1 (APE 1), as described in Example 2.
DETAILED DESCRIPTION
[0038] Methods for Generating Fragmented Polynucleotides with 3'
End Hydroxyl Groups
[0039] The invention provides novel methods, compositions, and kits
for fragmenting polynucleotides to generated polynucleotide
fragments with hydroxyl groups at their 3' ends. The methods,
compositions, and kits of the invention are useful for fragmenting
and labeling polynucleotides. These methods are suitable for, for
example, generation of labeled polynucleotide fragments, for use as
hybridization probes, or generation of polynucleotide fragments
which may be hybridized to a polynucleotide template and extended
with a template dependent polymerase.
[0040] In methods of the invention, a polynucleotide is cleaved at
or near an abasic site present in the polynucleotide. The abasic
site may be prepared by cleavage of a base portion of a
non-canonical nucleotide present in the polynucleotide, cleavage of
a base portion of a canonical nucleotide present in the
polynucleotide, or cleavage of a base portion of a methylated
nucleotide present in the polynucleotide. For methods in which an
abasic site is generated by cleavage of a base portion of a
non-canonical nucleotide, the spacing of non-canonical nucleotides
in the polynucleotide to be fragmented and labeled, relates to and
determines the size of fragments and intensity of labeling. This
feature permits control of fragment size and/or site of labeling by
use of conditions permitting controlled incorporation of
non-canonical nucleotide, for example, during synthesis of the
polynucleotide comprising the non-canonical nucleotide from a
polynucleotide template.
[0041] Cleavage at or near an abasic site is generally effected
with an agent or under conditions which do not generate a 3'
hydroxyl group, i.e., conditions that generate a "blocked" 3' end
that contains a non-nucleotide moiety, such as an aldehyde group
(e.g., the sugar residue remaining behind after generation of the
abasic site), at the 3' end.
[0042] Generally, a chemical fragmentation method is used, wherein
a polynucleotide fragment comprising a blocked 3' end is generated.
In some embodiments, chemical fragmentation at or near an abasic
site is effected with a polyamine, such as, for example,
N,N'-dimethylethylenediamine (DMED), which produces polynucleotide
fragments with blocked 3' ends. Cleavage with a polyamine most
commonly employs a .beta.- or .beta.-.gamma. elimination mechanism.
.beta.-elimination results in cleavage of the 3'-phosphodiester
bond and a remnant of the sugar moiety (an aldehyde) is attached to
the 3' end. Depending on the cleavage agent used, other
modifications of the 3' end are also possible, such as a
3'-phosphoglycolate group. Blocked 3' ends may be unblocked by
digestion with an enzyme capable of removing the attached blocking
moiety, such as a 3' to 5' exonuclease, preferably a non-processive
exonuclease, thereby producing polynucleotide fragments with a 3'
end hydroxyl group. In some embodiments, an enzyme that comprises
an exonuclease activity and does not comprise endonuclease activity
is used. In some embodiments, an enzyme that comprises both
exonuclease and endonuclease activities is used. In one embodiment,
an enzyme that comprises both exonuclease and endonuclease
activities is used under conditions in which the endonuclease
activity is substantially minimized or absent. In some embodiments,
endonuclease 4, exonuclease T, or the 3' to 5' exonuclease activity
of the apurinic/apyrimidinic endonuclease (APE 1) is used.
[0043] In some embodiments, polynucleotide fragments with unblocked
3' ends are labeled with an agent capable of labeling at 3'
hydroxyl groups of polynucleotides. In some embodiments, a template
independent polymerase is used for labeling. In one embodiment, the
template independent polymerase is terminal deoxynucleotidyl
transferase (TdT), an enzyme which is capable of attaching one or
more nucleotides (i.e., labeled nucleotides, unlabeled nucleotides,
or a mixture of labeled and unlabeled nucleotides) at a
polynucleotide 3' end hydroxyl group by extension of the
polynucleotide from the 3' end. "Labeled" or "detectable"
nucleotide or polynucleotide, as used herein, refers to a
nucleotide (or nucleotide analog thereof) or polynucleotide that is
directly or indirectly detectable. A nucleotide may comprise a
directly-detectable label such as, for example, a fluorophore
(e.g., cy dyes, alexa dyes, fluorescein, etc.), an enzyme, a
chromophore, or a radiolabel, or the nucleotide may comprise an
indirectly-detectable label such as a hapten which is detectable by
binding of a labeled second member of a specific binding pair, such
as, for example, biotin/avidin or streptavidin, antigen/antibody,
etc., and the label attached to the second member of the binding
pair may be, for example, a fluorophore, an enzyme, a chromophore,
or a radiolabel, or the second member of the binding pair may be
attached to a detectable particle. In one embodiment, the
nucleotide is a biotinylated nucleotide, such as, for example,
biotin 2',3'-dideoxy-UTP, and is detectable by binding of labeled
avidin or streptavidin.
[0044] In some embodiments, a polynucleotide fragment with an
unblocked 3' end, produced as described herein, is hybridized to a
polynucleotide template and extended from the 3' hydroxyl group
with a template-dependent polymerase, using labeled or unlabeled
nucleotides, or a mixture of labeled or unlabeled nucleotides. When
labeled polynucleotides are incorporated, a labeled polynucleotide
extension product is produced.
[0045] In some embodiments, a polynucleotide fragment with an
unblocked 3' end, produced as described herein, is ligated to
another polynucleotide with a ligase enzyme. If the polynucleotide
to which the polynucleotide fragment is ligated is labeled, a
labeled ligation product is produced.
[0046] In some embodiments, a polynucleotide fragment with an
unblocked 3' end, produced as described herein, is "tailed" using a
template-independent polymerase, wherein a "tail" of nucleotides,
i.e., labeled or unlabeled nucleotides or a mixture thereof, is
added at the 3' end of the fragment.
Generation of Abasic Sites in Polynucleotides Comprising
Non-Canonical Nucleotides
[0047] In one aspect, the invention provides methods for
fragmenting and labeling a polynucleotide comprising an abasic site
produced by incorporation of a non-canonical nucleotide. The
methods generally comprise generation of a polynucleotide
comprising a non-canonical nucleotide, cleavage of a base portion
of the non-canonical nucleotide present in the polynucleotide with
an agent (such as an enzyme) capable of cleaving a base portion of
the non-canonical nucleotide (whereby an abasic site is generated);
chemical cleavage of the phosphodiester backbone at or near the
abasic site with an agent or under conditions that do not generate
a 3' hydroxyl group, i.e., conditions that generate a
polynucleotide fragment having a blocked 3' end; digestion of
fragments with an enzyme capable of generating a 3' end with a 3'
hydroxyl group from a blocked 3' end; and optionally labeling at
the 3' hydroxyl group with an enzyme or agent capable of attaching
a label to the 3' hydroxyl group, whereby labeled polynucleotide
fragments are generated.
[0048] The methods of fragmenting and labeling a polynucleotide
generally comprise synthesis of a polynucleotide comprising a
non-canonical nucleotide from a polynucleotide template in the
presence of a non-canonical nucleotide, whereby a polynucleotide
comprising a non-canonical nucleotide(s) is generated.
[0049] Non-canonical nucleotides are known in the art and any
suitable non-canonical nucleotide can be used. In some embodiments,
two or more different non-canonical nucleotides are used, such that
a polynucleotide comprising two or more non-canonical nucleotides
is generated. Methods for synthesizing polynucleotides from a
polynucleotide template are known in the art and described herein,
and any suitable method can be used in the methods of the
invention. In some embodiments, synthesis of the polynucleotide
comprising the non-canonical nucleotides comprises SPIA.TM. (single
primer isothermal amplification; see Kurn, U.S. Pat. Nos. 6,251,639
and 6,692,918), Ribo-SPIA.TM. (see Kurn, U.S. Pat. No. 6,946,251),
PCR, primer extension, reverse transcription, strand displacement
amplification (SDA), multiple displacement amplification (MDA),
rolling circle amplification (RCA), nick translation based DNA
synthesis, DNA replication, and the like. The polynucleotide that
is synthesized can be single stranded, double-stranded or partially
double stranded, and either or both strands can comprise a
non-canonical nucleotide. In some embodiments, the polynucleotide
that is synthesized comprises a cDNA. The polynucleotide template
(from which the polynucleotide comprising a non-canonical
nucleotide is synthesized) is any template from which one desires
to produce polynucleotide fragments or labeled fragments thereof In
some embodiments, the template comprises RNA, mRNA, genomic DNA,
cDNA, or synthetic DNA. In other embodiments, the template
comprises a cDNA library, a subtractive hybridization library, or a
genomic library. In one embodiment, the polynucleotide comprising
the non-canonical nucleotide is synthesized using limited and/or
controlled incorporation of the non-canonical nucleotide, which
results in generation of a polynucleotide with a frequency or
proportion of non-canonical nucleotides such that, labeled
fragments of a desired size (or size range) are generated
(following production of an abasic site, cleavage of the
phosphodiester backbone at or near an abasic site with an agent or
under conditions in which a 3' end comprising a hydroxyl group is
not produced (i.e., production of a fragments with blocked hydroxyl
groups), generation of hydroxyl groups at the 3' ends on the
polynucleotide fragments (i.e., by unblocking the blocked 3' ends,
for example, with an enzyme comprising a 3' to 5' exonuclease
activity), and labeling of the polynucleotide fragments using
polymerase extension from the 3' hydroxyl groups in the presence of
a detectable nucleotide or ligation to a detectable polynucleotide
(e.g., extension at the 3' hydroxyl groups with a template
independent polymerase such as terminal transferase to incorporate
one or more detectable nucleotides at the 3' ends).
[0050] In some embodiments, a labeled primer is used during
synthesis of the polynucleotide comprising a non-canonical
nucleotide. In other embodiments, a primer comprising a
non-canonical nucleotide (such as dUTP) is used during synthesis of
the polynucleotide comprising a non-canonical nucleotide. In other
embodiments, the primer is a composite primer, said composite
primer comprising a RNA portion and a 3' DNA portion.
[0051] It is understood that a polynucleotide comprising a
non-canonical nucleotide can be a multiplicity (from small to very
large) of different polynucleotide molecules. Such populations can
be related in sequence (e.g., members of a gene family or
superfamily) or extremely diverse in sequence (e.g., generated from
all mRNA, generated from all genomic DNA, etc.). Polynucleotides
can also correspond to single sequences (which can be part or all
of a known gene, for example, a coding region, genomic portion,
etc.).
[0052] A base portion of the non-canonical nucleotide is cleaved by
an agent (such as an enzyme) capable of cleaving a base portion of
a non-canonical nucleotide. Such agents are known in the art and
described herein. In one embodiment, the agent capable of
specifically cleaving a base portion of a non-canonical nucleotide
is an N-glycosylase. In another embodiment, the agent is Uracil
N-Glycosylase (interchangeably termed "UNG" or "uracil DNA
glyosylase").
Generation of Abasic Sites in Polynucleotides Comprising Methylated
Nucleotides
[0053] In one aspect, the invention provides methods for
fragmenting and labeling a polynucleotide comprising an abasic site
produced by cleaving a base portion of a methylated nucleotide with
an agent capable of cleaving a base portion of the methylated
nucleotide to create an abasic site, whereby an abasic site is
generate. The methods generally comprise cleavage of a base portion
of a methylated nucleotide with an agent (such as an enzyme)
capable of cleaving a base portion of a methylated nucleotide
(whereby an abasic site is generated); chemical cleavage of the
phosphodiester backbone at or near the abasic site with an agent or
under conditions that do not generate a 3' hydroxyl group, i.e.,
conditions that generate a polynucleotide fragment with a blocked
3' end; digestion of fragments with an enzyme capable of generating
a polynucleotide containing a hydroxyl group at the 3' end; and
optionally labeling at the 3' end with an enzyme or agent capable
of attaching a label to the 3' hydroxyl group, whereby labeled
polynucleotide fragments are generated (3' end labeling).
Generation of Abasic Sites in Polynucleotides by Cleaving Base
Portions of Canonical Nucleotides
[0054] In one aspect, the invention provides methods for
fragmenting and labeling a polynucleotide comprising an abasic site
produced by cleaving a base portion of a canonical nucleotide with
an agent capable of cleaving a base portion of the canonical
nucleotide to create an abasic site, whereby an abasic site is
generated. The methods generally comprise cleavage of a base
portion of a canonical nucleotide with an agent (such as an enzyme)
capable of cleaving a base portion of a methylated nucleotide
(whereby an abasic site is generated); chemical cleavage of the
phosphodiester backbone at or near the abasic site with an agent or
under conditions that do not generate a 3' hydroxyl group, i.e.,
conditions that generate a polynucleotide fragment with a blocked
3' end; digestion of fragments with an enzyme capable of generating
a polynucleotide containing a hydroxyl group at the 3' end; and
optionally labeling at the 3' hydroxyl group with an enzyme or
agent capable of attaching a label to the 3' hydroxyl group,
whereby labeled polynucleotide fragments are generated.
Fragmentation at Abasic Sites to Produce Polynucleotide Fragments
with Blocked 3' Ends
[0055] In methods of the invention, the phosphodiester backbone of
a polynucleotide comprising an abasic site is cleaved at or near
the abasic site by an agent capable of cleaving the phosphodiester
backbone at or near an abasic site, such that two or more fragments
are produced. As used herein, "cleaving the backbone or
phosphodiester backbone" is also termed "fragmentation" or
"fragmenting." Fragmentation of the polynucleotide comprising an
abasic site is conducted with an agent or under conditions in which
polynucleotides comprising hydroxyl groups at their 3' ends are
substantially not produced. Generally, a chemical fragmentation
agent is used, producing polynucleotide fragments with blocked 3'
ends. In some embodiments, a polyamine, such as DMED, is used for
fragmentation.
[0056] Generally, cleavage occurs 3' to the abasic site (e.g.,
cleavage between the deoxyribose ring and 3'-phosphate group of the
abasic residue and the deoxyribose ring of the adjacent nucleotide,
generating a free 5' phosphate group on the deoxyribose ring of the
adjacent nucleotide), such that an abasic site is located at the 3'
end of the resulting fragment. In still other embodiments, more
complex forms of cleavage are possible, for example, cleavage such
that cleavage of the phosphodiester backbone and cleavage of a
portion of the abasic nucleotide results. Selection of reaction
conditions also permits control of the degree, level or
completeness of the fragmentation reactions. In some embodiments,
reaction conditions can be selected such that the cleavage reaction
is performed in the presence of a large excess of reagents and
allowed to run to completion with minimal concern about excessive
cleavage of the polynucleotide (i.e., while retaining a desired
fragment size, which may be determined by spacing of the
incorporated non-canonical nucleotide, during the synthesis step,
above). In other embodiments, reaction conditions are selected such
that fragmentation is not complete (in the sense that the backbone
at some abasic sites remains uncleaved (unfragmented), such that
polynucleotide fragments comprising more than one abasic site are
generated. Such fragments comprise internal (unfragmented) abasic
sites.
Unblocking of Blocked 3' Ends
[0057] The polynucleotide fragments generated by cleavage of the
phosphodiester backbone are contacted with an agent, such as an
enzyme comprising an exonuclease activity, that is capable of
generating a polynucleotide comprising a hydroxyl group at the 3'
end from a polynucleotide comprising a blocked 3' end. Preferably,
a non-processive exonuclease is used, such as, for example, APE 1.
The resulting polynucleotide fragments have unblocked 3' ends,
i.e., comprising a hydroxyl group at the 3' end.
[0058] In some embodiments, a polynucleotide fragment comprising a
3' end hydroxyl group, produced as described herein, is extended
with a template independent or template dependent polymerase. In
one embodiment, the polynucleotide fragment may be extended with a
template independent polymerase, such as a terminal transferase, to
incorporate one or more labeled nucleotide residue (or nucleotide
analogs thereof), one or more unlabeled nucleotide residue (or
nucleotide analogs thereof) or a mixture of labeled and unlabeled
nucleotide residues (or nucleotide analogs thereof), at the 3' end.
In another embodiment, the polynucleotide fragment is hybridized to
a polynucleotide template and extended by a template dependent
polymerase. In another embodiment, the polynucleotide fragment is
ligated to another polynucleotide with an enzyme comprising a
ligase enzyme.
Labeling of Polynucleotide Fragments at 3' Hydroxyl Groups
[0059] Agents capable of labeling at a 3' hydroxyl group of a
polynucleotide are known in the art. For example, a template
independent polymerase, such as TdT, can be used to attach one or
more labeled nucleotides at the 3' hydroxyl group (i.e., extend
from the 3' end). In some embodiments, the detectable moiety
(label) is directly or indirectly detectable. In some embodiments,
the detectable signal is amplified. In some embodiments, the
detectable moiety comprises an organic molecule. In other
embodiments, the detectable moiety comprises an antibody. In other
embodiments, the detectable signal is fluorescent. In other
embodiments, the detectable signal is enzymatically generated. In
one embodiment, the fragments are labeled by template independent
extension with TdT, using a labeled nucleotide triphosphate (or
labeled nucleotide analog thereof). In one embodiment, the labeled
nucleotide is a biotinylated nucleotide, such as, for example,
biotin 2',3'-dideoxy-UTP, biotin-dUTP, or biotin-UTP. Other labeled
nucleotides, e.g., dNTPs or NTPs, or terminator nucleotides such as
2',3'-dideoxy-NTPs), or combinations thereof, as well as
combinations of labeled and unlabeled nucleotides or
dideoxy-nucleotides, may also be used.
[0060] The methods of the invention include methods of using
polynucleotide fragments and labeled polynucleotides produced by
the methods of the invention (so-called "applications"). The
invention provides methods to characterize (for example, detect
presence or absence of and/or quantify) a sequence of interest by
analyzing fragmented and labeled products by
detection/quantification methods such as those based on array
technologies or solution phase technologies. In some embodiments,
the invention provides methods of detecting the presence or absence
of mutations.
[0061] In other embodiments, the invention provides methods of
producing a hybridization probe; hybridization using the
hybridization probes; detection using the hybridization probes;
characterizing and/or quantitating nucleic acid; preparing a
subtractive hybridization probe; comparative genomic hybridization;
and determining a gene expression profile, using the fragmented
nucleic acids generated by the methods of the invention.
General Techniques
[0062] The practice of the invention will employ, unless otherwise
indicated, conventional techniques of molecular biology (including
recombinant techniques), microbiology, cell biology, biochemistry,
and immunology, which are within the skill of the art. Such
techniques are explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook
et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984);
"Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in
Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular
Biology" (F. M. Ausubel et al., eds., 1987, and periodic updates);
"PCR: The Polymerase Chain Reaction", (Mullis et al., eds.,
1994).
[0063] Primers, oligonucleotides and polynucleotides employed in
the invention can be generated using standard techniques known in
the art.
Definitions
[0064] A "template sequence," or "template nucleic acid" or
"template" as used herein, is a polynucleotide comprising a
sequence of interest, for which synthesis of a complement
comprising a non-canonical nucleotide is desired. The template
sequence may be known or not known, in terms of its actual
sequence. In some instances, the terms "target," "template," and
variations thereof, are used interchangeably.
[0065] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA. The nucleotides can be deoxyribonucleotides, modified
nucleotides or bases, and/or their analogs, or any substrate that
can be incorporated into a polymer by DNA polymerase. Nucleotides
include canonical and non-canonical nucleotides and a
polynucleotide can comprise canonical and non-canonical
nucleotides. A polynucleotide may comprise modified (altered)
nucleotides, such as, for example, modification to the nucleotide
structure and or modification to the phosphodiester backbone. As
discussed herein modified nucleotide can be canonical nucleotide or
non-canonical (cleavable) nucleotides. It is understood, however,
that modified nucleotides that are not non-canonical (cleavable)
nucleotide under the reaction conditions used in the methods of the
invention, if present, generally should not affect the ability of
the polynucleotide to undergo cleavage of a base portion of
non-canonical nucleotide, such that an abasic site is generated,
and/or cleavage of a phosphodiester backbone at an abasic site,
such that fragments are generated, and/or immobilization of a
polynucleotide (or fragment thereof) to a substrate, as described
herein. If present, modification to the nucleotide structure, such
as methylated nucleotides may be imparted before or after assembly
of the polymer. The sequence of nucleotides may be interrupted by
non-nucleotide components. A polynucleotide may be further modified
after polymerization, such as by conjugation with a labeling
component. Other types of modifications include, for example,
"caps", substitution of one or more of the naturally occurring
nucleotides with an analog, internucleotide modifications such as,
for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies,
signal peptides, ply-L-lysine, etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). It is understood that internucleotide
modifications may, e.g., alter the efficiency and/or kinetics of
cleavage of the phosphodiester backbone (as when, for example a
phosphodiester backbone is cleaved at an abasic site, as described
herein). Further, any of the hydroxyl groups ordinarily present in
the sugars may be replaced, for example, by phosphonate groups,
phosphate groups, protected by standard protecting groups, or
activated to prepare additional linkages to additional nucleotides.
The 5' and 3' terminal OH can be phosphorylated or substituted with
amines or organic capping groups moieties of from 1 to 20 carbon
atoms. Other hydroxyls may also be derivatized to standard
protecting groups. Polynucleotides can also contain analogous forms
of ribose or deoxyribose sugars that are generally known in the
art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-
or 2'-azido-ribose, carbocyclic sugar analogs, .alpha.-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs. One or more phosphodiester linkages
may be replaced by alternative linking groups. These alternative
linking groups include, but are not limited to, embodiments wherein
phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"),
"(O)NR.sub.2 ("amidate"), P(O)R, P(O)OR', CO or CH.sub.2
("formacetal"), in which each R or R' is independently H or
substituted or unsubstituted alkyl (1-20 C) optionally containing
an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl
or araldyl. Not all linkages in a polynucleotide need be identical.
The preceding description applies to all polynucleotides referred
to herein, including DNA. It is understood, however, that modified
nucleotides and/or internucleotide linkages and/or, if present,
generally should not affect the ability of the polynucleotide to
undergo cleavage of a base portion of a non-canonical nucleotide,
such that an abasic site is generated, and/or the ability of a
polynucleotide to undergo cleavage of a phosphodiester backbone at
an abasic site, such that fragments are generated, and/or the
ability of a polynucleotide to be immobilized at an abasic site
(such as an abasic site at an end of a polynucleotide and/or an
abasic site that is not at an end of a polynucleotide) to a
surface, as described herein.
[0066] "Oligonucleotide," as used herein, generally refers to
short, generally single stranded, generally synthetic
polynucleotides that are generally, but not necessarily, less than
about 200 nucleotides in length. The terms "oligonucleotide" and
"polynucleotide" are not mutually exclusive. The description above
for polynucleotides is equally and fully applicable to
oligonucleotides.
[0067] A "primer," as used herein, refers to a nucleotide sequence
(a polynucleotide), generally with a free 3'-OH group, that
hybridizes with a template sequence (such as a template RNA, or a
primer extension product) and is capable of promoting
polymerization of a polynucleotide complementary to the template. A
"primer" can be, for example, an oligonucleotide. It can also be,
for example, a sequence of the template (such as a primer extension
product or a fragment of an RNA template created following RNase
cleavage of a template RNA-DNA complex) that is hybridized to a
sequence in the template itself (for example, as a hairpin loop),
and that is capable of promoting nucleotide polymerization. Thus, a
primer can be an exogenous (e.g., added) primer or an endogenous
(e.g., template fragment) primer.
[0068] A "complex" is an assembly of components. A complex may or
may not be stable and may be directly or indirectly detected. For
example, as is described herein, given certain components of a
reaction, and the type of product(s) of the reaction, existence of
a complex can be inferred. For purposes of this invention, a
complex is generally an intermediate with respect to the final
polynucleotide fragments, labeled polynucleotide, labeled
polynucleotide fragments, and/or immobilized polynucleotide or
fragment thereof.
[0069] A "fragment" of a polynucleotide or oligonucleotide is a
contiguous sequence of 2 or more bases. In other embodiments, a
fragment (also termed "region" or "portion") is any of about 3,
about 5, about 10, about 15, about 20, about 25, about 30 about 35
about 40, about 50, about 65, about 75, about 85, about 100, about
125, about 150, about 175, about 200, about 225, about 250, about
300, about 350, about 400, about 450, about 500, about 550, about
600, about 650 or more nucleotides in length. In some embodiments,
the fragments can be at least about 3, about 5, about 10, about 15,
about 20, about 25, about 30 about 35 about 40, about 50, about 65,
about 75, about 85, about 100, about 125, about 150, about 175,
about 200, about 225, about 250, about 300, about 350, about 400,
about 450, about 500, about 550, about 600, about 650 or more
nucleotides in length. In other embodiments, the fragments can be
less than about 3, about 5, about 10, about 15, about 20, about 25,
about 30 about 35 about 40, about 50, about 65, about 75, about 85,
about 100, about 125, about 150, about 175, about 200, about 225,
about 250, about 300, about 350, about 400, about 450, about 500,
about 550, about 600, about 650 or more nucleotides in length. In
some embodiment, these fragment lengths represent an average size
in the population of fragments generated using the methods of the
invention.
[0070] A "reaction mixture" is an assemblage of components, which,
under suitable conditions, react to form a complex (which may be an
intermediate) and/or a product(s).
[0071] "A", "an" and "the", and the like, unless otherwise
indicated include plural forms. "A" fragment means one or more
fragments. "A" non-canonical nucleotide means one or more
non-canonical nucleotides.
[0072] Conditions that "allow" or "permit" an event to occur or
conditions that are "suitable" for an event to occur, such as
polynucleotide synthesis, cleavage of a base portion of a
non-canonical nucleotide, cleavage of a phosphodiester backbone at
an abasic site, and the like, or "suitable" conditions are
conditions that do not prevent such events from occurring. Thus,
these conditions permit, enhance, facilitate, and/or are conducive
to the event. Such conditions, known in the art and described
herein, depend upon, for example, the nature of the polynucleotide
sequence, temperature, and buffer conditions. These conditions also
depend on what event is desired, such as polynucleotide synthesis,
cleavage of a base portion of a non-canonical nucleotide, cleavage
of a phosphodiester backbone at an abasic site, labeling an abasic
site, immobilizing a polynucleotide fragment or a polynucleotide,
etc.
[0073] "Microarray" and "array," as used interchangeably herein,
comprise a surface with an array, preferably ordered array, of
putative binding (e.g., by hybridization) sites for a biochemical
sample (target) which often has undetermined characteristics. In a
preferred embodiment, a microarray refers to an assembly of
distinct polynucleotide or oligonucleotide probes immobilized at
defined positions on a substrate. Arrays are formed on substrates
fabricated with materials such as paper, glass, plastic (e.g.,
polypropylene, nylon, polystyrene), polyacrylamide, nitrocellulose,
silicon and other metals, optical fiber or any other suitable solid
or semi-solid support, and configured in a planar (e.g., glass
plates, silicon chips) or three-dimensional (e.g., pins, fibers,
beads, particles, microtiter wells, capillaries) configuration.
Probes forming the arrays may be attached to the substrate by any
number of ways including (i) in situ synthesis (e.g., high-density
oligonucleotide arrays) using photolithographic techniques (see,
Fodor et al., Science (1991), 251:767-773; Pease et al., Proc.
Natl. Acad. Sci. U.S.A. (1994), 91:5022-5026; Lockhart et al.,
Nature Biotechnology (1996), 14:1675; U.S. Pat. Nos. 5,578,832;
5,556,752; and 5,510,270); (ii) spotting/printing at medium to
low-density (e.g., cDNA probes) on glass, nylon or nitrocellulose
(Schena et al, Science (1995), 270:467-470, DeRisi et al, Nature
Genetics (1996), 14:457-460; Shalon et al., Genome Res. (1996),
6:639-645; and Schena et al., Proc. Natl. Acad. Sci. U.S.A. (1995),
93:10539-11286); (iii) by masking (Maskos and Southern, Nuc. Acids.
Res. (1992), 20:1679-1684) and (iv) by dot-blotting on a nylon or
nitrocellulose hybridization membrane (see, e.g., Sambrook et al.,
Eds., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol.
1-3, Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y.)).
Probes may also be noncovalently immobilized on the substrate by
hybridization to anchors, by means of magnetic beads, or in a fluid
phase such as in microtiter wells or capillaries. The probe
molecules are generally nucleic acids such as DNA, RNA, PNA, and
cDNA but may also include proteins, polypeptides, oligosaccharides,
cells, tissues and any permutations thereof which can specifically
bind the target molecules.
[0074] The term "3'" generally refers to a region or position in a
polynucleotide or oligonucleotide 3' (downstream) from another
region or position in the same polynucleotide or
oligonucleotide.
[0075] The term "5'" generally refers to a region or position in a
polynucleotide or oligonucleotide 5' (upstream) from another region
or position in the same polynucleotide or oligonucleotide.
[0076] The term "3'-DNA portion," "3'-DNA region," "3'-RNA
portion," and "3'-RNA region," refer to the portion or region of a
polynucleotide or oligonucleotide located towards the 3' end of the
polynucleotide or oligonucleotide, and may or may not include the
3' most nucleotide(s) or moieties attached to the 3' most
nucleotide of the same polynucleotide or oligonucleotide. The 3'
most nucleotide(s) can be preferably from about 1 to about 50, more
preferably from about 10 to about 40, even more preferably from
about 20 to about 30 nucleotides.
[0077] As used herein, "canonical" nucleotide means a nucleotide
comprising one the four common nucleic acid bases adenine,
cytosine, guanine and thymine that are commonly found in DNA. The
term also encompasses the respective deoxyribonucleosides,
deoxyribonucleotides or 2'-deoxyribonucleoside-5'-triphosphates
that contain one of the four common nucleic acid bases adenine,
cytosine, guanine and thymine (though as explained herein, the base
can be a modified and/or altered base as discussed, for example, in
the definition of polynucleotide). As used herein, the base
portions of canonical nucleotides are generally not cleavable under
the conditions used in the methods of the invention.
[0078] As used herein, "non-canonical nucleotide" (interchangeably
called "non-canonical deoxyribonucleoside triphosphate") refers to
a nucleotide comprising a base other than the four canonical bases.
The term also encompasses the respective deoxyribonucleosides,
deoxyribonucleotides or 2'-deoxyribonucleoside-5'-triphosphates
that contain a base other than the four canonical bases. In the
context of this invention, nucleotides containing uracil (such as
dUTP), or the respective deoxyribonucleosides, deoxyribonucleotides
or 2'-deoxyribonucleoside-5'-triphosphates, are a non-canonical
nucleotides. As used herein, the base portions of non-canonical
nucleotides are capable of being, generally, specifically or
selectively cleaved (such that a nucleotide comprising an abasic
site is created) under the reaction conditions used in the methods
of the invention. As described herein, non-canonical nucleotides
are generally also capable of being incorporated into a
polynucleotide during synthesis of a polynucleotide (during e.g.,
primer extension and/or replication); capable of being generally,
specifically or selectively cleaved by an agent that cleaves a base
portion of a nucleotide, such that a polynucleotide comprising an
abasic site is generated; comprise a suitable internucleotide
connection (when incorporated into a polynucleotide) such that a
phosphodiester backbone at an abasic site (i.e., the non-canonical
nucleotide following cleavage of a base portion) is capable of
being cleaved by an agent capable of such cleavage; capable of
being labeled (following generation of an abasic site); and/or
capable of immobilization to a surface (following generation of an
abasic site), according to the methods described herein. It is
understood that the non-canonical nucleotide may, but does not
necessarily, require all of the features described above, depending
on the particular method of the invention in which the
non-canonical nucleotide is to be used. In some embodiments,
non-canonical nucleotides are altered and/or modified nucleotides
as described herein. Non-canonical nucleotide refers to a
nucleotide that is incorporated into a polynucleotide as well as to
a single nucleotide.
[0079] The term "analyte" as used herein refers to a substance to
be detected or assayed by the method of the present invention, for
example, a compound whose properties, location, quantity and/or
identity is desired to be characterized. Typical analytes may
include, but are not limited to proteins, peptides, nucleic acid
segments, cells, microorganisms and fragments and products thereof,
organic molecules, inorganic molecules, or any substance for which
immobilization sites for binding partner(s) can be developed. As
this disclosure clearly conveys, an analyte is a substrate.
[0080] As used herein, an "abasic site" refers to the site of
incorporation of the non-canonical nucleotide following treatment
with an agent capable of effecting cleavage of a base portion of
the non-canonical nucleotide. An abasic site (interchangeably
termed "AP site") can comprise a hemiacetal ring, and lacks a base
portion of the non-canonical nucleotide. As used herein, "abasic
site" encompasses any chemical structure remaining following
treatment of a canonical or non-canonical nucleotide (present in a
polynucleotide chain) with an agent (e.g., an enzyme, or heat or
basic conditions) capable of effecting cleavage of a base portion
of a non-canonical nucleotide. Thus, an abasic site as used herein
includes a modified sugar moiety attached to the 3' terminus of
nicked polynucleotide, as when, for example, endonuclease III or
OGG1 protein are used to cleave the base portion of the
non-canonical nucleotide. See, e.g., Kow, (2000) Methods 22,
164-169 (e.g., FIG. 4).
[0081] As used herein, cleavage of a backbone (e.g., phosphodiester
backbone) "at" an abasic site means cleavage of the phosphodiester
linkage 3' to the abasic site or 5' to the abasic site, or both. As
the disclosure herein conveys, "at" an abasic site refers to
proximate or near location (such as immediately 3', immediately
5'). In still other embodiments, more complex forms of cleavage are
possible, for example, cleavage such that cleavage of the
phosphodiester backbone and cleavage of (a portion of) the abasic
nucleotide results.
[0082] As used herein, a "label" (interchangeably called a
"detectable moiety") refers to a moiety that is associated or
linked with a polynucleotide (interchangeably called "labeling").
The labeled polynucleotide may be directly or indirectly detected,
generally through a detectable signal. The detectable moiety
(label) can be attached (or associated) either directly or through
a non-interfering linkage group with other moieties capable of
specifically associating with one or more sites to be labeled. The
detectable moiety (label) may be covalently or non-covalently
associated as well as directly or indirectly associated.
[0083] As used herein, a "blocked 3' end" refers to a 3' end of a
polynucleotide fragment that contains a moiety, such as an aldehyde
group, e.g., the sugar residue attached to the nucleotide base that
was removed to create an abasic site, which is left behind at the
3' end of the polynucleotide as a result of cleaving a
polynucleotide at the abasic site as described herein, rather than
a hydroxyl group at the 3' end. "Unblocking" of the 3' end refers
to removal of the non-nucleotide moiety, e.g., an aldehyde group,
resulting in a polynucleotide having a hydroxyl group at the 3'
end.
[0084] The following are examples of the methods of the invention.
It is understood that various other embodiments may be practiced,
given the general description provided herein. For example,
reference to using an agent capable of cleaving a base portion of a
non-canonical nucleotide means that any of the agents capable of
cleaving a base portion of the non-canonical nucleotide described
herein may be used.
Methods for Labeling and Fragmenting Nucleic Acids
[0085] The invention provides methods for generating fragments of
nucleic acids. The methods generally comprise cleavage of the
phosphodiester backbone of a polynucleotide comprising an abasic
site at or near the abasic site, whereby a polynucleotide fragment
comprising a blocked 3' end is generated; and contacting the
polynucleotide fragment with an enzyme capable of unblocking the
blocked 3' end of the fragment, whereby a polynucleotide fragment
comprising a 3' hydroxyl group is generated. The polynucleotide
comprising an abasic site may be produced by cleaving the base
portion of a non-canonical nucleotide, a canonical nucleotide, or a
methylated nucleotide. In some embodiments, the polynucleotide
comprising an abasic site is cleaved at the abasic site with a
polyamine, thereby producing a polynucleotide fragment comprising a
blocked 3' end, wherein the blocked 3' end comprises a sugar
aldehyde group from the nucleotide residue from which the abasic
site was generated. In some embodiments, the polynucleotide
fragment comprising a blocked 3' end is contacted with an enzyme
comprising a 3' to 5' exonuclease activity, thereby unblocking the
blocked 3' end (e.g., removing a sugar aldehyde group or other
non-nucleotide moiety from the 3' end) to produce a polynucleotide
fragment comprising a 3' hydroxyl group.
[0086] In one embodiment, the invention provides a method for
fragmenting a polynucleotide comprising a blocked 3' end,
comprising unblocking the blocked 3' end with an agent capable of
unblocking the blocked 3' end of a polynucleotide, for example, an
enzyme comprising a 3'-5' exonuclease activity as described herein.
The polynucleotide comprising a blocked 3' end may be produced, for
example, by cleaving a polynucleotide comprising an abasic site at
or near the abasic site with an agent, for example, a polyamine,
capable of cleaving a polynucleotide at or near an abasic site to
produce a polynucleotide fragment comprising a blocked 3' end.
[0087] In some embodiments, polynucleotide fragments comprising 3'
hydroxyl groups produced as described herein are labeled. In one
embodiment, the polynucleotide fragment is labeled by extension at
the 3' end with a template independent polymerase, resulting in
addition of one or more labeled nucleotides (or nucleotides
thereof) to the 3' end of the fragment. In one embodiment, the
polynucleotide fragment is labeled by hybridizing the
polynucleotide fragment to a polynucleotide template, extending
from the 3' end with a template dependent polymerase, and
incorporating labeled nucleotides into the extended polynucleotide
fragment. In one embodiment, the polynucleotide fragment is labeled
by ligation at the 3' end to a labeled polynucleotide with a ligase
enzyme.
[0088] In one embodiment, a polynucleotide fragment comprising a 3'
end hydroxyl group, produced as described herein, is extended from
the 3' end using a template independent polymerase to produce a
tailed 3' end which may comprises labeled and/or unlabeled
nucleotides. Tailing of the fragment comprising a 3' end hydroxyl
group may comprise polymerizing a plurality of dNTPs or ribo-NTPs
at the 3' end.
[0089] For simplicity, individual steps of the labeling and
fragmentation method are discussed below. It is understood,
however, that the steps may be performed simultaneously and/or in
varied order, as discussed herein.
Synthesis of a Polynucleotide Comprising a Non-Canonical
Nucleotide
[0090] In some embodiments, methods of the invention comprise
generation of a polynucleotide comprising an abasic site by
cleaving the base portion of a non-canonical nucleotide in a
polynucleotide comprising a non-canonical nucleotide. The
polynucleotide comprising a non-canonical nucleotide may be
produced by synthesizing a polynucleotide from a template in the
presence of at least one non-canonical nucleotide (interchangeably
termed "non-canonical deoxyribonucleoside triphosphate"), whereby a
polynucleotide comprising a non-canonical nucleotide is generated.
The frequency of incorporation of non-canonical nucleotides into
the polynucleotide relates to the size of fragment produced using
the methods of the invention because the spacing between
non-canonical nucleotides in the polynucleotide comprising a
non-canonical nucleotide, along with the reaction conditions used,
determines the approximate size of the fragments resulting from
generation of an abasic site from the non-canonical nucleotide and
cleavage of the backbone at the abasic site, as described
herein.
[0091] Generally, the polynucleotide is DNA, though, as noted
herein, the polynucleotide can comprise altered and/or modified
nucleotides, internucleotide linkages, ribonucleotides, etc. As
generally used herein, it is understood that "DNA" applies to
polynucleotide embodiments.
[0092] Methods for synthesizing polynucleotides, e.g., single and
double stranded DNA, from a template are well known in the art, and
include, for example, single primer isothermal amplification
(SPIA.TM.), Ribo-SPIA.TM., PCR, reverse transcription, primer
extension, limited primer extension, replication (including rolling
circle replication), strand displacement amplification (SDA), nick
translation, multiple displacement amplification (MDA), rolling
circle amplification (RCA) and, e.g., any method that results in
synthesis of the complement of a template sequence such that at
least one non-canonical nucleotide can be incorporated into a
polynucleotide. See, e.g., Kurn, U.S. Pat. No. 6,251,639; Kurn, WO
02/00938; Kurn, U.S. Pat. No. 6,946,251, Kurn, U.S. Pat. No.
6,692,918; Mullis, U.S. Pat. No. 4,582,877; Wallace, U.S. Pat. No.
6,027,923; U.S. Pat. Nos. 5,508,178; 5,888,819; 6,004,744;
5,882,867; 5,710,028; 6,027,889; 6,004,745; 5,763,178; 5,011,769;
see also Sambrook (1989) "Molecular Cloning: A Laboratory Manual",
second edition; Ausebel (1987, and updates) "Current Protocols in
Molecular Biology"; Mullis, (1994) "PCR: The Polymerase Chain
Reaction". One or more methods known in the art can be used to
generate a polynucleotide comprising a non-canonical nucleotide. It
is understood that the polynucleotide comprising a non-canonical
nucleotide can be single stranded or double stranded or partially
double stranded, and that one or both strands of a double stranded
polynucleotide can comprise a non-canonical nucleotide. For
convenience, "DNA" is used herein to describe (and exemplify) a
polynucleotide. Suitable methods include methods that result in one
single- or double-stranded polynucleotide comprising a
non-canonical nucleotide (for example, reverse transcription,
production of double stranded cDNA, a single round of DNA
replication), as well as methods that result in multiple single
stranded or double stranded copies or copies of the complement of a
template (for example, single primer isothermal amplification or
Ribo-SPIA.TM. or PCR). In one embodiment, a single-stranded
polynucleotide comprising a non-canonical nucleotide is synthesized
using single primer isothermal amplification. See Kurn, U.S. Pat.
Nos. 6,251,639 and 6,692,918.
[0093] Generally, the polynucleotide comprising a non-canonical
nucleotide is generated from a template in the presence of all four
canonical nucleotides and at least one non-canonical nucleotide
under reaction conditions suitable for synthesis of
polynucleotides, including suitable enzymes and primers, if
necessary. Reaction conditions and reagents, including primers, for
synthesizing the polynucleotide comprising a non-canonical
nucleotide are known in the art, and further discussed herein. As
described herein, non-canonical nucleotides are generally capable
of polymerization (i.e., are substrates for DNA polymerase), and
capable of being rendered abasic following treatment with a
suitable agent capable of generally, specifically or selectively
cleaving a base portion of a non-canonical nucleotide. Suitable
non-canonical nucleotides are well-known in the art, and include:
deoxyuridine triphosphate (dUTP), deoxyinosine triphosphate (dITP),
5-hydroxymethyl deoxycytidine triphosphate (5-OH-Me-dCTP). See,
e.g., Jendrisak, U.S. Pat. No. 6,190,865 B1; Mol. Cell Probes
(1992)251-6. Generally, in embodiments in which a polynucleotide
comprising an non-canonical nucleotide serves as a template for
further amplification (e.g., as when multiple copies of a double
stranded polynucleotides comprising a non-canonical nucleotide are
synthesized, e.g., by PCR amplification), a polynucleotide
comprising a non-canonical nucleotide must be capable of serving as
a template for further amplification.
[0094] It is understood that two or more different non-canonical
nucleotides can be incorporated into the polynucleotide synthesized
from the template by DNA polymerase, whereby a polynucleotide
comprising at least two different non-canonical nucleotides is
generated.
[0095] Conditions for limited and/or controlled incorporation of a
non-canonical nucleotide are known in the art. See, e.g.,
Jendrisak, U.S. Pat. No. 6,190,865 B1; Mol. Cell Probes (1992)
251-6; Anal. Biochem. (1993) 211:164-9; see also Sambrook (1989)
"Molecular Cloning: A Laboratory Manual", second edition; Ausebel
(1987, and updates) "Current Protocols in Molecular Biology". The
frequency (or spacing) of non-canonical nucleotides in the
resulting polynucleotide comprising a non-canonical nucleotide, and
thus the average size of fragments generated using the methods of
the invention (i.e., following cleavage of a base portion of a
non-canonical nucleotide, and cleavage of a phosphodiester backbone
at a non-canonical nucleotide), is controlled by variables known in
the art, including: frequency of nucleotide(s) corresponding to the
non-canonical nucleotide(s) in the template (or other measures of
nucleotide content of a sequence, such as average G-C content),
ratio of canonical to non-canonical nucleotide present in the
reaction mixture; ability of the polymerase to incorporate the
non-canonical nucleotide, relative efficiency of incorporation of
non-canonical nucleotide verses canonical nucleotide, and the like.
It is understood that the average fragmentation size also relates
to the reaction conditions used during fragmentation, as is further
discussed herein. The reaction conditions can be empirically
determined, for example, by assessing average fragment size
generated using the methods of the invention taught herein. The
level of labeling at an abasic site also relates to the frequency
of incorporation of non-canonical nucleotides, as is further
discussed herein.
[0096] Generally, a non-canonical base can be incorporated at about
every 5, 10, 15, 20, 25, 30, 40, 50, 65, 75, 85, 100, 123, 150,
175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 650 or more
nucleotides apart in the resulting polynucleotide comprising a
non-canonical nucleotide. In one embodiment, the non-canonical
nucleotide is incorporated about every 200 nucleotides, about every
100 nucleotide, or about every 50 nucleotide. In another
embodiment, the non-canonical nucleotide is incorporated about
every 50 to about 200 nucleotides. In some embodiments, a 1:5 ratio
of dUTP and dTTP is used in the reaction mixture.
[0097] The polynucleotide template (along which the polynucleotide
comprising a non-canonical nucleotide is synthesized) may be any
template from which labeled polynucleotide fragments are desired to
be produced. As is evident from the description herein, the labeled
polynucleotide fragments are the complement of the sequence of the
polynucleotide template. The template includes double-stranded,
partially double-stranded, and single-stranded nucleic acids from
any source in purified or unpurified form, which can be DNA (dsDNA
and ssDNA) or RNA, including tRNA, mRNA, rRNA, mitochondrial DNA
and RNA, chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures
thereof, genes, chromosomes, plasmids, the genomes of biological
material such as microorganisms, e.g., bacteria, yeasts, viruses,
viroids, molds, fungi, plants, animals, humans, and fragments
thereof. Obtaining and purifying nucleic acids use standard
techniques in the art. RNAs can be obtained and purified using
standard techniques in the art. A DNA template (including genomic
DNA template) can be transcribed into RNA form, which can be
achieved using methods disclosed in Kurn, U.S. Pat. No. 6,251,639
B1, and by other techniques (such as expression systems) known in
the art. RNA copies of genomic DNA would generally include
untranscribed sequences generally not found in mRNA, such as
introns, regulatory and control elements, etc. DNA copies of an RNA
template can be synthesized using methods described in Kurn, U.S.
Pat. No. 6,946,251 or other techniques known in the art). Synthesis
of polynucleotide comprising a non-canonical nucleotide from a
DNA-RNA hybrid can be accomplished by denaturation of the hybrid to
obtain a ssDNA and/or RNA, cleavage with an agent capable of
cleaving RNA from an RNA/DNA hybrid, and other methods known in the
art. The template can be only a minor fraction of a complex mixture
such as a biological sample and can be obtained from various
biological material by procedures well known in the art. The
template can be known or unknown and may contain more than one
desired specific nucleic acid sequence of interest, each of which
may be the same or different from each other. Therefore, the
methods of the invention are useful not only for producing one
specific polynucleotide comprising a non-canonical nucleotide, but
also for producing simultaneously more than one different specific
polynucleotides comprising a non-canonical nucleotide. The template
DNA can be a sub-population of nucleic acids, for example, a
subtractive hybridization probe, total genomic DNA, restriction
fragments, a cDNA library, cDNA prepared from total mRNA, a cloned
library, or amplification products of any of the templates
described herein. In some cases, the initial step of the synthesis
of the complement of a portion of a template nucleic acid sequence
is template denaturation. The denaturation step may be thermal
denaturation or any other method known in the art, such as alkali
treatment.
[0098] For simplicity, the polynucleotide comprising a
non-canonical nucleotide is described as a single nucleic acid. It
is understood that the polynucleotide can be a single
polynucleotide, or a population of polynucleotides (from a few to a
multiplicity to a very large multiplicity of polynucleotides). It
is further understood that a polynucleotide comprising a
non-canonical nucleotide can be a multiplicity (from small to very
large) of different polynucleotide molecules. Such populations can
be related in sequence (e.g., member of a gene family or
superfamily) or extremely diverse in sequence (e.g., generated from
all mRNA, generated from all genomic DNA, etc.). Polynucleotides
can also correspond to single sequence (which can be part or all of
a known gene, for example a coding region, genomic portion, etc.).
Methods, reagents, and reaction conditions for generating specific
polynucleotide sequences and multiplicities of polynucleotide
sequences are known in the art.
[0099] Suitable methods of synthesis of a polynucleotide comprising
a non-canonical nucleotide are generally template-dependent (in the
sense that polynucleotide comprising a non-canonical nucleotide is
synthesized along a polynucleotide template, as generally described
herein). It is understood that non-canonical nucleotides can be
incorporated into a polynucleotide as a result of
template-independent methods. For example, one or more primer(s)
can be designed to comprise one or more non-canonical nucleotides.
See, e.g., Richards, U.S. Pat. Nos. 6,037,152, 5,427,929, and
5,876,976. As discussed herein, inclusion of at least one
non-canonical nucleotide in a primer results in cleavage of a
base-portion of a non-canonical nucleotide and labeling at the
abasic site (i.e., following generation of an abasic site, as
described herein), thus generating a polynucleotide fragment or a
labeled polynucleotide fragment comprising a portion of the primer.
Inclusion of a non-canonical nucleotide in a primer may be
particularly suitable for methods such as single primer isothermal
amplification. See Kurn, U.S. Pat. No. 6,251,639 B1; Kurn, WO
02/00938; Kurn, U.S. Patent Publication No. 2003/0087251 A1.
Non-canonical nucleotide(s) can also be added to a polynucleotide
by template-independent methods such as tailing or ligation of a
second polynucleotide comprising a non-canonical nucleotide.
Methods for tailing and ligation are well-known in the art.
Cleavage of a Base Portion of a Nucleotide to Create an Abasic
Site
[0100] In methods for fragmentation of polynucleotides as described
herein, a polynucleotide comprising an abasic site is cleaved at or
near the abasic site to generate a polynucleotide fragment with a
blocked 3' end, and contacted with an enzyme capable of unblocking
the blocked 3' end to generate a polynucleotide fragment with a 3'
hydroxyl group. The polynucleotide comprising an abasic site may be
generated by cleaving the base portion of a nucleotide to create an
abasic site. In various embodiments, the nucleotide from which the
base portion is cleaved to create an abasic site is a non-canonical
nucleotide, a canonical nucleotide, or a methylated nucleotide.
Cleaving a Base Portion of a Non-Canonical Nucleotide to Create an
Abasic Site
[0101] A polynucleotide comprising a non-canonical nucleotide is
treated with an agent, such as an enzyme, capable of generally,
specifically, or selectively cleaving a base portion of the
non-canonical deoxyribonucleoside to create an abasic site. As used
herein, "abasic site" encompasses any chemical structure remaining
following removal of a base portion (including the entire base)
with an agent capable of cleaving a base portion of a nucleotide,
e.g., by treatment of a non-canonical nucleotide (present in a
polynucleotide chain) with an agent (e.g., an enzyme, acidic
conditions, or a chemical reagent) capable of effecting cleavage of
a base portion of a non-canonical nucleotide. In some embodiments,
the agent (such as an enzyme) catalyzes hydrolysis of the bond
between the base portion of the non-canonical nucleotide and a
sugar in the non-canonical nucleotide to generate an abasic site
comprising a hemiacetal ring and lacking the base (interchangeably
called "AP" site), though other cleavage products are contemplated
for use in the methods of the invention. Suitable agents and
reaction conditions for cleavage of base portions of non-canonical
nucleotides are known in the art, and include: N-glycosylases (also
called "DNA glycosylases" or "glycosidases") including Uracil
N-Glycosylase ("UNG"; specifically cleaves dUTP) (interchangeably
termed "uracil DNA glyosylase"), hypoxanthine-N-Glycosylase, and
hydroxy-methyl cytosine-N-glycosylase; 3-methyladenine DNA
glycosylase, 3- or 7-methylguanine DNA glycosylase,
hydroxymethyluracil DNA glycosylase; T4 endonuclease V. See, e.g.,
Lindahl, PNAS (1974) 71(9):3649-3653; Jendrisak, U.S. Pat. No.
6,190,865 B1. In one embodiment, UNG is used to cleave a base
portion of the non-canonical nucleotide.
[0102] Generally, cleavage of base portions of non-canonical
nucleotides is general, specific or selective cleavage, in the
sense that the agent (such as an enzyme) capable of cleaving a base
portion of a non-canonical nucleotide generally, specifically or
selectively cleaves the base portion of a particular non-canonical
nucleotide, whereby greater than about 98%, about 95%, about 90%,
about 85%, or about 80% of the base portions cleaved are base
portions of non-canonical nucleotides. However, extent of cleavage
can be less. Thus, reference to specific cleavage is exemplary.
General, specific or selective cleavage is desirable for control of
the fragment size in the methods of generating labeled
polynucleotide fragments of the invention (i.e., the fragments
generated by cleavage of the backbone at an abasic site).
Generally, reaction conditions are selected such that the reaction
in which the abasic site(s) are created can run to completion.
[0103] In some embodiments, the polynucleotide comprising a
non-canonical nucleotide is purified following synthesis of the
non-canonical polynucleotide (to eliminate, for example, residual
free non-canonical nucleotides that are present in the reaction
mixture). In other embodiments, there is no intermediate
purification between the synthesis of the polynucleotide comprising
the non-canonical nucleotide and subsequent steps (such as cleavage
of a base portion of the non-canonical nucleotide and cleavage of a
phosphodiester backbone at the abasic site).
[0104] As noted herein, for convenience, cleavage of a base portion
of a non-canonical nucleotide (whereby an abasic site is generated)
has been described as a separate step. It is understood that this
step may be performed simultaneously with synthesis of the
polynucleotide comprising a non-canonical nucleotide (as described
above), cleavage of the backbone at an abasic site (fragmentation)
and/or labeling at an abasic site.
[0105] It is understood that the choice of non-canonical nucleotide
can dictate the choice of enzyme to be used to cleave the base
portion of that non-canonical enzyme, to the extent that particular
non-canonical nucleotides are recognized by particular enzymes that
are capable of cleaving a base portion of the non-canonical
nucleotide.
Cleaving a Base Portion of a Canonical Nucleotide to Create an
Abasic Site
[0106] In another aspect, the invention comprises use of an agent,
such as an enzyme, that cleaves a base portion of a canonical
nucleotide, to generate a polynucleotide comprising an abasic site.
In some embodiments, the agent is not capable of cleaving a
methylated nucleotide.
[0107] In some embodiments, the agent is an enzyme. In one
embodiment, the enzyme is cytosine deaminase. See Sohail et al, NAR
2003, 31: 2990-94. Cytosine deaminase catalyzes the deamination of
cytosine, such that dUTP is generated. Cleavage of a base portion
of dUTP is necessary to generate the abasic site. Thus, the
invention encompasses use of (a) an agent (such as cytosine
deaminase) that modifies a nucleotide (such as dCTP), whereby dUTP
is generated, in conjunction with (b) an agent (such as an enzyme,
such as UNG) that cleaves a base portion of dUTP, whereby an abasic
site is generated. Methods for cleaving a base portion of dUTP are
known in the art. See, e.g., Lindahl, PNAS (1974) 71(9):3649-3653;
Jendrisak, U.S. Pat. No. 6,190,865 B1; U.S. Pat. No. 5,035,996;
U.S. Pat. No. 5,418,149; Sartori et al (2002) EMBO J 21:3182-3191.
As used herein, "in conjunction" encompasses simultaneous treatment
(e.g., when cytosine deaminase and UNG cleavage occurs in the same
reaction mixture) and/or treatment at different times (e.g., when
cytosine deaminase and UNG treatment is conducted
sequentially).
[0108] Generally, cleavage of the base portion of a canonical
nucleotide is general, specific or selective cleavage (in the sense
that the agent (such as an enzyme) capable of cleaving a base
portion of a canonical nucleotide generally, specifically or
selectively cleaves the base portion of a particular canonical
nucleotide, whereby about any of 98%, 95%, 90%, 85%, or 80% of the
base portions cleaved are base portions of canonical nucleotides.
However, extent of cleavage can be less. Thus, reference to
specific cleavage is exemplary.
[0109] It is understood that the frequency (or spacing) of abasic
sites in the resulting polynucleotide comprising an abasic site
(following cleavage of a base portion of a unmethylated nucleotide,
and thus the average size of fragments generated using the methods
of the invention (i.e., following cleavage of a phosphodiester
backbone at an abasic site), is controlled by variables known in
the art, including: frequency of the target canonical nucleotide(s)
from which an abasic site will be generated in the polynucleotide
(or other measures of nucleotide content of a sequence, such as
average G-C content), length of the polynucleotide, and the
reaction conditions used during generation of abasic site.
Cleaving a Base Portion of a Methylated Nucleotide to Create an
Abasic Site
[0110] In aspects involving cleavage of a base portion of a
methylated nucleotide to generate a polynucleotide comprising an
abasic site, the polynucleotide comprising a methylated nucleotide
(in some embodiments, suspected of comprising a methylated
nucleotide) is treated with an agent, such as an enzyme, capable of
generally, specifically, or selectively cleaving a base portion of
the methylated deoxyribonucleoside to create an abasic site. As
used herein, "abasic site" encompasses any chemical structure
remaining following removal of a base portion (including the entire
base) of a methylated nucleotide with an agent capable of cleaving
a base portion of a methylated nucleotide, e.g., by treatment of a
methylated nucleotide (present in a polynucleotide chain) with an
agent (e.g., an enzyme) capable of effecting cleavage of a base
portion of a methylated nucleotide. In some embodiments, the agent
(such as an enzyme) catalyzes hydrolysis of the bond between the
base portion of the methylated nucleotide and a sugar in the
methylated nucleotide to generate an abasic site comprising a
hemiacetal ring and lacking the base (interchangeably called "AP"
site), though other cleavage products are contemplated for use in
the methods of the invention. Generally, the methods involving
cleavage of a base portion of a methylated nucleotide are suitable
for use with polynucleotides comprising a low frequency of
methylated nucleotides (i.e., generally, not hypermethylated
regions, such a CpG islands and the like), though other uses are
contemplated.
[0111] The polynucleotide comprising (in some embodiments,
suspected of comprising) a methylated nucleotide is treated with an
agent, such as an enzyme, capable of generally, specifically, or
selectively cleaving a base portion of the methylated
deoxyribonucleoside to create an abasic site. As used herein,
"abasic site" encompasses any chemical structure remaining
following removal of a base portion (including the entire base) of
a methylated nucleotide with an agent capable of cleaving a base
portion of a methylated nucleotide, e.g., by treatment of a
methylated nucleotide (present in a polynucleotide chain) with an
agent (e.g., an enzyme, acidic conditions, or a chemical reagent)
capable of effecting cleavage of a base portion of a methylated
nucleotide. In some embodiments, the agent (such as an enzyme)
catalyzes hydrolysis of the bond between the base portion of the
methylated nucleotide and a sugar in the canonical nucleotide to
generate an abasic site comprising a hemiacetal ring and lacking
the base (interchangeably called "AP" site), though other cleavage
products are contemplated for use in the methods of the
invention.
[0112] In some embodiments, the methylated nucleotide is
5-methylcytosine. In some embodiments, 5-methylcytosine is present
as a methylated CpG dinucleotide. The CpG dinucleotide may be fully
or hemi-methylated. In other embodiments, the methylated nucleotide
is 3-methyladenine. In other embodiments, the methylated nucleotide
is 7-methyladenine and/or 3-methylguanine.
[0113] Suitable agents and reaction conditions for cleavage of base
portions of methylated nucleotides are known in the art, and
include: 5-methylcytosine DNA glycosylase (5-MCDG), which cleaves
the base portion of 5-methylcytosine (5-MeC) from the DNA backbone
(Wolffe et al., Proc. Nat. Acad. Sci. USA 96:5894-5896, 1999);
3-methyladenosine-DNA glycosylase I, which cleaves the base portion
of 3-methyl adenosine from the DNA backbone (see, e.g. Hollis et al
(2000) Mutation Res. 460: 201-210); and/or 3-methyladenosine DNA
glycosylase II, which cleaves the base portion of
3-methyladenosine, 7-methylguanine, 7-methyladenosine,
and/3-methylguanine from the DNA backbone. See McCarthy et al
(1984) EMBO J. 3:545-550. Multifunctional and mono-functional forms
of 5-MCDG have been described. See Zhu et al., Proc. Natl. Acad.
Sci. USA 98:5031-6, 2001; Zhu et al., Nuc. Acid Res.
28:4157-4165,2000; and Nedderrnann et al., J.B.C. 271:12767-74,
1996 (describing bifunctional 5-MCDG; Vairapandi & Duker,
Oncogene 13:933-938, 1996; Vairapandi et al., J. Cell. Biochem.
79:249-260, 2000 (describing mono-functional enzyme comprising
5-MCDG activity). In some embodiments, 5-MCDG preferentially
cleaves fully methylated polynucleotide sites (e.g., CpG
dinucleotides), and in other embodiments, 5-MCDG preferentially
cleaves a hemi-methylated polynucleotide. For example,
mono-functional human 5-methylcytosine DNA glycosylase cleaves DNA
specifically at fully methylated CpG sites, and is relatively
inactive on hemimethylated DNA (Vairapandi & Duker, supra;
Vairapandi et al., supra). By contrast, chick embryo
5-methylcytosine-DNA glycosylase has greater activity directed to
hemimethylated methylation sites. In some embodiments, the activity
of 5-MCDG is potentiated (increased or enhanced) with accessory
factors, such as recombinant CpG-rich RNA, ATP, RNA helicase
enzyme, and proliferating cell nuclear antigen (PCNA). See U.S.
Patent Publication No. 20020197639 A1. One or more agents may be
used. In some embodiments, the one or more agents cleave a base
portion of the same methylated nucleotide. In other embodiments,
the one or more agents cleave a base portion of different
methylated nucleotides. Treatment with two or more agents may be
sequential or simultaneous.
[0114] As is evident, in some embodiments, dUTP is generated as an
intermediate and cleavage of a base portion of dUTP is necessary to
generate the abasic site. Methods for cleaving a base portion of
dUTP are known in the art. See, e.g., Lindahl, PNAS (1974)
71(9):3649-3653; Jendrisak, U.S. Pat. No. 6,190,865 B1; U.S. Pat.
No. 5,035,996; U.S. Pat. No. 5,418,149; Sartori et al (2002) EMBO J
21:3182-3191. Thus, in some embodiments, an agent that cleaves a
base portion of a methylated nucleotides (such as an enzyme, such
as 5-MCDG) is used in conjunction with UNG to generate an abasic
site from the methylated nucleotide. As used herein, "in
conjunction" encompasses simultaneous treatment (e.g., when 5-MCDG
and UNG cleavage occurs in the same reaction mixture) and/or
treatment at different times (e.g., when 5-MCDG and UNG treatment
is conducted sequentially).
[0115] In some embodiments, cleavage of the base portion of the
methylated nucleotides is general, specific or selective cleavage
(in the sense that the agent (such as an enzyme) capable of
cleaving a base portion of a methylated nucleotide generally,
specifically or selectively cleaves the base portion of a
particular methylated nucleotide), whereby about any of 98%, 95%,
90%, 85%, or 80% of the base portions cleaved are base portions of
methylated nucleotides. However, extent of cleavage can be less.
Thus, reference to specific cleavage is exemplary.
[0116] It is understood that the frequency (or spacing) of abasic
sites in the resulting polynucleotide comprising an abasic site
(following cleavage of a base portion of a methylated nucleotide,
and thus the average size of fragments generated using the methods
of the invention (i.e., following cleavage of a phosphodiester
backbone at an abasic site), is controlled by variables known in
the art, including: frequency of methylated nucleotide(s) in the
polynucleotide (or other measures of nucleotide content of a
sequence, such as average G-C content), length of the
polynucleotide comprising a methylated nucleotide, and the reaction
conditions used during generation of abasic site, as is farther
discussed herein.
Cleaving the Backbone at or Near the Abasic Site to Generate a
Polynucleotide Fragment with a Blocked 3' End
[0117] The backbone of the polynucleotide comprising an abasic site
is cleaved at or near the abasic site with an agent that generates
a polynucleotide fragment with a blocked 3' end. It is understood
that cleavage of the base portion of a nucleotide to create an
abasic site and cleavage of the polynucleotide backbone can be
performed simultaneously. For convenience, however, these reactions
are described as separate steps.
[0118] Following generation of an abasic site by cleavage of the
base portion of a nucleotide, for example, a non-canonical
nucleotide present in the polynucleotide, the backbone of the
polynucleotide is cleaved at or near the abasic site, for example,
the site of incorporation of a non-canonical nucleotide (also
termed the abasic site, following cleavage of the base portion of
the non-canonical nucleotide), with an agent capable of effecting
cleavage of the backbone at the abasic site to generate a
polynucleotide fragment comprising a blocked 3' end. Cleavage of
the polynucleotide backbone (also termed "fragmentation") results
in at least two fragments (depending on the number of abasic sites
present in the polynucleotide comprising an abasic site, and the
extent of cleavage), one of which does not comprise a blocked 3'
end.
[0119] Suitable agents (for example, an enzyme, a chemical and/or
reaction conditions such as heat) capable of cleavage of the
backbone at an abasic site to generate a polynucleotide fragment
with a blocked 3' end are well known in the art, and include: heat
treatment and/or chemical treatment (including basic conditions,
acidic conditions, alkylating conditions, or amine mediated
cleavage of abasic sites, (see e.g., McHugh and Knowland, Nucl.
Acids Res. (1995) 23(10):1664-1670; Bioorgan. Med. Chem (1991)
7:2351; Sugiyama, Chem. Res. Toxicol. (1994) 7: 673-83; Horn, Nucl.
Acids. Res., (1988) 16:11559-71). As used herein, "agent"
encompasses reaction conditions such as heat. In another
embodiment, cleavage is with a polyamine, such as
N,N'-dimethylethylenediamine (DMED). See, e.g. McHugh and Knowland,
supra.
[0120] Generally, cleavage is between the nucleotide immediately 3'
to the abasic residue and the abasic residue. As is well known in
the art, cleavage can be 3' to the abasic site (e.g., cleavage
between the deoxyribose ring and 3'-phosphate group of the abasic
residue and the deoxyribose ring of the adjacent nucleotide,
generating a free 5' phosphate group on the deoxyribose ring of the
adjacent nucleotide), such that an abasic site is located at the 3'
end of the resulting fragment. Treatment under basic conditions or
with amines (such as N,N'-dimethylethylenediamine) results in
cleavage of the phosphodiester backbone immediately 3' to the
abasic site to produce a polynucleotide fragment with a blocked 3'
end. In addition, more complex forms of cleavage are also possible,
for example, cleavage such that cleavage of the phosphodiester
backbone and cleavage of (a portion of) the abasic nucleotide
results. For example, under certain conditions, cleavage using
chemical treatment and/or thermal treatment may comprise a
.beta.-elimination step which results in cleavage of a bond between
the abasic site deoxyribose ring and its 3' phosphate, generating a
reactive .alpha.,.beta.-unsaturated aldehyde which can be labeled
or can undergo further cleavage and cyclization reactions. See,
e.g. Sugiyama, Chem. Res. Toxicol. (1994) 7: 673-83; Horn, Nucl.
Acids. Res., (1988) 16:11559-71. It is understood that more than
one method of cleavage can be used, including two or more different
methods which result in multiple, different types of cleavage
products comprising blocked 3' ends.
[0121] Generally, cleavage of the backbone at an abasic site is
general, specific or selective cleavage, whereby greater than about
98%, about 95%, about 90%, about 85%, or about 80% of the cleavage
is at an abasic site. However, extent of cleavage can be less.
Thus, reference to specific cleavage is exemplary. General,
specific or selective cleavage is desirable for control of the
fragment size in the methods of generating labeled polynucleotide
fragments of the invention. In some embodiments, reaction
conditions can be selected such that the cleavage reaction is
performed in the presence of a large excess of reagents and allowed
to run to completion with minimal concern about excessive cleavage
of the polynucleotide (i.e., while retaining a desired fragment
size, which may be determined by spacing of incorporated
non-canonical nucleotides, during the synthesis step, above). In
other embodiments, extent of cleavage can be less, such that
polynucleotide fragments are generated comprising an abasic site at
an end and an abasic site(s) within or internal to the
polynucleotide fragment (i.e., not at an end).
[0122] As noted herein, in embodiments in which an abasic site is
generated by cleavage of a base portion of a non-canonical
nucleotide in a polynucleotide synthesized in the presence of a
non-canonical nucleotide, the frequency of incorporation of
non-canonical nucleotides into the polynucleotide relates to the
size of fragment produced using the methods of the invention
because the spacing between non-canonical nucleotides in the
polynucleotide comprising a non-canonical nucleotide, as well as
the reaction conditions selected, determines the approximate size
of the resulting fragments (following cleavage of a base portion of
a non-canonical nucleotide, whereby an abasic site is generated,
and cleavage of the backbone at the abasic site as described
herein).
[0123] In methods of the invention, suitable fragment sizes are
generally about 5, 10, 15, 20, 25, 30, 40, 50, 65, 75, 85, 100,
123, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600,
650 or more nucleotides in length. In some embodiments, the
fragment is about 200 nucleotides, about 100 nucleotides, or about
50 nucleotides in length. In another embodiment, the size of a
population of fragments is about 50 to 200 nucleotides. It is
understood that the fragment size is approximate, particularly when
populations of fragments are generated, because the incorporation
of a non-canonical nucleotide (which relates to the fragment size
following cleavage) will vary from template to template, and also
between copies of the same template. Thus, fragments generated from
same starting material (such as a single polynucleotide template)
may have different (and/or overlapping) sequence, while still
having the same approximate size or size range.
[0124] Following cleavage of the polynucleotide backbone at the
abasic site, every fragment will comprise one abasic site (if
cleavage is completely efficient), except for the 3'-most fragment,
which will lack an abasic site. All other fragments will comprise a
3' abasic site (a blocked 3' end).
Unblocking of the Blocked 3' End to Generate a Polynucleotide
Fragment with a 3' Hydroxyl Group
[0125] A polynucleotide fragment comprising a blocked 3' end,
prepared as described herein, is contacted with an enzyme capable
of unblocking the blocked 3' end, whereby a polynucleotide fragment
comprising a 3' hydroxyl group is generated. In some embodiments,
the enzyme capable of unblocking the blocked 3' end comprises a 3'
to 5' exonuclease activity, generally a non-processive activity. In
some embodiments, the enzyme that comprises a 3' to 5' exonuclease
activity does not comprise an endonuclease activity, although an
enzyme comprising an endonuclease activity may also be used, under
conditions in which the endonuclease activity is minimized or
absent. In some embodiments, the enzyme comprising a 3' to 5'
exonuclease activity is selected from the group consisting of
endonuclease 4, exonuclease T, and APE 1.
[0126] Various 3'-5' exonucleases may be used for removal of the 3'
blocking group following fragmentation of a polynucleotide
comprising an abasic site with an agent, such as a polyamine, for
example, DMED, that produces a fragment with a blocked 3' end. A
review of 3'-5' exonucleases is presented in Shevelev et al. (2002)
Nature Reviews Molecular Cell Biology 3:367-376. Many DNA repair
related exonucleases have been discovered in recent years in
addition to APE 1 and homologous AP nucleases in other eukaryotes
and prokaryotes. Of special interest are the TREX1 and two 3'-5'
exonucleases capable of unblocking a blocked polynucleotide 3'
terminus, as described in Mazur et al., J. Biol. Chem.
276(20):17033-17029. Another effective exonuclease is APE 2, which
efficiently removes 3' blocking groups from polynucleotide 3'
termini, as described in Burkovics et al. (2006) Nucleic Acids Res.
34(9):2508-2515.
[0127] In some embodiments, an important feature of the 3'-5'
exonuclease is the ability to remove a blocking group at the 3'
terminus of a single stranded polynucleotide (such as a single
stranded amplification product). In some embodiments, the
exonuclease is non-processive. An example of such a non-processive
exonuclease is human TREX2 3'-5' exonuclease, as described in
Perrino et al., J. Biol. Chem. 280(15): 15212-15218.
Polymerase Extension or Ligation of Polynucleotide Fragments with
3' Hydroxyl Groups
[0128] A polynucleotide fragment with an unblocked 3' end, prepared
as described herein, may be extended from the hydroxyl group at the
3' end by a template independent or template dependent polymerase
or may be ligated at the 3' end to another polynucleotide with a
ligase enzyme.
[0129] In one embodiment, a polynucleotide fragment with an
unblocked 3' end, prepared as described herein, is extended from
the 3' hydroxyl group with a template independent polymerase, such
as TdT, to incorporate one or more nucleotides, for example, one or
more detectable nucleotides, at the 3' end of the polynucleotide
fragment. In some embodiments, a labeled nucleotide is
incorporated. In one embodiment, the labeled nucleotide is a
biotinylated nucleotide, such as a biotinylated triphosphate (NTP),
deoxynucleotide triphosphate (dNTP), or dideoxynucleotide
triphosphate (ddNTP). For example, biotin 2'3'-dideoxy-UTP or
biotin 2',3'-dideoxy-CTP may be incorporated at the 3' end of the
polynucleotide fragment with TdT. In another embodiment, the
labeled nucleotide comprises a fluorophore (e.g., cy dyes, alexa
dyes, fluorescein, and other fluorophores known in the art). In
other embodiments, the incorporated detectable nucleotide comprises
an enzyme, a chromophore, a radiolabel, or a hapten which is
detectable by binding of a labeled second member of a binding pair,
such as, for example, biotin/avidin or streptavidin,
antigen/antibody, and the label attached to the second member of
the binding pair may comprise, for example, a fluorophore, an
enzyme, a chromophore, a radiolabel, or may be attached to a
detectable particle. In one embodiment, the labeled nucleotide is a
biotinylated nucleotide, for example, biotin 2',3'-dideoxy-UTP or
2',3'-dideoxy-CTP, and is detectable by binding of labeled avidin
or streptavidin. In some embodiments, a polynucleotide fragment
prepared in accordance with methods described herein is tailed with
unlabeled nucleotides or a mixture of labeled and unlabeled
nucleotides with a template independent polymerase such as TdT.
[0130] In another embodiment, a polynucleotide fragment with an
unblocked 3' end, prepared as described herein, is hybridized to a
polynucleotide template and extended from the 3' hydroxyl group
with a template dependent polymerase. In one embodiment, the
polynucleotide fragment with an unblocked 3' end is used as a
primer to initiate synthesis of a polynucleotide complementary to
the template. In one embodiment, the polynucleotide fragment is
extended in the presence of one or more labeled nucleotides, such
as one or more nucleotides attached to a member of a binding pair
that is detectable by binding of a labeled second member of the
binding pair, as described above, to produce a detectable
polynucleotide.
[0131] In another embodiment, a polynucleotide fragment with an
unblocked 3' end, prepared as described herein, is ligated at the
3' hydroxyl group to another polynucleotide using a ligase enzyme.
In some embodiments, the polynucleotide to which the polynucleotide
fragment is ligated comprises one or more detectable nucleotides as
described above, resulting in a detectable ligated polynucleotide
comprising the polynucleotide fragment with the unblocked 3' end
and the polynucleotide to which the fragment was ligated.
[0132] As discussed above, a "label" can be directly detectable, or
the label can be indirectly detectable, such as, for example, when
the label is covalently or non-covalently associated with another
moiety which is itself detectable. For example, biotin can be
attached to nucleotide, which may be detected by binding to
detectable avidin or streptavidin. In another example, an antibody
(that can be detectably labeled) binds to a cognate antigen that is
attached to a nucleotide. In some embodiments, the label comprises
an organic molecule, a hapten, or a particle (such as a polystyrene
bead). In some embodiments, the label is detected using antibody
binding, biotin binding, or via fluorescence or enzyme activity. In
some embodiments, the detectable signal is amplified.
[0133] In some embodiments, labeled polynucleotide fragments are
produced which each comprise a single label, for example,
incorporation of a detectable nucleotide at the 3' hydroxyl group
with terminal transferase. This aspect is useful in quantitating
level of hybridization, because signal is proportional to number of
bound fragments, and does not relate to the length of the
hybridizing fragment or the number of labels per fragment. Thus,
hybridization intensity can generally be directly compared,
regardless of fragment length. This offers an advantage over prior
art methods in which nucleic acid fragments are labeled with
multiple detectable moieties, e.g., incorporation of labeled
nucleotides, and other methods of directly and indirectly detecting
incorporated nucleotides. These methods generally result in
multiple labels per hybridizing fragment, and thus are generally
less suitable for quantitative applications. Multiple labels per
nucleic acid can result in quenching, and potential interference
with hybridization kinetics (due to the presence of multiple
labeled moieties per nucleic acid).
[0134] Methods of signal detection are known in the art. Signal
detection may be visual or utilize a suitable instrument
appropriate to the particular label used, such as a spectrometer,
fluorimeter, or microscope. For example, where the label is a
radioisotope, detection can be achieved using, for example, a
scintillation counter, or photographic film as in autoradiography.
Where a fluorescent label is used, detection may be by exciting the
fluorochrome with the appropriate wavelength of light and detecting
the resulting fluorescence, such as by microscopy, visual
inspection or photographic film, fluorometer, CCD cameras, scanner
and the like. Where enzymatic labels are used, detection may be by
providing appropriate substrates for the enzyme and detecting the
resulting reaction product. For example, many substrates of
horseradish peroxidase, such as o-phenylenediamine, give colored
products. Simple calorimetric labels can usually be detected by
visual observation of the color associated with the label; for
example, conjugated colloidal gold is often pink to reddish, and
beads appear the color of the bead. Instruments suitable for high
sensitivity detection are known in the art.
[0135] It is understood that the polynucleotide or polynucleotide
fragments can be additionally labeled using other methods known in
the art, such as incorporation of labeled nucleotide analogs during
synthesis of a polynucleotide comprising a non-canonical
nucleotide, from which a polynucleotide comprising an abasic site
is generated. In addition, following cleavage of the phosphodiester
backbone of the polynucleotide comprising an abasic site, the 3'
most fragment will lack an abasic site, (in embodiments in which
the fragmentation reaction goes to completion). However, if
polynucleotide synthesis step requires primer(s), a labeled
primer(s) can be used such that the resulting fragment comprising a
primer is labeled. Suitable labels and methods of labeling primers
are known. In addition, a primer comprising a non-canonical
nucleotide can be used. Following generation of an abasic site,
cleavage of the phosphodiester backbone at the abasic site, and
labeling at the abasic site, the fragment comprising at least a
portion of the primer will be labeled.
Reaction Conditions and Detection
[0136] Appropriate reaction media and conditions for carrying out
the methods of the invention include those that permit cleavage of
a polynucleotide comprising an abasic site with an agent capable of
cleaving a polynucleotide to produce a polynucleotide fragment with
a blocked 3' end, and unblocking of a blocked 3' end with an agent
capable of unblocking the 3' end of a polynucleotide to produce a
polynucleotide comprising a 3' end hydroxyl group.
[0137] Appropriate reaction media and conditions for carrying out
the cleavage of the phosphodiester backbone at an abasic site
according to the methods of the invention are those that permit
cleavage of the phosphodiester backbone at or near an abasic site
to produce a polynucleotide fragment comprising a blocked 3' end.
Such media and conditions are known to persons of skill in the art,
and are described in various publications, such as Bioorgan. Med.
Chem (1991) 7:2351; Sugiyama, Chem. Res. Toxicol. (1994) 7: 673-83;
Horn, Nucl. Acids. Res., (1988) 16:11559-71); Lindahl, PNAS (1974)
71(9):3649-3653; Jendrisak, U.S. Pat. No. 6,190,865 B1, Shida,
Nucleic Acids Res. (1996) 24(22):4572-76; Srivastava, J. Biol Chem.
(1998) 273(13):21203-209; Carey, Biochem. (1999) 38:16553-60; Chem
Res Toxicol (1994) 7:673-683. A reaction mixture suitable for
simultaneous UNG treatment and N,N'-dimethylethylenediamine
treatment is described in Example 4 of U.S. Patent Application No.
2004/0005614.
[0138] In another example, nucleic acids containing abasic sites
are heated in a buffer solution containing an amine, for example,
25 mM Tris-HCl and 1-5 mM magnesium ions, for 10-30 minutes at
70.degree. C. to 95.degree. C. Alternatively, 1.0 M piperidine (a
base) is added to polynucleotide comprising an abasic site which
has been precipitated with ethanol and vacuum dried. The solution
is then heated for 30 minutes at 90.degree. C. and lyophilized to
remove the piperidine. In another example, cleavage is effected by
treatment with basic solution, e.g., 0.2 M sodium hydroxide at
37.degree. for 15 minutes. See Nakamura (1998) Cancer Res.
58:222-225. In yet another example, incubation at 37 C with 100 mM
N,N'-dimethylethylenediamine acetate, pH 7.4 is used to cleave. See
McHugh and Knowland, (1995) Nucl. Acids Res. 23(10) 1664-1670.
[0139] In some embodiments, nucleic acid synthesis is performed to
produce the polynucleotide to be fragmented. Appropriate media and
conditions are known to persons of skill in the art, and are
described in various publications, such as U.S. Pat. Nos.
6,190,865; 5,554,516; 5,716,785; 5,130,238; 5,194,370; 6,090,591;
5,409,818; 5,554,517; 5,169,766; 5,480,784; 5,399,491; 5,679,512;
PCT Pub. No. WO99/42618; Mol. Cell Probes (1992) 251-6, and Anal.
Biochem. (1993) 211:164-9. For example, a buffer may be Tris
buffer, although other buffers can also be used as long as the
buffer components are non-inhibitory to enzyme components of the
methods of the invention. The pH is preferably from about 5 to
about 11, more preferably from about 6 to about 10, even more
preferably from about 7 to about 9, and most preferably from about
7.5 to about 8.5. The reaction medium can also include bivalent
metal ions such as Mg.sup.2+ or Mn.sup.2+, at a final concentration
of free ions that is within the range of from about 0.01 to about
15 mM, and most preferably from about 1 to 10 mM. The reaction
medium can also include other salts, such as KCl or NaCl, that
contribute to the total ionic strength of the medium. For example,
the range of a salt such as KCl is preferably from about 0 to about
125 mM, more preferably from about 0 to about 100 mM, and most
preferably from about 0 to about 75 mM. The reaction medium can
further include additives that could affect performance of the
amplification reactions, but that are not integral to the activity
of the enzyme components of the methods. Such additives include
proteins such as BSA, single strand binding proteins (e.g., T4 gene
32 protein), and non-ionic detergents such as NP40 or Triton.
Reagents, such as DTT, that are capable of maintaining enzyme
activities can also be included. Such reagents are known in the
art. Where appropriate, an RNase inhibitor (such as Rnasin) that
does not inhibit the activity of the RNase employed in the method
(if any) can also be included. Any aspect of the methods of the
invention can occur at the same or varying temperatures. The
synthesis reactions (particularly, primer extension other than the
first and second strand cDNA synthesis steps, and strand
displacement) can be performed isothermally, which avoids the
cumbersome thermocycling process. The synthesis reaction is carried
out at a temperature that permits hybridization of the
oligonucleotides (primer) of the invention to the template
polynucleotide and primer extension products, and that does not
substantially inhibit the activity of the enzymes employed. The
temperature can be in the range of preferably about 25.degree. C.
to about 85.degree. C., more preferably about 30.degree. C. to
about 80.degree. C., and most preferably about 37.degree. C. to
about 75.degree. C. In some embodiments that include RNA
transcription, the temperature for the transcription steps is lower
than the temperature(s) for the preceding steps. In these
embodiments, the temperature of the transcription steps can be in
the range of preferably about 25.degree. C. to about 85.degree. C.,
more preferably about 30.degree. C. to about 75.degree. C., and
most preferably about 37.degree. C. to about 70.degree. C.
[0140] In embodiments in which the polynucleotide comprising an
abasic site is produced from a polynucleotide comprising a
non-canonical nucleotide, Nucleotides, including non-canonical
nucleotides (or other nucleotide analogs), that can be employed for
synthesis of the nucleic acid comprising a non-canonical nucleotide
in the methods of the invention are provided in the amount of from
preferably about 50 to about 2500 .mu.M, more preferably about 100
to about 2000 .mu.M, even more preferably about 200 to about 1700
.mu.M, and most preferably about 250 to about 1500 .mu.M. The
oligonucleotide components of the synthesis reactions of the
invention are generally in excess of the number of template nucleic
acid sequence to be replicated. They can be provided at about or at
least about any of the following: 10, 10.sup.2, 10.sup.4, 10.sup.6,
10.sup.8, 10.sup.10, 10.sup.12 times the amount of target nucleic
acid. Composite primers can be provided at about or at least about
any of the following concentrations: 50 nM, 100 nM, 500 nM, 1000
nM, 2500 nM, 5000 nM.
[0141] Optionally, the polynucleotide comprising a non-canonical
nucleotide can be treated with hydroxylamine (or any other suitable
agent) to remove any aldehydes that may have formed spontaneously
in the nucleic acid. See, e.g., Makrogiorgos, WO00/39345.
[0142] For convenience, the synthesis of a polynucleotide
comprising a non-canonical nucleotide, and the cleavage of a base
portion of that polynucleotide by an enzyme capable of cleaving a
base portion of the non-canonical nucleotide, and the cleavage of
the phosphodiester backbone at the abasic site, are described as
separate steps. It is understood that these steps may be performed
simultaneously, except (generally) in the case when a
polynucleotide comprising a non-canonical nucleotide must be
capable of serving as a template for further amplification (as in
exponential methods of amplification, e.g. PCR).
[0143] Appropriate reaction media and conditions for carrying out
the cleavage of a base portion of a non-canonical nucleotide
according to the methods of the invention are those that permit
cleavage of a base portion of a non-canonical nucleotide. Such
media and conditions are known to persons of skill in the art, and
are described in various publications, such as Lindahl, PNAS (1974)
71(9):3649-3653; Jendrisak, U.S. Pat. No. 6,190,865 B1; U.S. Pat.
No. 5,035,996; U.S. Pat. No. 5,418,149. For example, buffer
conditions can be as described above with respect to polynucleotide
synthesis. In one embodiment, UNG (Epicentre Technologies, Madison
Wisc.) is added to a nucleic acid synthesis reaction mixture, and
incubated at 37.degree. C. for 20 minutes. In one embodiment, the
reaction conditions are the same for the synthesis of a
polynucleotide comprising a non-canonical nucleotide and the
cleavage of a base portion of the non-canonical nucleotide. In
another embodiment, different reaction conditions are used for
these reactions. In some embodiments, a chelating regent (e.g.
EDTA) is added before or concurrently with UNG in order to prevent
the polymerase from extending the ends of the cleavage
products.
[0144] In some embodiments, some of the components for performing a
method as described herein are added simultaneously at various
timepoints. In one embodiment, components for cleaving a base
portion of a nucleotide to generate a polynucleotide comprising an
abasic site and components for cleaving the phosphodiester backbone
of the polynucleotide comprising the abasic site may be combined
for simultaneous reaction. In one embodiment, components for
cleaving a base portion of a nucleotide to generate a
polynucleotide comprising an abasic site, components for cleaving
the phosphodiester backbone of the polynucleotide comprising the
abasic site, and components for unblocking a blocked 3' end may be
combined for simultaneous reaction. In one embodiment, components
for cleaving the phosphodiester backbone of a polynucleotide
comprising the abasic site, and components for unblocking a blocked
3' end may be combined for simultaneous reaction. Such components
may be added in any order at appropriate timepoints. Such
timepoints can be readily identified by a person of skill in the
art. In these embodiments, the reaction conditions and components
may be varied between the different reactions.
[0145] The fragmenting or fragmenting and labeling process can be
stopped at various timepoints, and resumed at a later time. Said
timepoints can be readily identified by a person of skill in the
art. Methods for stopping the reactions are known in the art,
including, for example, cooling the reaction mixture to a
temperature that inhibits enzyme activity or heating the reaction
mixture to a temperature that destroys an enzyme. Methods for
resuming the reactions are also known in the art, including, for
example, raising the temperature of the reaction mixture to a
temperature that permits enzyme activity or replenishing a
destroyed (depleted) enzyme or other reagent. In some embodiments,
one or more of the components of the reactions is replenished prior
to, at, or following the resumption of the reactions.
Alternatively, the reaction can be allowed to proceed (i.e., from
start to finish) without interruption.
[0146] The reaction can be allowed to proceed without purification
of intermediate reaction products. Alternatively, products can be
purified at various timepoints, conditions for which can be readily
identified by a person of skill in the art.
Compositions
[0147] The invention also provides compositions and kits used in
the methods described herein. The compositions may comprise any
component(s), reaction mixture(s) and/or intermediate(s) described
herein, as well as any combination thereof. For example, in one
embodiment, the invention provides a composition comprising an
agent capable of cleaving a base portion of a nucleotide to
generate an abasic site in a polynucleotide, an agent capable of
cleaving a phosphodiester backbone at or near an abasic site to
produce a polynucleotide fragment with a blocked 3' end, and an
enzyme capable of unblocking a blocked 3' end to generate a
polynucleotide comprising a 3' hydroxyl group. In one embodiment,
the agent capable of cleaving a base portion of a nucleotide to
generate an abasic site is an N-glycosylase, for example, UNG. In
one embodiment, the agent capable of cleaving a phosphodiester
backbone at or near the abasic site is a polyamine, for example,
DMED. In one embodiment, the enzyme capable of unblocking a blocked
3' end comprises a 3'-5' exonuclease activity, preferably a
non-processive exonuclease activity, for example, endonuclease 4,
exonuclease T, or APE 1. Compositions of the invention may also
comprise buffers, co-factors, or other components for carrying out
the reactions of the methods described herein.
[0148] The invention also provides a composition comprising a
polynucleotide fragment produced by a method as described herein
and a template independent polymerase, a template dependent
polymerase, or a ligase. In one embodiment, the invention provides
a composition comprising a polynucleotide fragment produced as
described herein, a template independent polymerase, for example,
TdT, and a labeled nucleotide, an unlabeled nucleotide, or a
mixture or labeled and unlabeled nucleotides. In one embodiment,
the invention provides a composition comprising a polynucleotide
fragment produced as described herein, a template dependent
polymerase, and a polynucleotide template to which the
polynucleotide fragment is capable of hybridizing, optionally
further comprising nucleotides for polymerization, e.g., labeled,
unlabeled, or a mixture of labeled and unlabeled nucleotides. In
one embodiment, the composition comprises a complex comprising the
polynucleotide fragment hybridized to a polynucleotide template. In
one embodiment, the invention provides a composition comprising a
polynucleotide fragment prepared as described herein, a ligase
enzyme, and a polynucleotide to which the polynucleotide fragment
is desired to be ligated.
[0149] The compositions are generally in lyophilized or in a
suitable medium, such as aqueous form (if appropriate), preferably
in a suitable buffer.
[0150] The invention also provides polynucleotide fragments and
labeled polynucleotide fragments produced by any of the methods
described herein, and compositions comprising such fragments.
Accordingly, the invention provides a population of fragmented or
fragmented and labeled polynucleotides, which are produced by any
of the methods described herein (or compositions comprising the
products).
[0151] The invention also provides reaction mixtures (or
compositions comprising reaction mixtures) which contain various
combinations of components described herein. Examples of reaction
mixtures have been described. In one embodiment, the reaction
mixture comprises a polynucleotide comprising an abasic site, and
an agent (such as UNG) that is capable of cleaving a base portion
from a non-canonical nucleotide, an agent (such as an amine, such
as N,N'-dimethylethylenediamine) capable of cleaving the
phosphodiester back at an abasic site, and an enzyme capable of
unblocking a blocked 3' end of a polynucleotide. In one embodiment,
the invention provides a reaction mixture comprising (a) UNG; (b)
N,N'-dimethylethylenediamine; and (c) APE 1.
Kits
[0152] The invention also provides kits for carrying out the
methods of the invention. Accordingly, a variety of kits are
provided in suitable packaging. The kits may contain instructions
for carrying out any of the methods described herein for production
of polynucleotide fragments or labeled polynucleotide fragments, or
for one or more of the following applications using polynucleotide
fragments prepared as described herein: methods of producing a
hybridization probe; characterizing and/or quantitating nucleic
acid; detecting a mutation; preparing a subtractive hybridization
probe; detection (using a hybridization probe); and determining a
gene expression profile, using the fragmented nucleic acids
generated by the methods of the invention. The kits of the
invention comprise one or more containers comprising any
combination of the components described herein, and the following
are examples of such kits.
[0153] In one embodiment, a kit of the invention comprises an agent
capable of cleaving a base portion of a nucleotide to generate
abasic site in a polynucleotide, an agent capable of cleaving a
phosphodiester backbone of a polynucleotide at or near an abasic
site to produce a polynucleotide fragment with a blocked 3' end,
and an enzyme capable of unblocking the blocked 3' end to produce a
polynucleotide fragment comprising a 3' end hydroxyl group. The kit
may further comprise an agent capable of labeling the
polynucleotide fragment comprising a 3' end hydroxyl group and/or a
label to be incorporated into an extension or ligation product of
the polynucleotide fragment. In one embodiment, the kit further
comprises an enzyme capable of extending the 3' end of the
polynucleotide fragment in a template independent or template
dependent manner and optionally further comprises nucleotide
substrates for such enzymes, either labeled or unlabeled or a
mixture of labeled and unlabeled nucleotides. In one embodiment,
the kit further comprises a ligase enzyme. In one embodiment, the
kit comprises UNG, DMED, and APE 1, and optionally further
comprises TdT. In one embodiment, the kit further comprises
components for synthesis of a polynucleotide to be fragmented, such
as a primer, for example, a composite primer, and/or nucleotides,
for example, canonical and/or non-canonical nucleotides.
[0154] Kits may also include one or more suitable buffers (as
described herein) or any other necessary reagents for carrying out
the reactions of the methods described herein. One or more reagents
in the kit can be provided as a dry powder, usually lyophilized,
including excipients, which on dissolution will provide for a
reagent solution having the appropriate concentrations for
performing any of the methods described herein. Each component can
be packaged in separate containers or some components can be
combined in one container where cross-reactivity and shelf life
permit.
[0155] The kits of the invention may optionally include a set of
instructions, generally written instructions, although electronic
storage media (e.g., magnetic diskette or optical disk) containing
instructions are also acceptable, relating to the use of components
of the methods of the invention for the intended methods of the
invention, and/or, as appropriate, for using the products for
purposes such as, for example preparing a hybridization probe,
expression profiling, preparing a microarray, or characterizing a
nucleic acid. The instructions included with the kit generally
include information as to reagents (whether included or not in the
kit) necessary for practicing the methods of the invention,
instructions on how to use the kit, and/or appropriate reaction
conditions. Instructions may be in the form of printed media,
electronic media, or a reference to a website address where
instructions may be obtained.
[0156] The component(s) of the kit may be packaged in any
convenient, appropriate packaging. The components may be packaged
separately, or in one or multiple combinations.
[0157] The relative amounts of the various components in the kits
can be varied widely to provide for concentrations of the reagents
that substantially optimize the reactions that need to occur to
practice the methods disclosed herein and/or to further optimize
the sensitivity of any assay.
Tailing of the Fragmented Polynucleotide and Subsequent Isothermal
Single Primer Amplification (SPIA.TM.)
[0158] Fragmented polynucleotides may be tailed and subsequently
amplified using the previously-described SPIA.TM. amplification
procedure (U.S. Pat. Nos. 6,251,639 and 6,692,918).
[0159] Single stranded cDNA may be produced in the presence of
non-canonical nucleotides, for example, amplified from RNA or DNA
(e.g., amplified from RNA by the previously-described Ribo-SPIA.TM.
method (U.S. Pat. No. 6,946,251)) or amplified from DNA by the
SPIA.TM. method (U.S. Pat. Nos. 6,251,639 and 6,692,918), or
generated without amplification by random priming or poly-A
initiated reverse transcription using primers with or without a
5'-end tails (which can be RNA, for example, in a chimeric primer,
or DNA). Single stranded cDNA is produced in the presence of
non-canonical nucleotides and subsequently fragmented using a
method of the invention as described herein. The fragmented single
stranded DNA generated by the method of the invention comprises a
3'-hydroxyl terminus which can be extended by the template
independent DNA synthesis using terminal transferase. Tailing of
DNA using terminal transferase is well known in the art. The
addition of homopolymeric tails, with deoxyribonucleotides has been
previously described and is widely used for manipulation of cDNA
(see, e.g., G Deng et al. (1981) Nucleic Acids Res. 9(16):
4173-4188; Schmidt et al., Nucleic Acids Res. 24(9) 1789-1791;
Albuquerque-Silva et al., Nucleic Acids Research 26(13): 3314-3316;
U.S. Pat. No. 6,406,890). Tailing at the 3'-end of a polynucleotide
comprising a 3' end hydroxyl group using terminal transferase is
widely used in molecular biology and commercial reagents and kits
for various manipulations, including cDNA cloning, are readily
available.
[0160] It is desirable to generate cDNA copies of single stranded,
fragmented cDNA for further analysis, especially for analysis based
on hybridization to probes which are designed for hybridization to
the second strand cDNA (hybridization to cDNA which is the same
sense as mRNA). It is also desirable to linearly amplify second
strand cDNA fragments. The generation of fragmented amplified cDNA
comprising 3'-hydroxyl groups is described herein. Tailing of the
fragmented amplified cDNA with terminal transferase can be achieved
by methods described in the above references and well known in the
art. It is desirable to limit the length of the tail, which can be
achieved by controlling the reaction conditions, the amount of the
incorporated dNTP or rNTP or the use of a mixture of dNTP in the
presence of a terminator such as a ddNTP. Tailing of fragmented
cDNA products (produced from a single mRNA or multiplicity of mRNA
or total RNA) results in the generation of a whole representative
population of fragmented cDNA (libraries) with a common consensus
3'-end sequence.
[0161] These products may be amplified using a first chimeric
primer comprising a 3'-DNA portion that is hybridizable to the 3'
tail sequence of the tailed fragmented cDNA and a 5'-RNA sequence
which is not hybridizable to the fragmented cDNA products. DNA
polymerase comprising both DNA-dependent RNA-dependent DNA
polymerase activities may be used to extend the 3' ends of the
fragmented and tailed cDNA along the hybridized chimeric primer and
the 3'-end of the hybridized primer along the hybridized fragmented
and tailed cDNA, to generate double stranded DNA products with an
RNA/DNA heteroduplex at one end. The DNA-dependent DNA polymerase
and RNA-dependent DNA polymerase may be activities of the same
enzyme or of two different enzymes. Extension of the tailed
fragmented polynucleotide along the first chimeric primer requires
that the 3' end of the fragmented and tailed polynucleotide be
unblocked. Therefore, it is desirable to tail the fragmented
polynucleotide with nucleotides that do not serve as terminators.
This requirement dictates the type of tailing reaction mixtures and
conditions used for the tailing reaction. Amplification of this
product may proceed by the addition of a second amplification
chimeric primer that contains a sequence homologous to the sequence
of the RNA portion of the first chimeric primer, RNase H, and a DNA
polymerase with strand displacement activity, as previously
described (U.S. Pat. Nos. 6,251,239 and 6,692,918). Extension of
the first primer along the fragmented polynucleotide is not
essential to the process, and thus a 3'-blocked first chimeric
primer may also be useful for this process.
[0162] The multiplicity of copies of the fragmented cDNA as
described above is useful for further analysis and characterization
by any of the methods described below. The single stranded
amplification products can be labeled by incorporation of labeled
nucleotides, tailing of the single stranded amplification products
with labeled nucleotides using terminal transferase, and
incorporation of nucleotides which can be labeled post-DNA
synthesis, such as, for example, aminoallyl-dUTP or various
non-enzymatic methods for labeling of nucleic acids (for example,
ULS labeling, Kreatech).
Applications using the Labeling and/or Fragmentation and/or
Immobilization Methods of the Invention
[0163] The methods and compositions of the invention can be used
for a variety of purposes. For purposes of illustration, methods of
producing a hybridization probe, characterizing and/or quantitating
nucleic acid, detecting a mutation, preparing a subtractive
hybridization probe, detection (using the hybridization probe), and
determining a gene expression profile, using the labeled and/or
fragmented nucleic acids generated by the methods of the invention,
are described.
[0164] Immobilized polynucleotides, for example on a microarray,
prepared according to any of the methods of the invention, are also
useful for methods of analyzing and characterizing nucleic acids,
including methods of hybridizing nucleic acids, methods of
characterizing and/or quantitating nucleic acids, methods of
detecting a mutation in a nucleic acids, and methods of determining
a gene expression profile, as described below, and these
applications likewise apply to immobilized polynucleotides.
Method of Producing a Hybridization Probe
[0165] Labeled polynucleotide fragments obtained by the methods of
the invention are useful as hybridization probes. Accordingly, in
one aspect, the invention provides methods for nucleic acid
hybridization, comprising using a labeled polynucleotide fragment
as a hybridization probe, wherein the labeled polynucleotide
fragment is produced using a method as described herein. In one
embodiment, the invention provides a method for generating
hybridization probes, comprising generating labeled polynucleotides
using any of the methods described herein, and using the labeled
polynucleotides as a hybridization probe. In another embodiment,
the invention provides methods for generating a hybridization
probe, comprising generating labeled polynucleotide fragments using
any of the methods described herein, and using the labeled
polynucleotide fragments as a hybridization probe. The labeled
polynucleotide fragments can be produced from any template known in
the art, including RNA, DNA, genomic DNA (including global genomic
DNA amplification), and libraries (including cDNA, genomic or
subtractive hybridization library). The invention also provides
methods of hybridizing using the hybridization probes described
herein.
Characterization of Nucleic Acids
[0166] The labeled and/or fragmented nucleic acids obtained by the
methods of the invention are amenable to further
characterization.
[0167] The fragmented nucleic acids, or labeled fragments thereof
(i.e., products of any of the methods described herein), can be
analyzed using, for example, probe hybridization techniques known
in the art, such as Southern and Northern blotting, and hybridizing
to probe arrays. They can also be analyzed by electrophoresis-based
methods, such as differential display and size characterization,
which are known in the art.
[0168] In one embodiment, the methods of the invention are utilized
to analyze polynucleotide fragments, wherein the polynucleotide
fragments are generated using a method as described herein. In one
embodiment, the invention provides a method for analyzing
polynucleotides, comprising generating polynucleotide fragments,
e.g., labeled polynucleotide fragments, according to a method as
described herein, and contacting the polynucleotide fragments with
a probe. The polynucleotide fragments can be produced from any
template known in the art, including RNA, DNA, genomic DNA
(including global genomic DNA amplification), or amplified products
thereof, and libraries (including cDNA, genomic or subtractive
hybridization library).
[0169] In one embodiment, the methods of the invention are utilized
to generate fragmented polynucleotides which are analyzed (for
example, detection and/or quantification) by contacting them with,
for example, microarrays (of any suitable substrate, which includes
glass, chips, plastic), beads, or particles, that comprise suitable
probes such as cDNA and/or oligonucleotide probes. Thus, the
invention provides methods to characterize (for example, detect
and/or quantify and/or identify) a fragmented and labeled
polynucleotide by analyzing the labeled products, for example, by
hybridization of the labeled products to, for example, probes
immobilized at, for example, specific locations on a solid or
semi-solid substrate, probes immobilized on defined particles
(including beads, such as Bead Array, Illumina), or probes
immobilized on blots (such as a membrane), for example arrays, or
arrays of arrays. Immobilized probes include immobilized probes
generated by the methods described herein, and also include at
least the following: cDNA and synthetic oligonucleotides, which can
be synthesized directly on the substrate.
[0170] Other methods of analyzing labeled products are known in the
art, such as, for example, by contacting them with a solution
comprising probes, followed by extraction of complexes comprising
the labeled products and probes from solution. The identity of the
probes provides characterization of the sequence identity of the
products, and thus by extrapolation can also provide
characterization of the identity of a template from which the
products were prepared (for example, the identity of an RNA in a
solution). For example, hybridization of the labeled products is
detectable, and the amount of specific labels that are detected is
proportional to the amount of the labeled products prepared from a
specific RNA sequence of interest. This measurement is useful for,
for example, measuring the relative amounts of the various RNA
species in a sample, which are related to the relative levels of
gene expression, as described herein. The amount of labeled
products (as indicated by, for example, detectable signal
associated with the label) hybridized at defined locations on an
array can be indicative of the detection and/or quantification of
the corresponding template RNA species in the sample.
[0171] Methods of characterization include sequencing by
hybridization (see, e.g., Dramanac, U.S. Pat. No. 6,270,961) and
global genomic hybridization (also termed comparative genome
hybridization) (see, e.g., Pinkel, U.S. Pat. No. 6,159,685).
[0172] In another aspect, the invention provides a method of
quantitating fragmented polynucleotides comprising use of an
oligonucleotide (probe) of defined sequence (which may be
immobilized, for example, on a microarray).
Mutation Detection Utilizing the Methods of the Invention
[0173] The fragmented polynucleotides generated according to the
methods of the invention are also suitable for analysis for the
detection of any alteration in the template nucleic acid sequence
(from which the fragmented polynucleotides are synthesized), as
compared to a reference nucleic acid sequence which is identical to
the template nucleic acid sequence other than the sequence
alteration. The sequence alterations may be sequence alterations
present in the genomic sequence or may be sequence alterations
which are not reflected in the genomic DNA sequences, for example,
alterations due to post transcriptional alterations, and/or mRNA
processing, including splice variants. Sequence alterations
(interchangeably called "mutations") include deletion,
substitution, insertion and/or transversion of one or more
nucleotide.
[0174] Accordingly, the invention provides methods of detecting
presence or absence of a mutation in a template, comprising
analyzing a polynucleotide fragment generated using a method as
described herein, whereby presence or absence of a mutation is
detected. In one embodiment, the method comprises: (a) generating a
polynucleotide fragment, e.g., a labeled polynucleotide fragment,
by any of the methods described herein; and (b) analyzing the
polynucleotide fragment whereby presence or absence of a mutation
is detected. In some embodiments, the polynucleotide fragment is
compared to a labeled reference template, or fragments thereof.
Analyzing the polynucleotide fragment, whereby presence or absence
of a mutation is detected, can be performed by any method known in
the art. In some embodiments, probes for detecting mutations are
provided as a microarray.
[0175] Any alteration in the test nucleic acid sequence, such as
base substitution, insertions or deletion, could be detected using
this method. The method is expected to be useful for detection of
specific single base polymorphism, SNP, and the discovery of new
SNPs.
[0176] Other art recognized methods of analysis for the detection
of any alteration in the template nucleic acid sequence, as
compared to a reference nucleic acid sequence, are suitable for use
in the methods of the present invention. For example, essentially
any hybridization-based method of detection of mutations is
suitable for use with the fragmented polynucleotides produced by
the methods of the invention.
Determination of Gene Expression Profile
[0177] The fragmented polynucleotides, e.g., labeled polynucleotide
fragments, produced by the methods of the invention are
particularly suitable for use in determining the levels of
expression of one or more genes in a sample. As described above,
fragmented polynucleotides can be detected and quantified by
various methods, as described herein and/or known in the art. Since
RNA is a product of gene expression, the levels of the various RNA
species, such as mRNAs, in a sample is indicative of the relative
expression levels of the various genes (gene expression profile).
Thus, determination of the amount of RNA sequences of interest
present in a sample, as determined by quantifying products (for
example amplification products) of the sequences, provides for
determination of the gene expression profile of the sample
source.
[0178] Accordingly, the invention provides methods of determining
gene expression profile in a sample, said method comprising:
amplifying single stranded (or double stranded) product from at
least one RNA sequence of interest in the sample; generating an
abasic site in the amplified product; fragmenting the
polynucleotide comprising the abasic site according to the methods
described herein; and determining amount of fragmented
polynucleotide produced from each RNA sequence of interest, wherein
each said amount is indicative of amount of each RNA sequence of
interest in the sample, whereby the expression profile in the
sample is determined.
[0179] Accordingly, the invention provides of determining gene
expression profile in a sample, comprising determining the amount
of a polynucleotide fragment produced from a polynucleotide
template as described herein, wherein the amount of a fragment is
indicative of the amount of the polynucleotide template in the
sample from which the template was derived, whereby a gene
expression profile in the sample is determined. In one embodiment,
the method comprises: (a) generating a polynucleotide fragment,
e.g., a labeled polynucleotide fragment, from at least one
polynucleotide template in the sample using any of the methods
described herein; and (b) determining amount of polynucleotide
fragment produced from of each polynucleotide template, wherein
each said amount is indicative of amount of each polynucleotide
template in the sample, whereby the gene expression profile in the
sample is determined.
[0180] It is understood that amount of fragmented polynucleotide
produced (and thus the amount of product) may be determined using
quantitative and/or qualitative methods. Determining amount of
fragmented polynucleotides includes determining whether fragmented
polynucleotides are present or absent. Thus, an expression profile
can include information about presence or absence of one or more
RNA sequence of interest. "Absent" or "absence" of product, and
"lack of detection of product" as used herein includes
insignificant, or de minimus levels.
[0181] The methods of expression profiling are useful in a wide
variety of molecular diagnostics, and especially in the study of
gene expression in essentially any cell (including a single cell)
or cell population. A cell or cell population (e.g. a tissue) may
be from, for example, blood, brain, spleen, bone, heart, vascular,
lung, kidney, pituitary, endocrine gland, embryonic cells, tumors,
or the like. Expression profiling is also useful for comparing a
control (normal) sample to a test sample, including test samples
collected at different times, including before, after, and/or
during development, a treatment, and the like.
Methods of Preparing a Subtractive Hybridization Probe
[0182] Fragmented polynucleotides, e.g., labeled fragmented
polynucleotides produced by methods of the invention are
particularly suitable for use in preparation of subtractive
hybridization probes. For example, two nucleic acid populations,
one sense and one antisense, can be allowed to mix together with
one population present in molar excess ("driver"). Sequence present
in both populations will form hybrids, while sequences present in
only one population remain single-stranded. Thereafter, various
well-known techniques are used to separate the unhybridized
molecules representing differentially expressed sequences. See,
e.g., Hamson et al., U.S. Pat. No. 5,589,339; Van Gelder, U.S. Pat.
No. 6,291,170.
Comparative Hybridization
[0183] In another aspect, the invention provides methods for
comparative hybridization (such as comparative genomic
hybridization), said method comprising: (a) preparing a first
population of polynucleotide fragments from a first template
polynucleotide sample using any of the methods described herein;
(b) comparing hybridization of the first population to at least one
probe with hybridization of a second population of labeled
polynucleotides or fragments thereof. In some embodiments, the at
least one probe is a chromosomal spread. In still other
embodiments, the at least one probe is provided as a microarray. In
some embodiments, the first and second population comprise
detectably different labels. In other embodiments, a second
population of polynucleotide fragments is prepared from a second
polynucleotide sample using any of the methods described herein. In
some embodiments, comparing comprises determining amount of the
products, whereby the amount of the first and second polynucleotide
templates is quantified.
[0184] In some embodiments, comparative hybridization comprises
preparing a first population of labeled polynucleotide fragments
according to any of the methods described herein, wherein the
template from which the first population is synthesized is genomic
DNA. A second population of labeled polynucleotides (to which the
first population is desired to be compared) is prepared from a
second genomic DNA template. The first and second populations are
labeled with different labels. The hybridized first and second
populations are mixed, and hybridized to an array or chromosomal
spread. The different labels are detected and compared.
[0185] The following Examples are provided to illustrate, but not
limit, the invention.
Examples
Example 1
Removal of Blocked 3' Termini and Progressive Degradation of Single
Stranded DNA with Processive 3' to 5' Exonuclease
[0186] Amplified single stranded cDNA comprising dUTP was prepared
by isothermal RNA amplification using the Ovation Biotin System
(NuGEN Technologies) according to the manufacturer's instruction.
Amplified cDNA products generated by amplification of a few total
RNA samples (Universal Human RNA, Stratagene, 20 ng each) were
purified and pooled. The pooled purified cDNA was used throughout
the examples below.
[0187] Pooled cDNA (5 ug) was mixed with UNG (USB, 4 units) in
reaction buffer containing 32 mM DMED, and incubated at 37.degree.
C. for 30 minutes. As shown previously (U.S. Application No.
2004/0005614), treatment with UNG results in the removal of the
base portion of dU residues and formation of abasic sites. DMED
cleaves the backbone to generate fragmented DNA with 3'-modified
termini. The blocked 3'-end can be removed by a 3' to
5'-exonucleases to generate a 3' hydroxyl group. Insofar as the aim
of this procedure is to generate fragmented DNA with 3'-OH termini,
it is desirable to use a non processive exonuclease so as to assure
limited hydrolysis of the fragmented DNA. The nuclease activities
of exonuclease 1 (Exo1), exonuclease 3 (Exo3), exonuclease T
(ExoT), endonuclease D (Endo4), and APE 1 (NEB) were tested. The
UNG and DMED treated amplified cDNA was purified (DyeEx, Qiagen)
and the purified product was incubated with the various enzymes in
the respective reaction buffers (as per the manufacturer
instructions). Following 30 min. incubation at 37.degree. C., the
products were purified and the size of the treated products was
analyzed electrophoretically (BioAnalyzer, Agilent). The more
processive the 3' to 5' exonuclease activity of a given enzyme, the
greater the expected result in reduced size and reduction in
quantity of the fragmented single stranded cDNA population. As
shown in FIG. 3A, processive exonuclease activity of Exo1 and Exo3
resulted in reduced product recovery and reduced size of the
recovered fragmented cDNA, as compared to that of the UNG and DMED
fragmented cDNA substrate. The non processive 3'-exonuclease
activity of endonuclease 4, exonuclease T, and APE 1, on the other
hand, resulted in product distribution size similar to the input
fragmented cDNA (UNG and DMED treated amplified cDNA). Further
evidence for the non processive 3'-deblocking activity of these
enzymes was obtained by the demonstration of the ability to end
label the fragmented cDNA treated with these enzymes by
template-independent extension of the 3-OH termini of the cDNA by
terminal transferase, as described in Example 2 below.
[0188] APE 1 is a multifunctional repair enzyme comprising an
endonuclease activity for the excision of abasic sites and
subsequent fragmentation of the DNA template to generate fragments
with 3-OH termini, on the one hand, and a 3' to 5' exonuclease
activity for the removal of the blocked 3'-end of damaged DNA
template. As shown in FIG. 3B, treatment of amplified cDNA
comprising abasic sites (amplified cDNA treated with the specific
glycosylase, UNG), with APE 1 (40units) led to partial
fragmentation of the cDNA template, as compared to the size
distribution of DMED fragmented amplified cDNA comprising abasic
sites. The relative inefficiency of APE 1 endonuclease activity for
full fragmentation of the amplified cDNA template comprising abasic
site resulted in the requirement of a large amount of the enzyme.
Full fragmentation of the template DNA was achieved in reactions
carried out with 500 to 1000 units of APE 1 (data not shown). The
3' to 5' exonuclease activity of the enzyme is very efficient, and
the non processive exonuclease activity enables the de-blocking of
the 3'-end of template cDNA fragmented by the combined treatment
with UNG (to generate abasic sites) and DMED (to fragment the DNA
backbone while leaving an aldehyde group at the 3'termini). Use of
the exonuclease activity of the enzyme for the generation of
fragments with 3 '-OH termini was validated by the ability to end
label the fragmented and de-blocked template by terminal
transferase template-independent extension with labeled nucleotide
(biotin end labeling) as described in Example 2 below.
Example 2
Generation of Fragmented cDNA with 3'-OH Termini which are Suitable
for Labeling by Template Independent Extension using Terminal
Deoxynucleotidyl Transferase (TdT) and Labeled Nucleotide
[0189] Pooled amplified cDNA comprising dU residues was prepared as
described in Example 1. Treatment of the pooled cDNA with UNG and
DMED, as described in Example 1, was used to generate fragmented
cDNA with blocked 3'-OH termini. A cDNA fragment with a blocked 3'
terminus can be labeled with an aldehyde reactive conjugate of a
desired label to yield fragmented and labeled cDNA target suitable
for microarray based analysis (U.S. Application No. 2004/0005614;
Dafforn et al. (2004) "BioTechniques 37:854-857; Kurn et al. (2005)
Clinical Chemistry 51:1973-1981). However, extension of the
fragmentation process to polymerase extension based end labeling
(e.g., terminal transferase template independent labeling) requires
deblocking of the 3'-termini. As discussed in Example 1, various
non processive 3'-to-5'exonucleases were tested for the ability to
deblock the blocked 3' termini of such fragments. The validation of
the ability to generate suitable substrates for end labeling was
obtained by end labeling of the pooled fragmented and nuclease
treated targets for end labeling with TdT (terminal
deoxynucleotidyl transferase) and biotin labeled ddUTP. End
labeling of the targets was assessed by hybridization of the
targets to high density Human Focus GeneChip arrays (Affymetrix).
Various array analysis parameters obtained with the various targets
are shown in Table 1.
[0190] End labeling of the various fragmented cDNA products with
TdT was carried out under the following conditions: The reactions
were carried out in 1.times. NEB buffer #4 (50 mM potassium
acetate, 20 mM Tris-Acetate, 10 mM magnesium acetate, 1 mM DTT, pH
7.9), 0.25 mM CoCl2, in the presence of 0.5 nmol Biotin
2',3'-dideoxy-UTP (Roche), and 40U TdT, in a total volume of 50
.mu.l. The TdT reactions were carried out at 37.degree. C. for 60
min. followed by TdT inactivation (70.degree. C. for 15 min). The
reaction products were added to a GeneChip hybridization mixture.
Hybridization, wash, signal generation and array scanning were
carried out as per the manufacturer instructions.
TABLE-US-00001 TABLE 1 Array Analysis Parameters Scaling Back- %
(3'/5') Array Targets Raw Q Factor ground Present GAPDH 1 UNG,
DMED, TdT, ddUTP 0.88 276.1 30.4 4.3 1.40 2 UNG, APE1(30U), TdT,
ddUTP 0.93 44.1 32.3 32.3 1.84 3 UNG, DMED, DyeEx ExoT, TdT, 0.99
60.6 31.6 25.5 2.20 ddUTP 4 UNG, DMED, DyeEx Endo4, TdT, 1.19 24.6
34.8 38.3 2.13 ddUTP 5 UNG, DMED, DyeEx APE10U, 1.04 16.7 28.2 47.2
2.25 TdT, ddUTP 6 UNG, DMED, DyeEx APE50U. 1.16 17.1 33.9 45.4 1.90
TdT, ddUTP 7 UNG, DMED, APE10U, TdT, 0.92 30.9 26.3 41 2.25 ddUTP 8
UNG, DMED100 mM, pH 7.4, Mg 1.06 8.3 31.9 56.8 1.54 0.4 mM, APE10U,
TdT, ddUTP 9 UNG, DMED100 mM, pH 7.4, Mg 1.01 6.5 31.1 60.4 1.47 2
mM, APE10U, TdT, ddUTP 10 UNG, DMED34 mM, pH 7.4, Mg 1.07 4.0 31.7
64.0 1.50 4 mM, APE10U, TdT, ddUTP
Results
[0191] Array 1: The target was generated by Biotin 3'-end labeling
of fragmented targets which were not further treated with
exonuclease to unblock the 3'termini. The high Scaling Factor and
low percent of genes called Present (% Present), represent poor
labeling and are consistent with the inability to extend the
3'-blocked target for end labeling.
[0192] Removal of the abasic site generated by UNG by APE 1, which
cleaves the phosphodiester bond 5' to the abasic site sugar,
generating a nick with 5' sugar phosphate (dRP) and 3' hydroxyl
group at the 3'-end, enabling the biotin labeling of the fragmented
cDNA targets by TdT. The endonuclease activity results in lower
Scaling Factor and higher % Present results as compared to Array 1.
However, this array performance is not optimal as the cDNA fragment
sizes are lager than DMED cuts (as seen in FIG. 3B). The results
indicate that the endonuclease activity of APE 1 is not sufficient
to efficiently fragment the cDNA comprising abasic sites to the
proper size required for efficient hybridization to the high
density GeneChip arrays (Affymetrix)
[0193] Arrays 3 and 4: Purified cDNA comprising dU residues was
fragmented by the action of UNG and DMED and further treated with
exo T or the exonuclease activity of Endo 4, respectively. The
results indicate efficient exonuclease activity of these
nonprocessive exonucleases so as to unblock the 3'-termini and
enable end-labeling by TdT and ddUTP.
[0194] Arrays 5 and 6: Similar labeling efficiency was enabled by
unblocking of the DMED fragmented cDNA by the exonuclease activity
of APE 1 when present at either 10 or 50 units per reaction.
[0195] Array 7: The reaction condition was the same as for the
target hybridized to array 5, except that UNG generation of abasic
sites, DMED fragmentation of the cDNA at the abasic sites and
unblocking of the 3'-end of the fragmented cDNA by APE 1 were
carried out in a single reaction mixture (30 mi. at 37.degree. C.).
The array results obtained with these reaction conditions provided
a comparison for arrays 8, 9, and 10 as generation of abasic sites,
fragmentation, and unblocking of 3'end occurred in a single
reaction.
[0196] Arrays 8, 9 and 10: Reaction conditions for the generation
of fragmented cDNA with 3'-OH termini were assessed. The best
performance (as per the ability to efficiently biotin-end-label the
products by TdT for improved array results) was observed with
conditions for target hybridized to array 10. As described for
targets hybridized to arrays 8 and 9, UNG, DMED and APE 1 were
reacted in a single reaction mixture. The general superior
performance for this group of arrays is attributed to the reaction
buffer condition that favored 3'-to 5'-exonuclease activity of APE
1. The buffer condition changes included lower Mg.sup.2+
concentration, no Na.sup.+, and the reaction pH at 7.4. (Chou et
al. (2003) J. Biol. Chem. 278(20):18289-96.
[0197] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entireties for
all purposes and to the same extent as if each individual
publication, patent, or patent application were specifically and
individually indicated to be so incorporated by reference.
[0198] Although the foregoing invention has been described in some
detail by way of illustration and examples for purposes of clarity
of understanding, it will be apparent to those skilled in the art
that certain changes and modifications may be practiced without
departing from the spirit and scope of the invention. Therefore,
the description should not be construed as limiting the scope of
the invention, which is delineated by the appended claims.
Sequence CWU 1
1
11123DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1nnnnnnnnnn nnnnnnnnnn nnn
23223DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2nnnnnnnnnn nnnnnnnnnn nnn
2335DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 3nnnnn 546DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4nnnnnn 657DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 5nnnnnnn
765DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 6nnnnn 574DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7nnnn 486DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 8nnnnnn
695DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 9nnnnn 5106DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10nnnnnn 6117DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 11nnnnnnn 7
* * * * *