U.S. patent application number 12/619664 was filed with the patent office on 2011-12-15 for methods, kits and compositions pertaining to the suppression of the detectable probe binding to randomly distributed repeat sequences genomic nucleic acid.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to JENS J. HYLDIG-NIELSEN, KIRSTEN VANG NIELSON, BRETT F. WILLIAMS.
Application Number | 20110306520 12/619664 |
Document ID | / |
Family ID | 23263855 |
Filed Date | 2011-12-15 |
United States Patent
Application |
20110306520 |
Kind Code |
A1 |
NIELSON; KIRSTEN VANG ; et
al. |
December 15, 2011 |
Methods, Kits and Compositions Pertaining to the Suppression of the
Detectable Probe Binding to Randomly Distributed Repeat Sequences
Genomic Nucleic Acid
Abstract
This invention is directed to methods, kits, non-nucleotide
probes as well as other compositions pertaining to the suppression
of binding of detectable nucleic acid probes to undesired
nucleotide sequences of genomic nucleic acid in assays designed to
determine target genomic nucleic acid.
Inventors: |
NIELSON; KIRSTEN VANG;
(BRONSHO, DK) ; HYLDIG-NIELSEN; JENS J.; (MOSS
BEACH, CA) ; WILLIAMS; BRETT F.; (EUGENE,
OR) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
CARLSBAD
CA
|
Family ID: |
23263855 |
Appl. No.: |
12/619664 |
Filed: |
November 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10255434 |
Sep 24, 2002 |
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12619664 |
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60324499 |
Sep 24, 2001 |
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Current U.S.
Class: |
506/16 ; 506/18;
530/409 |
Current CPC
Class: |
C12Q 1/6837 20130101;
C12Q 1/6837 20130101; C12Q 2600/156 20130101; C12Q 1/6832 20130101;
C12Q 1/6832 20130101; C12Q 1/6876 20130101; C12Q 2537/162 20130101;
C12Q 2525/151 20130101; C12Q 2525/107 20130101; C12Q 2525/186
20130101; C12Q 2525/107 20130101; C12Q 2525/186 20130101; C12Q
2537/162 20130101; C12Q 2525/107 20130101; C12Q 2525/186 20130101;
C12Q 1/6832 20130101 |
Class at
Publication: |
506/16 ; 506/18;
530/409 |
International
Class: |
C40B 40/10 20060101
C40B040/10; C07K 2/00 20060101 C07K002/00; C40B 40/06 20060101
C40B040/06 |
Claims
1-17. (canceled)
18. A mixture of two or more peptide nucleic acid probes of
different nucleobase sequence, wherein the two or more probes are
16 to 50 nucleobases in length, and each probe comprises at least
sixteen consecutive nucleobases that are greater than ninety
percent homologous to a fraction of SEQ ID NO: 1 or SEQ ID NO:
2.
19-36. (canceled)
37. A composition comprising: a) genomic nucleic acid comprising
one or more segments of randomly distributed repeat sequence; and
b) two or more peptide nucleic acid probes of different nucleobase
sequence hybridized to at least a fraction of the one or more
segments of randomly distributed repeat sequence of the genomic
nucleic acid, wherein each of the two or more probes is 16 to 50
nucleobases in length and each comprises at least sixteen
consecutive nucleobases that are greater than eighty percent
homologous to a fraction of SEQ ID NO: 1 or SEQ ID NO: 2.
38. A composition comprising: a) a detectable nucleic acid probe of
at least 100 nucleobases that has been derived from genomic nucleic
acid and that contains one or more segments of randomly distributed
repeat sequence; and b) two or more peptide nucleic acid probes of
different nucleobase sequence hybridized to at least a fraction of
the one or more segments of randomly distributed repeat sequence of
the detectable nucleic acid probe, wherein each of the two or more
probes is 16 to 50 nucleobases in length and each comprises at
least sixteen consecutive nucleobases that are greater than eighty
percent homologous to a fraction of SEQ ID NO: 1 or SEQ ID NO:
2.
39-90. (canceled)
91. A reagent kit comprising: a) a mixture of two or more peptide
nucleic acid probes of different nucleobases sequence, wherein the
two or more probes are 16 to 50 nucleobases in length, and wherein
each probe comprises at least sixteen consecutive nucleobases that
are at least eighty percent homologous to a fraction of SEQ ID NO:
1 and SEQ ID NO:2; and b) reagents and compositions for performing
an assay to determine hybridization of one or more detectable
nucleic acid probes to one or more targets in genomic nucleic acid
of a sample.
92-96. (canceled)
97. A kit comprising: a) a mixture of two or more peptide nucleic
acid probes of different nucleobases sequence, wherein the two or
more probes are 16 to 50 nucleobases in length, and wherein each
probe comprises a segment of at least ten consecutive nucleobases
that are at least eighty percent homologous to a sequence selected
from the group consisting of: SEQ ID NOS: 3-13 and 15-25; and b)
reagents and compositions for performing an assay to determine
hybridization of one or more detectable nucleic acid probes to one
or more targets in genomic nucleic acid of a sample.
98-102. (canceled)
103. A combination of two or more peptide nucleic acid probes of
differing sequence each consisting of a nucleobase sequence from 16
to 50 nucleobases in length wherein each probe comprises at least
sixteen consecutive nucleobases that are at least eighty percent
homologous to one or more sequences selected from the group
consisting of SEQ ID NOS: 3-13.
104. A peptide nucleic acid probe, comprising one or more of the
nucleobase sequences selected from the group consisting of SEQ ID
NOS: 3, 5-9, 11-13 and 15-25, wherein the peptide nucleic acid
probe is 16 to 50 nucleobases in length.
105. The peptide nucleic acid probe of claim 104, wherein the
peptide nucleic acid subunits of the peptide nucleic acid probe
have the formula: ##STR00005## wherein: each J is the same or
different and is selected from the group consisting of: H, R.sup.1,
OR', SR.sup.1, NHR.sup.1, NR.sup.1.sub.2, F, CI, Br and I; each K
is the same or different and is selected from the group consisting
of: O, S, NH and NR.sup.1; each R.sup.1 is the same or different
and is an alkyl group comprising one to five carbon atoms that may
optionally contain a heteroatom or a substituted or unsubstituted
aryl group; each A is selected from the group consisting of a
single bond, a group of the formula; --(CJ.sub.2).sub.s- and a
group of the formula; --(CJ.sub.2).sub.sC(O)--, wherein J is
defined above and each s is an integer from one to five; each t is
1 or 2; each u is 1 or 2; and each L is the same or different and
is selected from the group consisting of: J, dabcyl, fluorescein,
adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine,
2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,
hypoxanthine, p seudoisocyto sine, 2-thiouracil, 2-thiothymidine,
other naturally occurring nucleobase analogs, other non-naturally
occurring nucleobases and substituted and unsubstituted aromatic
moieties.
106. The peptide nucleic acid probe of claim 104, wherein the
peptide nucleic acid subunits of the peptide nucleic acid probe
comprise a naturally or nonnaturally occurring nucleobase attached
to an aza nitrogen of an N-[2-(aminoethyl)]glycine backbone through
a methylene carbonyl linkage.
107. A composition, comprising: a) one or more detectable nucleic
acid probes; and b) the peptide nucleic acid probe of claim
104.
108. The composition of claim 107, wherein at least one detectable
nucleic acid probe is at least 100 nucleobases in length.
109. A composition, comprising: a) genomic nucleic acid; and b) the
peptide nucleic acid probe of claim 104.
110. The composition of claim 109, wherein the genomic nucleic acid
comprises one or more segments of randomly distributed repeat
sequence.
111. A kit, comprising: a) the peptide nucleic acid probe of claim
110; and b) reagents and compositions for performing an assay to
determine hybridization of one or more detectable nucleic acid
probes to one or more targets in genomic nucleic acid of a
sample.
112. A mixture of about 5 to about 50 peptide nucleic acid probes
of different nucleobase sequence, wherein each peptide nucleic acid
probe is at least sixteen subunits in length and comprises a
nucleobase sequence of at least ten consecutive nucleobases that
are at least eighty percent homologous to SEQ ID NO: 1 or SEQ ID
NO: 2, without regard to whether the subunits containing the at
least ten consecutive nucleobases are separated by one or more
linkers.
113. The mixture of peptide nucleic acid probes of claim 112,
wherein the peptide nucleic acid subunits of at least one peptide
nucleic acid probe have the formula: ##STR00006## wherein: each J
is the same or different and is selected from the group consisting
of: H, R.sup.1, OR', SR.sup.1, NHR.sup.1, NR.sup.1.sub.2, F, CI, Br
and I; each K is the same or different and is selected from the
group consisting of: O, S, NH and NR.sup.1; each R.sup.1 is the
same or different and is an alkyl group comprising one to five
carbon atoms that may optionally contain a heteroatom or a
substituted or unsubstituted aryl group; each A is selected from
the group consisting of a single bond, a group of the formula;
--(CJ.sub.2).sub.s- and a group of the formula;
--(CJ.sub.2).sub.sC(O)--, wherein J is defined above and each s is
an integer from one to five; each t is 1 or 2; each u is 1 or 2;
and each L is the same or different and is selected from the group
consisting of: J, dabcyl, fluorescein, adenine, cytosine, guanine,
thymine, uridine, 5-methylcytosine, 2-aminopurine,
2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,
pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally
occurring nucleobase analogs, other non-naturally occurring
nucleobases and substituted and unsubstituted aromatic
moieties.
114. The mixture of peptide nucleic acid probes of claim 112,
wherein the peptide nucleic acid subunits of at least one peptide
nucleic acid probe comprise a naturally or non-naturally occurring
nucleobase attached to an aza nitrogen of an
N42-(aminoethyl)]glycine backbone through a methylene carbonyl
linkage.
115. A composition, comprising: a) one or more detectable nucleic
acid probes; and b) the mixture of peptide nucleic acid probes of
claim 112.
116. The composition of claim 115, wherein at least one detectable
nucleic acid probe is at least 100 nucleobases in length.
117. A composition, comprising: a) genomic nucleic acid; and b) the
mixture of peptide nucleic acid probes of claim 112.
118. The composition of claim 117, wherein the genomic nucleic acid
comprises one or more segments of randomly distributed repeat
sequence.
119. A kit, comprising: a) the mixture of peptide nucleic acid
probes of claim 112; and b) reagents and compositions for
performing an assay to determine hybridization of one or more
detectable nucleic acid probes to one or more targets in genomic
nucleic acid of a sample.
120. A mixture of about 5 to about 50 peptide nucleic acid probes
of different nucleobase sequence, wherein each peptide nucleic acid
probe is at least sixteen subunits in length and comprises a
nucleobase sequence of at least ten consecutive nucleobases that
are at least eighty percent homologous to a sequence selected from
the group consisting of: SEQ ID NOS: 3-26, without regard to
whether the subunits containing the at least ten consecutive
nucleobases are separated by one or more linkers.
121. The mixture of peptide nucleic acid probes of claim 120,
wherein the peptide nucleic acid subunits of at least one peptide
nucleic acid probe have the formula: ##STR00007## wherein: each J
is the same or different and is selected from the group consisting
of: H, R.sup.1, OR', SR.sup.1, NHR.sup.1, NR.sup.1.sub.2, F, CI, Br
and I; each K is the same or different and is selected from the
group consisting of: O, S, NH and NR.sup.1; each R.sup.1 is the
same or different and is an alkyl group comprising one to five
carbon atoms that may optionally contain a heteroatom or a
substituted or unsubstituted aryl group; each A is selected from
the group consisting of a single bond, a group of the formula;
--(CJ.sub.2).sub.s- and a group of the formula;
--(CJ.sub.2).sub.sC(O)--, wherein J is defined above and each s is
an integer from one to five; each t is 1 or 2; each u is 1 or 2;
and each L is the same or different and is selected from the group
consisting of: J, dabcyl, fluorescein, adenine, cytosine, guanine,
thymine, uridine, 5-methylcytosine, 2-aminopurine,
2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,
pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally
occurring nucleobase analogs, other non-naturally occurring
nucleobases and substituted and unsubstituted aromatic
moieties.
122. The mixture of peptide nucleic acid probes of claim 120,
wherein the peptide nucleic acid subunits of at least one peptide
nucleic acid probe comprise a naturally or non-naturally occurring
nucleobase attached to an aza nitrogen of an
N-[2-(aminoethyD]glycine backbone through a methylene carbonyl
linkage.
123. A composition, comprising: a) one or more detectable nucleic
acid probes; and b) the mixture of peptide nucleic acid probes of
claim 120.
124. The composition of claim 123, wherein at least one detectable
nucleic acid probe is at least 100 nucleobases in length.
125. A composition, comprising: a) genomic nucleic acid; and b) the
mixture of peptide nucleic acid probes of claim 121.
126. The composition of claim 125, wherein the genomic nucleic acid
comprises one or more segments of randomly distributed repeat
sequence.
127. A kit, comprising: a) the mixture of peptide nucleic acid
probes of claim 121; and b) reagents and compositions for
performing an assay to determine hybridization of one or more
detectable nucleic acid probes to one or more targets in genomic
nucleic acid of a sample.
128. A mixture of about 5 to about 50 peptide nucleic acid probes
of different nucleobase sequence, wherein each peptide nucleic acid
probe is at least sixteen subunits in length and comprises a
nucleobase sequence selected from the group consisting of: SEQ ID
NOS: 3-14, without regard to whether the subunits containing the at
least sixteen nucleobases are separated by one or more linkers.
129. The mixture of peptide nucleic acid probes of claim 128,
wherein each peptide nucleic acid probe comprises a nucleobase
sequence at least sixteen nucleobases in length selected from the
group consisting of: SEQ ID NOS: 3-13 and the sequence
TT(k)TTTTT(k)TTTLysOLysOTTT(k)TTTTT(k)TT, wherein each k is a
D-lysine; Lys is L-lysine; and each 0 is 8-amino-3,6-dioxaoctanoic
acid, without regard to whether the subunits containing the at
least sixteen nucleobases are separated by one or more linkers.
130. The mixture of peptide nucleic acid probes of claim 128,
wherein the peptide nucleic acid subunits of at least one peptide
nucleic acid probe have the formula: ##STR00008## wherein: each J
is the same or different and is selected from the group consisting
of: H, R.sup.1, OR', SR.sup.1, NHR.sup.1, NR.sup.1.sub.2, F, CI, Br
and I; each K is the same or different and is selected from the
group consisting of: O, S, NH and NR.sup.1; each R.sup.1 is the
same or different and is an alkyl group comprising one to five
carbon atoms that may optionally contain a heteroatom or a
substituted or unsubstituted aryl group; each A is selected from
the group consisting of a single bond, a group of the formula;
--(CJ.sub.2).sub.s- and a group of the formula;
--(CJ.sub.2).sub.sC(O)--, wherein J is defined above and each s is
an integer from one to five; each t is 1 or 2; each u is 1 or 2;
and each L is the same or different and is selected from the group
consisting of: J, dabcyl, fluorescein, adenine, cytosine, guanine,
thymine, uridine, 5-methylcytosine, 2-aminopurine,
2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,
pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally
occurring nucleobase analogs, other non-naturally occurring
nucleobases and substituted and unsubstituted aromatic
moieties.
131. The mixture of peptide nucleic acid probes of claim 128,
wherein the peptide nucleic acid subunits of at least one peptide
nucleic acid probe comprise a naturally or non-naturally occurring
nucleobase attached to an aza nitrogen of an
N42-(aminoethyl)]glycine backbone through a methylene carbonyl
linkage.
132. A composition, comprising: a) one or more detectable nucleic
acid probes; and b) the mixture of peptide nucleic acid probes of
claim 128.
133. The composition of claim 132, wherein at least one detectable
nucleic acid probe is at least 100 nucleobases in length.
134. A composition, comprising: a) genomic nucleic acid; and b) the
mixture of peptide nucleic acid probes of claim 128.
135. The composition of claim 134, wherein the genomic nucleic acid
comprises one or more segments of randomly distributed repeat
sequence.
136. A kit, comprising: a) the mixture of peptide nucleic acid
probes of claim 128; and b) reagents and compositions for
performing an assay to determine hybridization of one or more
detectable nucleic acid probes to one or more targets in genomic
nucleic acid of a sample.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of United States
Provisional Patent Application Ser. No. 60/324,499, filed on Sep.
24, 2001; herein incorporated by reference for any and all
purposes.
BACKGROUND
[0002] 1. Field of the Invention
[0003] This invention pertains to the field of molecular
cytogenetics and more specifically this invention pertains methods,
kits and compositions being used to suppress the binding of
detectable nucleic acid probes to undesired sequences, such as
randomly distributed repeat sequences, in genomic nucleic acid.
[0004] 2. Background
[0005] Nucleic acid hybridization is a fundamental process in
molecular biology. Probe-based assays are useful in the detection,
identification, quantitation and/or analysis of nucleic acids.
Nucleic acid probes have long been used to analyze samples for the
presence of nucleic acid from bacteria, fungi, virus or other
organisms and are also useful in examining genetically-based
disease states or clinical conditions of interest. Nonetheless,
nucleic acid probe-based assays have been slow to achieve
commercial success. This lack of commercial success is, at least
partially, the result of difficulties associated with specificity,
sensitivity and/or reliability.
[0006] Fluorescence in-situ hybridization (FISH) has become an
important tool for determining the number, size and/or location of
specific DNA sequences in mammalian cells. Typically, the
hybridization reaction fluorescently stains the target sequences so
that their location, size and/or number can be determined using
fluorescence microscopy, flow cytometry or other suitable
instrumentation. DNA sequences ranging from whole genomes down to
several kilobases can be studied using current hybridization
techniques in combination with commercially available
instrumentation.
[0007] In Comparative Genomic Hybridization (CGH) whole genomes are
stained and compared to normal reference genomes for the detection
of regions with aberrant copy number. In the m-FISH technique
(multi color FISH) each separate normal chromosome is stained by a
separate color (Eils et al, Cytogenetics Cell Genet 82: 160-71
(1998)). When used on abnormal material, the probes will stain the
aberrant chromosomes thereby deducing the normal chromosomes from
which they are derived (Macville M et al., Histochem Cell Biol.
108: 299-305 (1997)). Specific DNA sequences, such as the ABL gene,
can be reliably stained using probes of only 15 kb (Tkachuk et al.,
Science 250: 559-62 (1990)). FISH-based staining is sufficiently
distinct such that the hybridization signals can be seen both in
metaphase spreads and in interphase nuclei. Single and multicolor
FISH, using nucleic acid probes, have been applied to different
clinical applications generally known as molecular cytogenetics,
including prenatal diagnosis, leukemia diagnosis, and tumor
cytogenetics.
[0008] A large component of the human genome comprises repeat
sequences. Heat denaturation and reannealing studies on DNA of
higher organisms have distinguished three populations of eukaryotic
DNA; a quickly reannealing component representing 25% of total DNA,
an intermediate component that represents 30% of the total DNA, and
a slow component that represents 45% of the total DNA (Britten et
al., Science 161: 529-540 (1968)). Sequence analysis has shown that
the slow component is made up by single-copy sequences, which
include protein encoding genes, while the fast and intermediate
components represents repetitive sequences. The fast component
contains small (a few nucleotides long), highly repetitive DNA
sequences, which are usually found in tandem while the intermediate
component contain the interspersed repetitive DNA (Novick et al.,
Human Genome Bioscience, 46(1): 32-41 (1996) and Brosius J.,
Science 251: 753 (1991)). The repetitive units of the intermediate
component are interspersed within the genome and is the major
reason that large genomic nucleic acid probes (i.e. >100 bp)
derived from genomic nucleic acid are not well suited for
hybridization analysis.
[0009] Interspersed repeated sequences are classified as either
SINEs (short interspersed repeated sequences) or LINEs (Kroenberg
et al., Cell, 53: 391-400 (1988)). In primates, each of these
classes are dominated by a single DNA sequence family, both of
which are classified as retrosponos (Rogers J., International
Review of Cytology, 93: 187-279 (1985)). The major human SINEs are
the Alu-repeat DNA sequence family. The Alu-repeat DNA family
members are characterized by a consensus sequence of approximately
280 to 300 by which consist of two similar sequences arranged as a
head to tail dimer. Approximately one million copies of the Alu
repeat sequence are estimated to be present per haploid human
genome, thereby representing about ten percent of the genome
(Ausubel et al., Current Protocols In Molecular Biology, John Wiley
& Sons, Inc., 1996)). That estimate is consistent with the
recent sequence determination of the human chromosome 21 and 22.
These reports demonstrate that Alu repeats cover 9.48% and 16.80%
of the DNA, respectively (Hattori et al. Nature, 405: 311-319
(2000) and Dunham I. et al., Nature, 402: 489-495 (1999)).
[0010] Alu elements have amplified in the human genome through
retroposition over the past 65 million years and have been
organized into a wealth of overlapping subfamilies based on
diagnostic mutations shared by subfamily members (See For Example:
Batzer et al., J. Mol. Evol., 42: 3-6 (1996)). Batzer et al.
described a consensus nomenclature for Alu repeats sequences;
representing the oldest (J), intermediate (S) and young (Y) family
branches. Only the Y family branch is still transcriptional active
but it is very small as each of the defined a5, a8 and b8 subfamily
members have produced less than 2000 elements (Sherry et al.,
Genetics, 147: 1977-1982 (1997)). It has been calculated that of
the primate Alu repeat family branches, approximately one-fifth
belong to the J family and four-fifths to the S family (Britten, R.
J., Proc. Natl. Acad. Sci. USA, 91: 6148-6150 (1994). The S family
is dominated by the Sx subfamily as it represents more than 50% of
the total S family branch.
[0011] In addition to SINEs and LINEs, there are several other
types of repeats that are known to exist in genomic nucleic acid of
humans as well as in other organisms. Chromosome telomeres are
repeat sequences that appear to exist only, or else predominately,
at the termini of all chromosomes. They are believed to shorten
during the life of an organism and may play a role in the aging of
an organism (See: Landsorp, P., WIPO Patent Application No.
WO97/14026). Likewise, chromosome centromeres contain distinct
repeat sequences that exist only, or else predominately, in the
central (centromere) region of a chromosome. Certain of the
centromere repeat sequences can be detected in all chromosomes of
an organism whilst other repeat sequences are unique to a
particular chromosome and can be used to identify specific
chromosomes (Taneja et al., Genes, Chromosomes & Cancer, 30:
57-63 (2001)).
[0012] Telomere and centromere repeat sequences differ from
interspersed repetitive sequence, such as SINE and LINEs, in that
the telomere and centromere repeat sequences are localized within a
certain region of the chromosome. By comparison, SINEs and LINEs,
which are referred to herein as randomly distributed repeat
sequences, are dispersed randomly throughout the entire genome
(Ullu E., TIBS: 216-219 (June, 1982)). Thus, as used herein, the
term "randomly distribute repeat sequence" is intended to refer to
repeat sequences that occur randomly within all, or essentially
all, genomic nucleic acid of an organism. These include, but are
not limited to, Alu-repeats, Kpn-repeats, di-nucleotide repeats,
tri-nucleotide repeats, tetra-nucleotide repeats, penta-nucleotide
repeats, hexa-nucleotide repeats, all of which are more generally
classified as SINEs or LINEs.
[0013] Detection of specific nucleic acid sequences by in situ
hybridization using non-radioactive labels has been applied for
almost twenty years. As stated above however, the randomly
distributed repeat sequences, such as SINEs and LINEs, are
particularly problematic for the production of specific nucleic
acid probes that are derived from large clones because the probes
will inevitably comprise randomly distributed repeat sequence. The
problem arises because the nucleic acid probes will have the
randomly distributed repeat sequence contained therein, thereby
facilitating hybridization between the randomly distributed repeat
sequences of the probes and natural genomic nucleic acid found
within all chromosomes. Because the detectable probes hybridize
specifically to the target, as well as to repeat sequence that is
randomly found in the genomic nucleic acid, there is a high degree
of background signal that is produced.
[0014] Refinement of non-radioactive detection and visualization
methods resulted in improved detection limits and thereby allowed
the localization of large single-copy sequences (Landegent et al.,
Nature, 317: 175-177 (1985)). In this study it was necessary to
construct a mixture of seven subclones (a total of 22.3 kb derived
from a cosmid DNA clone containing the 3' end of the Tg gene) in
order to eliminate highly repeated sequences present in the
original genomic cosmid DNA. Although this was an improvement, a
more attractive strategy, based on direct use of large genomic
cloned segments in combination with Cot1 DNA, has been described.
The use of Cot1 DNA eliminates background signal, caused by highly
repetitive sequences, by introducing a competitive hybridization
process (Landegent et al., Hum. Genet., 77: 366-370 (1987); U.S.
Pat. No. 5,447,841, issued to Gray et al.; and U.S. Pat. No.
6,203,977 B1 issued to Ward et al.).
[0015] Cot1 DNA is a heterogeneous mixture of genomic nucleic acid
that is prepared by degrading total human DNA and processing the
resulting material to thereby select for genomic nucleic acid
fragments that are enriched in the repeat sequences (Britten et
el., Methods Enzymol 29: 363-418 (1986)). Although the use of Cot1
DNA has been proven to be effective in suppressing undesired
binding of detectable nucleic acid fragments of greater that 100 by
to target genomic nucleic acid, there are several disadvantages to
this method. One such disadvantage pertains to the preparation of
the Cot1 DNA itself. Specifically, because the process relies on
the availability of total human DNA, the starting material is
itself not highly defined and is likely to vary from sample to
sample. Moreover, the processing methods are likely to produce
material that varies from batch to batch; this result being
somewhat dependent upon the variability of the starting material
and somewhat dependent upon the variability of the production
process itself. Additionally, the Cot1 DNA is a heterogeneous
mixture of fragments that is impossible to completely characterize
and define. Hence, the batch to batch variability, as well as the
inability to characterize the Cot1 DNA product, leaves substantial
room for improvement. The present invention addresses these, as
well as other, limitations of the art.
[0016] Despite its name, Peptide Nucleic Acid (PNA) is neither a
peptide, a nucleic acid nor is it an acid. Peptide Nucleic Acid
(PNA) is a non-naturally occurring polyamide (pseudopeptide) that
can hybridize to nucleic acid (DNA and RNA) with sequence
specificity (See: U.S. Pat. No. 5,539,082 and Egholm et al., Nature
365: 566-568 (1993)). Because they hybridize to nucleic acid with
sequence specificity, PNA oligomers have become commonly used in
probe based applications for the analysis of nucleic acids.
[0017] Being a non-naturally occurring molecule, unmodified PNA is
not known to be a substrate for the enzymes that are known to
degrade peptides or nucleic acids. Therefore, PNA should be stable
in biological samples, as well as have a long shelf-life. Unlike
nucleic acid hybridization, which is very dependent on ionic
strength, the hybridization of a PNA with a nucleic acid is fairly
independent of ionic strength and is favored at low ionic strength,
conditions that strongly disfavor the hybridization of nucleic acid
to nucleic acid (Egholm et al., Nature, at p. 567). Because of
their unique properties, it is clear that PNA is not the equivalent
of a nucleic acid in either structure or function.
[0018] Labeled PNA probes have been used for the analysis of rRNA
in ISH and FISH assays (See: WO95/32305 and WO97/18325). Labeled
PNA probes have also been used in the analysis of mRNA (e.g. Kappa
& Lambda Light Chain; Thisted M. et al., Cell Vision 3: 358-363
(1996)) and viral nucleic acid (e.g. EBV; Just T et al., J. Vir.
Methods: 73: 163-174 (1998)). A labeled PNA probe has also been
used to detect human X chromosome specific sequences in a PNA-FISH
format (See: WO97/18325, now U.S. Pat. No. 5,888,733). The analysis
of chromosome aberrations using PNA probes has also been disclosed
(See: WO99/57309). The ISH based analysis of eukaryotic chromosomes
and cells, using polyamide nucleic acids, has also been suggested
(See: U.S. Pat. No. 5,888,734).
[0019] Labeled peptide nucleic acids have been described for the
analysis of both telomere and centromere repeat sequences in
genomic nucleic acid (Lansdorp, P., WO97/14026). Likewise, labeled
peptide nucleic acid oligomers have been used in the analysis of
individual human chromosomes in a multiplex PNA-FISH assay (Taneja
et al., Genes, Chromosomes & Cancer, 30: 57-63 (2001).
Similarly, the analysis of trinucleotide repeats in chromosomal
DNA, using appropriate labeled PNA probes, has also been suggested
(See: WO97/14026). Subsequently, DNA and PNA probes were used to
examine cells for genetic defects associated with the expansion of
trinucleotide repeats that manifest as the disease known as human
myotonic dystrophy (See: Taneja, Biotechniques, 24: 472-476
(1998)). In all cases, labeled PNA probes were used to detect the
specific target nucleic acid repeat sequences.
[0020] PNA oligomers comprising the triplet repeat sequence CAG
have also been used for the selective isolation of
transcriptionally active chromatin restriction fragments (See:
Boffa et al., Proc. Nat'l. Acad. Sci. USA, 92: 1901-1905
(1995)).
[0021] Peptide nucleic acid oligomers have also been used to
suppress the binding of detectable probes to non-target sequences
(See: U.S. Pat. No. 6,110,676). Importantly however, there is no
specific description, suggestion or teaching of using peptide
nucleic acid oligomers to suppress the binding of detectable
nucleic acid probes to undesired randomly distributed repeat
sequences of genomic nucleic acid.
SUMMARY OF THE INVENTION
[0022] Generally, this invention is directed to methods, kits,
non-nucleotide probes as well as other compositions pertaining to
the suppression of binding of detectable nucleic acid probes to
undesired nucleotide sequences of genomic nucleic acid in assays
designed to determine target genomic nucleic acid. In many cases,
the most problematic undesired nucleotide sequences are SINEs and
LINEs (i.e. randomly distributed repeat sequence), which include,
but are not limited to, Alu-repeats, Kpn-repeats, di-nucleotide
repeats, tri-nucleotide repeats, tetra-nucleotide repeats,
penta-nucleotide repeats and hexa-nucleotide repeats.
[0023] In one embodiment, this invention pertains to a
non-nucleotide probe of at least sixteen nucleobase containing
subunits in length having an aggregate nucleobase sequence that is
at least eighty percent homologous to a sixteen nucleotide segment
of randomly distributed repeat sequence of genomic nucleic acid. By
homologous, we mean nucleobase sequence homology. By aggregate
nucleobase sequence we mean the nucleobase sequence comprising the
aggregate of nucleobase containing subunits of the probe even if
separated by one or more linkers. The nucleobase sequence of the
non-nucleotide probe can be substantially or completely homologous
to a fraction, or part, of either: (i) a known unit repeat of
Alu-repeat sequence; or (ii) a consensus sequence of a known unit
repeat of Alu-repeat sequence. The nucleobase sequence of the
non-nucleotide probe can be at least eighty percent homologous to a
sixteen nucleotide segment of the consensus unit repeat of
Alu-repeat selected from the group consisting of: Seq. Id. No. 1
and Seq. Id. No. 2 (See: Table 1). The non-nucleotide probe can be
a peptide nucleic acid.
[0024] In another embodiment, this invention pertains to a
non-nucleotide probe containing an aggregate nucleobase sequence of
at least ten consecutive nucleobases that is at least eighty
percent homologous to the nucleobase sequences selected from the
group consisting of: Seq. Id. No. 3, Seq. Id. No. 4, Seq. Id. No.
5, Seq. Id. No. 6, Seq. Id. No. 7, Seq. Id. No. 8, Seq. Id. No. 9,
Seq. Id. No. 10, Seq. Id. No. 11, Seq. Id. No. 12, Seq. Id. No. 13,
Seq. Id. No. 14, Seq. Id. No. 15, Seq. Id. No. 16, Seq. Id. No. 17,
Seq. Id. No. 18, Seq. Id. No. 19, Seq. Id. No. 20, Seq. Id. No. 21,
Seq. Id. No. 22, Seq. Id. No. 23, Seq. Id. No. 24, Seq. Id. No. 25
and Seq. Id. No. 26 (See: Table 1). These particular sequences, or
their complements, have been determined to be highly effective at
suppressing the binding of a detectable nucleic acid probe to
undesired chromosomes or chromosome regions in an assay for
detecting the ERBB2 (alias HER2) or MLL target nucleic acid
sequence in genomic nucleic acid (See: Examples 4 and 5). The ten
consecutive nucleobases can be either: (i) at least ninety percent
homologous to the identified sequences; or (ii) exactly homologous
to the identified sequences. The probe can be identical in
nucleobase sequence to any one of the identified sequences. The
non-nucleotide probe can be a peptide nucleic acid oligomer.
[0025] In still another embodiment, this invention pertains to a
mixture of two or more non-nucleotide probes wherein each probe
contains an aggregate nucleobase sequence that is at least eighty
percent homologous to a sixteen nucleotide segment of randomly
distributed repeat sequence of genomic nucleic acid. The randomly
distributed repeat sequence can be a SINE or LINE. SINEs and LINEs
can be selected from the group consisting of: Alu-repeats,
Kpn-repeats, di-nucleotide repeats, tri-nucleotide repeats,
tetra-nucleotide repeats, penta-nucleotide repeats and
hexa-nucleotide repeats. The non-nucleotide probes can be peptide
nucleic acid oligomers. The mixture of probes can further comprise
one or more detectable nucleic acid probes.
[0026] In yet another embodiment, this invention pertains to a
composition comprising genomic nucleic acid containing one or more
segments of randomly distributed repeat sequence. The randomly
distributed repeat sequence can be a SINE or LINE. SINEs and LINES
can be selected from the group consisting of: Alu-repeats,
Kpn-repeats, di-nucleotide repeats, tri-nucleotide repeats,
tetra-nucleotide repeats, penta-nucleotide repeats and
hexa-nucleotide repeats. The composition can further comprise two
or more different non-nucleotide probes that sequence specifically
hybridized to at least a fraction, or part, of the one or more
segments of randomly distributed repeat sequence of the genomic
nucleic acid. Hence, the composition can be the hybrid of the
segment of randomly distributed repeat sequence and the two or more
non-nucleotide probes. The non-nucleotide probes can be peptide
nucleic acid oligomers.
[0027] Because it may be desirable to provide the mixture of
non-nucleotide probes in the same container as the detectable
nucleic acid probes, and because the detectable nucleic acid probes
can possess segments of nucleobase sequence that are derived from
the randomly distributed repeat sequences, this invention is
further directed to a composition comprising a detectable nucleic
acid probe of at least 100 by that has been derived from genomic
nucleic acid and that contains one or more segments of randomly
distributed repeat sequence. The randomly distributed repeat
sequences can be SINEs or LINEs. The SINEs or LINEs can be selected
from the group consisting of: Alu-repeats, Kpn-repeats,
di-nucleotide repeats, tri-nucleotide repeats, tetra-nucleotide
repeats, penta-nucleotide repeats and hexa-nucleotide repeats. The
composition can further comprise two or more different
non-nucleotide probes sequence specifically hybridized to at least
a fraction of the one or more segments of randomly distributed
repeat sequence of the detectable nucleic acid probe. The
non-nucleotide probes can be peptide nucleic acid oligomers.
[0028] In still another embodiment, this invention is directed to a
method for suppressing the binding of one or more detectable
nucleic acid probes, that are greater than 100 by and that have
been derived from genomic nucleic acid, to one or more undesired
sequences in an assay for determining target genomic nucleic acid
of a sample. The method comprises contacting the sample with a
mixture of two or more non-nucleotide probes wherein each probe
contains an aggregate nucleobase sequence that is at least eighty
percent homologous to a segment of randomly distributed repeat
sequence of genomic nucleic acid. According to the method, the
sample is also contacted with the one or more detectable nucleic
acid probes. The target genomic nucleic acid of the sample can then
determined by determining the hybridization of the one or more
detectable nucleic acid probes to the target genomic nucleic acid
of the sample wherein the presence, absence or amount of
hybridization of the detectable nucleic acid probe to the target
genomic nucleic acid is representative of the presence, absence or
amount of target genomic nucleic acid in the sample. The
non-nucleotide probes can be peptide nucleic acid oligomers.
[0029] Thus, in yet another embodiment, this invention pertains to
comparing a sample of genomic nucleic acid with that of a control
sample using a genomic nucleic acid reference array. The method
comprises providing a sample of genomic nucleic acid to be tested,
providing a control of genomic nucleic acid, wherein the control
and the sample are differentially labeled, providing a genomic
nucleic acid reference array, and providing a mixture of two or
more non-nucleotide probes wherein each probe contains an aggregate
nucleobase sequence that is at least eighty percent homologous to a
sixteen nucleotide segment of randomly distributed repeat sequence
of genomic nucleic acid. The method further comprises treating the
sample and control genomic nucleic acid, the array or both the
sample and control genomic nucleic acid and the array with the
mixture of non-nucleotide probes under suitable hybridization
conditions. The array can then be contacted with the treated
mixture of sample and control genomic nucleic acid under suitable
hybridization conditions. The intensities of the signals from the
differential labels on the array, caused by hybridization of the
probes to genomic nucleic acid, can then be compared to thereby
determine one or more variations in copy numbers of sequences in
the sample as compared with the relative copy numbers of
substantially identical sequences in the control.
[0030] In still another embodiment, this invention is directed to a
method for determining non-nucleotide probes that hybridize to
randomly distributed repeat sequences and that are suitable for
suppressing the binding of a detectable nucleic acid probe, that is
greater than 100 by in length and that is derived from genomic
nucleic acid, to one or more undesirable sequences in an assay for
determining target genomic nucleic acid of a sample. The method
comprises designing possible nucleobase sequences of non-nucleotide
probes using sequence alignment of known randomly distributed
repeat sequences and then preparing labeled non-nucleotide probes
having said possible nucleobase sequences. According to the method,
genomic nucleic acid of a sample that contains the target genomic
nucleic acid can be treated with the labeled non-nucleotide probes
under suitable hybridization conditions. The relative signal of the
hybridized labeled probes of the many different possible nucleobase
sequences can then be determined. Based upon the signal intensity
data, the probe or probes that exhibit the strongest signal, as a
result of binding to the genomic nucleic acid, can be selected and
tested to thereby determine whether or not they are suitable for
suppressing the binding of a detectable nucleic acid probe of
greater than 100 by in length that is derived from genomic nucleic
acid to one or more non-target sequences in an assay for
determining target genomic nucleic acid of a sample. In order to
test the probe or probes, they can be re-synthesized in unlabeled
form and then tested using the method for suppressing the binding
of detectable probes to undesired sequences as described above. The
non-nucleotide probes can be peptide nucleic acid oligomers.
[0031] In still another embodiment, this invention is directed to a
reagent kit comprising a mixture of two or more non-nucleotide
probes containing at least sixteen consecutive nucleobases that are
at least eighty percent homologous to a fraction of the unit repeat
Alu-repeat consensus sequence selected from the group consisting
of: Seq. Id. No. 1 or Seq. Id. No. 2. The kit further comprises
other reagents, compositions and/or instructions suitable for
performing an assay to thereby determine genomic nucleic acid of a
sample. The reagent kit can further comprise one or more detectable
nucleic acid probes of greater than 100 by in length and that are
derived from genomic nucleic acid. The one or more detectable
nucleic acid probes can be provided in the container that contains
the mixture of two or more non-nucleotide probes.
[0032] In yet still another embodiment, this invention is directed
to a kit comprising a mixture of two or more non-nucleotide probes
wherein at least one probe contains a segment of at least ten
consecutive nucleobases that are at least eighty percent homologous
to the Alu-repeat sequences selected from the group consisting of:
Seq. Id. No. 3, Seq. Id. No. 4, Seq. Id. No. 5, Seq. Id. No. 6,
Seq. Id. No. 7, Seq. Id. No. 8, Seq. Id. No. 9, Seq. Id. No. 10,
Seq. Id. No. 11, Seq. Id. No. 12, Seq. No. 13, Seq. Id. No. 14,
Seq. Id. No. 15, Seq. Id. No. 16, Seq. Id. No. 17, Seq. Id. No. 18,
Seq. Id. No. 19, Seq. Id. No. 20, Seq. Id. No. 21, Seq. Id. No. 22,
Seq. Id. No. 23, Seq. Id. No. 24, Seq. Id. No. 25 and Seq. Id. No.
26 (See: Table 1). The kit further comprises other reagents,
compositions and/or instructions for performing a assay to thereby
determine genomic nucleic acid of a sample. The kit can further
comprise one or more detectable nucleic acid probes of greater than
100 by in length and that are derived from genomic nucleic acid. In
a most preferred embodiment, one or more detectable nucleic acid
probes can be provided in the container that contains the mixture
of two or more non-nucleotide probes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 is an illustration of the positioning of probes on
the individual strands within complementary strands of a unit
repeat of an Alu-repeat consensus sequence.
[0034] FIGS. 2-1, 2-2 and 2-3 are together output from sequence
alignment software.
[0035] FIGS. 3A-1, 3A-2, 3B-1 and 3B-2 are microscope generated
images of interphase nuclei and metaphase spread of human
chromosomes treated with labeled PNA oligomers that either obtained
a high R-banding score (3A-1 & 3A-2) or that just passed the
defined lower limit for a suitable R-banding pattern score (3B-1
& 3B-2).
[0036] FIG. 4A, 4B, 4C and 4D are microscope generated images of a
metaphase spread of human chromosomes treated with labeled PNA
oligomers designed from the J and S family consensus (4A & 4B)
or the Y family consensus (4C & 4D).
[0037] FIGS. 5A, 5B and 5C are microscope generated images of
interphase nuclei and metaphase spread of human chromosomes treated
with detectable HER-2 nucleic acid probe and either: 1) a blocking
mixture of unlabeled PNA oligomers (5A); 2) Cot1 DNA blocker (5B)
or is otherwise not treated with a blocking reagent (5C).
[0038] FIGS. 6A, 6B and 6C are microscope generated images of
interphase nuclei and metaphase spread of human chromosomes treated
with detectable MLL nucleic acid probe and either: 1) a blocking
mixture of unlabeled PNA oligomers (6A); 2) Cot1 DNA blocker (6B)
or is otherwise not treated with a blocking reagent (6C).
[0039] FIGS. 7A, 7B and 7C are microscope generated images of
paraffin embedded tissue sections of a breast carcinoma treated
with detectable HER2 nucleic acid probe and either: 1) a blocking
mixture of unlabeled PNA oligomers (7A); 2) Cot-1 DNA blocker (7B)
or is otherwise not treated with a blocking reagent (7C).
[0040] FIGS. 8A, 8B, 8C and 8D are microscope generated images of
interphase nuclei and metaphase spreads treated with detectable
HER2 nucleic acid probe and either: 1) no other probes (8A); 2) a
mixture of Alu PNA Blocking Probes (8B); Chromosome 17 PNA Probes
8C); or Mu PNA Blocking Probes and Chromosome 17 PNA Probes
(8D).
[0041] FIGS. 9A, 9B, 9C, 9D, 9E and 9F are microscope generated
images of interphase nuclei and metaphase spreads treated to
determine the CyclinD1 gene.
[0042] FIGS. 10A, 10B, 10C, 10D, 10E and 10F are microscope
generated images of interphase nuclei and metaphase spreads treated
to determine the c-MYC gene.
[0043] FIGS. 11A, 11B, 11C, 11D, 11E and 11F are microscope
generated images of interphase nuclei and metaphase spreads treated
to determine the EGFR gene.
[0044] FIGS. 12A, 12B, 12C, 12D, 12E, 12F, 12G, 12H and 12I are
microscope generated images of interphase nuclei and metaphase
spreads treated to determine the TOP2A deletion.
[0045] FIGS. 13A, 13B, 13C, 13D, 13E and 13F are microscope
generated images of interphase nuclei and metaphase spreads treated
to determine the TEL gene.
[0046] FIGS. 14A, 14B, 14C, 14D, 14E and 14F are microscope
generated images of interphase nuclei and metaphase spreads treated
to determine the E2A gene.
[0047] FIGS. 15A, 15B, 15C, 15D, 15E and 15F are microscope
generated images of interphase nuclei and metaphase spreads treated
to determine the BCR gene.
[0048] FIGS. 16A, 16B, 16C, 16D, 16E and 16F are microscope
generated images of interphase nuclei and metaphase spreads treated
to determine the IGH gene.
[0049] FIGS. 17A, 17B and 17C are microscope generated images of
interphase nuclei and metaphase spreads treated to determine the
IGL gene.
[0050] FIGS. 18A, 18B and 18C are microscope generated images of
interphase nuclei and metaphase spreads treated to determine the
IGK gene.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions:
[0051] For the purposes of interpreting this specification the
following definitions shall apply and whenever appropriate, terms
used in the singular shall also include the plural and vice versa.
[0052] a. As used herein, "nucleobase" means those naturally
occurring and those non-naturally occurring heterocyclic moieties
commonly known to those who utilize nucleic acid technology or
utilize peptide nucleic acid technology to thereby generate
polymers that can sequence specifically bind to nucleic acids.
Non-limiting examples of suitable nucleobases include: adenine,
cytosine, guanine, thymine, uracil, 5-propynyl-uracil,
2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,
2-thiouracil and 2-thiothymine, 2-aminopurine,
N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,
N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and
N8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitable
nucleobases include those nucleobases illustrated in FIGS. 2(A) and
2(B) of Buchardt et al. (U.S. Pat. No. 6,357,163). [0053] b. As
used herein, "sequence specifically" means hybridization by base
pairing through hydrogen bonding. Non-limiting examples of standard
base pairing includes adenine base pairing with thymine or uracil
and guanine base pairing with cytosine. Other non-limiting examples
of base-pairing motifs include, but are not limited to: adenine
base pairing with any of:
[0054] 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 2-thiouracil or
2-thiothymine; guanine base pairing with any of: 5-methylcytosine
or pseudoisocytosine; cytosine base pairing with any of:
hypoxanthine, N9-(7-deaza-guanine) or N9-(7-deaza-8-aza-guanine);
thymine or uracil base pairing with any of: 2-aminopurine,
N9-(2-amino-6-chloropurine) or N9-(2,6-diaminopurine); and
N8-(7-deaza-8-aza-adenine), being a universal base, base pairing
with any other nucleobase, such as for example any of: adenine,
cytosine, guanine, thymine, uracil, 5-propynyl-uracil,
2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,
2-thiouracil and 2-thiothymine, 2-aminopurine,
N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,
N9-(7-deaza-guanine) or N9-(7-deaza-8-aza-guanine) (See: Seela et
al., Nucl. Acids, Res.: 28(17): 3224-3232 (2000)). [0055] c. As
used herein, "nucleobase sequence" means all or a segment of
nucleobase-containing subunits in an oligomer or polymer.
Non-limiting examples of suitable polymers or polymers segments
that comprise "nucleobase sequence" or "nucleobase sequences"
include oligodeoxynucleotides (e.g. DNA), oligoribonucleotides
(e.g. RNA), peptide nucleic acids (PNA), nucleic acid analogs
and/or nucleic acid mimics. [0056] d. As used herein, "target
sequence" or "target genomic nucleic acid" is a nucleobase sequence
sought to be determined. The nucleobase sequence can be a
subsequence of a nucleic acid molecule of interest (e.g. a
chromosome). [0057] e. As used herein, "nucleic acid" is a
nucleobase sequence-containing oligomer, polymer, or polymer
segment, having a backbone formed solely from nucleotides, or
analogs thereof. Preferred nucleic acids are DNA and RNA. For the
avoidance of doubt, a peptide nucleic acid (PNA) oligomer is not a
nucleic acid since it is not formed from nucleotides or analogs
thereof. [0058] f. As used herein, "nucleotide" means any of
several compounds that consist of a ribose or deoxyribose sugar
joined to a purine or pyrimidine base and to a phosphate group.
Nucleotides are the basic structural subunits of nucleic acids
(e.g. RNA and DNA). [0059] g. As used herein, "analog" or "nucleic
acid analog" means an oligomer, polymer, or polymer segment
composed of at least one modified nucleotide, or subunits derived
directly a modification of nucleotides. [0060] h. As used herein,
"mimic" of "nucleic acid mimic" means a non-nucleotide polymer.
[0061] i. As used herein, a "non-nucleotide polymer" or
"non-nucleotide probe" is a nucleobase sequence-containing
oligomer, polymer, or polymer segment that does not comprise
nucleotides. A most preferred non-nucleotide polymer is a peptide
nucleic acid (PNA) oligomer. [0062] j. As used herein, "peptide
nucleic acid" or "PNA" means any oligomer or polymer comprising two
or more PNA subunits (residues), including, but not limited to, any
of the oligomer or polymer segments referred to or claimed as
peptide nucleic acids in U.S. Pat. Nos. 5,539,082, 5,527,675,
5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855,
5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,470
6,201,103, 6,228,982 and 6,357,163; all of which are herein
incorporated by reference. The term "peptide nucleic acid" or "PNA"
shall also apply to any oligomer or polymer segment comprising two
or more subunits of those nucleic acid mimics described in the
following publications: Lagriffoul et al., Bioorganic &
Medicinal Chemistry Letters, 4: 1081-1082 (1994); Petersen et al.,
Bioorganic & Medicinal Chemistry Letters, 6: 793-796 (1996);
Diderichsen et al., Tett. Lett. 37: 475-478 (1996); Fujii et al.,
Bioorg. Med. Chem. Lett. 7: 637-627 (1997); Jordan et al., Bioorg.
Med. Chem. Lett. 7: 687-690 (1997); Krotz et al., Tett. Lett. 36:
6941-6944 (1995); Lagriffoul et al., Bioorg. Med. Chem. Lett. 4:
1081-1082 (1994); Diederichsen, U., Bioorganic & Medicinal
Chemistry Letters, 7: 1743-1746 (1997); Lowe et al., J. Chem. Soc.
Perkin Trans. 1, (1997) 1: 539-546; Lowe et al., J. Chem. Soc.
Perkin Trans. 11: 547-554 (1997); Lowe et al., J. Chem. Soc. Perkin
Trans. 1 1:5 55-560 (1997); Howarth et al., J. Org. Chem. 62:
5441-5450 (1997); Altmann, K-H et al., Bioorganic & Medicinal
Chemistry Letters, 7: 1119-1122 (1997); Diederichsen, U.,
Bioorganic & Med. Chem. Lett., 8: 165-168 (1998); Diederichsen
et al., Angew. Chem. Int. Ed., 37: 302-305 (1998); Cantin et al.,
Tett. Lett., 38: 4211-4214 (1997); Ciapetti et al., Tetrahedron,
53: 1167-1176 (1997); Lagriffoule et al., Chem. Eur. J., 3: 912-919
(1997); Kumar et al., Organic Letters 3(9): 1269-1272 (2001); and
the Peptide-Based Nucleic Acid Mimics (PENAMs) of Shah et al. as
disclosed in WO96/04000.
[0063] In certain embodiments, a "peptide nucleic acid" or "PNA" is
an oligomer or polymer segment comprising two or more covalently
linked subunits of the formula:
##STR00001##
wherein, each J is the same or different and is selected from the
group consisting of H, R.sup.1, OR.sup.1, SR.sup.1, NHR.sup.1,
NR.sup.1.sub.2, F, Cl, Br and I. Each K is the same or different
and is selected from the group consisting of O, S, NH and NR.sup.1.
Each R.sup.1 is the same or different and is an alkyl group having
one to five carbon atoms that may optionally contain a heteroatom
or a substituted or unsubstituted aryl group. Each A is selected
from the group consisting of a single bond, a group of the formula;
--(CJ.sub.2).sub.s-- and a group of the formula;
--(CJ.sub.2).sub.sC(O)--, wherein, J is defined above and each s is
a whole number from one to five. Each t is 1 or 2 and each u is 1
or 2. Each L is the same or different and is independently selected
from: adenine, cytosine, guanine, thymine, uracil,
5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine,
pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine,
N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,
N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and
N8-(7-deaza-8-aza-adenine), other naturally occurring nucleobase
analogs or other non-naturally occurring nucleobases.
[0064] In certain other embodiments, a PNA subunit consists of a
naturally occurring or non-naturally occurring nucleobase attached
to the N-.alpha.-glycine nitrogen of the N-[2-(aminoethyl)]glycine
backbone through a methylene carbonyl linkage; this currently being
the most commonly used form of a peptide nucleic acid subunit.
[0065] k. As used herein, the terms "label", "reporter moiety" or
"detectable moiety" are interchangeable and refer to moieties that
can be attached to an oligomer or polymer to thereby render the
oligomer or polymer detectable by an instrument or method. [0066]
l. As used herein, "stained" means that individual organisms,
chromosomes or chromosome fragments or segments are directly or
indirectly marked with a detectable moiety as a result of the
sequence specific hybridization thereto of one or more detectable
probes. [0067] m. As used herein, "unit repeat" means the basic
unit of nucleobase sequence that is repeated in a randomly
distributed repeat sequence. [0068] n. As used herein, "block",
"oligomer block" or "block oligomer" are interchangeable and all
mean a PNA oligomer that is designed and available to be ligated to
a second appropriately modified PNA oligomer to thereby prepare an
elongated PNA oligomer. Oligomers or blocks that are
ligated/condensed may be unlabeled, labeled with one or more
reporter moieties and/or comprise one or more protected or
unprotected functional groups. With respect to an elongated
oligomer, "block" can also be used to refer to a part of the
elongated oligomer that originates from an oligomer block used to
form the elongated oligomer. The elongated oligomer also may be
used as a block in a ligation/condensation reaction that further
elongates the PNA oligomer.
2. Description of the Invention
I. General:
Production, Purification & Labeling of Detectable Nucleic Acid
Probes
[0069] To amplify a specific DNA sequence by cloning, the DNA can
be inserted into a vector and both insert and vector can then be
amplified inside appropriate host cells. The amplified DNA can then
extracted. Commonly used vectors include bacterial plasmids,
cosmids, PACs, BACs, and YACs.
[0070] The purified DNA can be labeled with different methods, e.g.
enzymatic or chemical. The most frequently used method is Nick
translation. The Nick translation reaction can employ two enzymes,
Dnase I which produces the "nicks" in the double-stranded DNA and
DNA polymerase, which incorporates labeled nucleotides along both
strands of the DNA duplex. Any labeling method known to those in
the art can be used for labeling the nucleic acid probe as used in
this invention.
PNA Synthesis:
[0071] Methods for the chemical assembly of PNAs are well known
(See: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331,
5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459,
5,891,625, 5,972,610, 5,986,053, 6,107,470, 6,201,103, 6,228,982
and 6,357,163; all of which are herein incorporated by reference
(Also see: PerSeptive Biosystems Product Literature)). As a general
reference for PNA synthesis methodology also please see: Nielsen et
al., Peptide Nucleic Acids; Protocols and Applications, Horizon
Scientific Press, Norfolk England (1999).
[0072] Chemicals and instrumentation for the support bound
automated chemical assembly of peptide nucleic acids are now
commercially available. Both labeled and unlabeled PNA oligomers
are likewise available from commercial vendors of custom PNA
oligomers. Chemical assembly of a PNA is analogous to solid phase
peptide synthesis, wherein at each cycle of assembly the oligomer
possesses a reactive alkyl amino terminus that is condensed with
the next synthon to be added to the growing polymer.
[0073] PNA may be synthesized at any scale, from submicromole to
millimole, or more. PNA can be conveniently synthesized at the 2
.mu.mole scale, using Fmoc(Bhoc), tBoc/Z, or MmT protecting group
monomers on an Expedite Synthesizer (Applied Biosystems) using a
XAL or PAL support. Alternatively the Model 433A Synthesizer
(Applied Biosystems) with MBHA support can be used. Moreover, many
other automated synthesizers and synthesis supports can be
utilized. Because standard peptide chemistry is utilized, natural
and non-natural amino acids can be routinely incorporated into a
PNA oligomer. Because a PNA is a polyamide, it has a C-terminus
(carboxyl terminus) and an N-terminus (amino terminus). For the
purposes of the design of a hybridization probe suitable for
antiparallel binding to the target sequence (the preferred
orientation), the N-terminus of the probing nucleobase sequence of
the PNA probe is the equivalent of the 5'-hydroxyl terminus of an
equivalent DNA or RNA oligonucleotide.
[0074] PNA oligomers can also be prepared by the ligation of
shorter oligomers, with (See: WO02/072865) or without (See: U.S.
Ser. No. 60/409/220) the introduction of a linker contained
therebetween the oligomer blocks.
PNA Labeling/Modification:
[0075] Non-limiting methods for labeling PNAs are described in U.S.
Pat. Nos. 6,110,676, 6,280,964, 6,355,421, WO99/21881, U.S. Pat.
No. 6,361,942, WO99/49293 and U.S. Pat. No. 6,441,152 (all of which
are herein incorporated by reference), the examples section of this
specification or are otherwise well known in the art of PNA
synthesis and peptide synthesis. Methods for labeling PNA are also
discussed in Nielsen et al., Peptide Nucleic Acids; Protocols and
Applications, Horizon Scientific Press, Norfolk, England (1999).
Non-limiting methods for labeling PNA oligomers are discussed
below.
[0076] Because the synthetic chemistry of assembly is essentially
the same, any method commonly used to label a peptide can often be
adapted to effect the labeling a PNA oligomer. Generally, the
N-terminus of the oligomer or polymer can be labeled by reaction
with a moiety having a carboxylic acid group or activated
carboxylic acid group. One or more spacer moieties can optionally
be introduced between the labeling moiety and the nucleobase
containing subunits of the oligomer. Generally, the spacer moiety
can be incorporated prior to performing the labeling reaction. If
desired, the spacer may be embedded within the label and thereby be
incorporated during the labeling reaction.
[0077] Typically the C-terminal end of the polymer can be labeled
by first condensing a labeled moiety or functional group moiety
with the support upon which the PNA oligomer is to be assembled.
Next, the first nucleobase containing synthon of the PNA oligomer
can be condensed with the labeled moiety or functional group
moiety. Alternatively, one or more spacer moieties (e.g.
8-amino-3,6-dioxaoctanoic acid; the "O-linker") can be introduced
between the label moiety or functional group moiety and the first
nucleobase subunit of the oligomer. Once the molecule to be
prepared is completely assembled, labeled and/or modified, it can
be cleaved from the support deprotected and purified using standard
methodologies.
[0078] For example, the labeled moiety or functional group moiety
can be a lysine derivative wherein the .epsilon.-amino group is a
protected or unprotected functional group or is otherwise modified
with a reporter moiety. The reporter moiety could be a fluorophore
such as 5(6)-carboxyfluorescein, Dye1, Dye2 or a quencher moiety
such as 4-((4-(dimethylamino)phenyl)azo)benzoic acid(dabcyl).
Condensation of the lysine derivative with the solid support can be
accomplished using standard condensation (peptide) chemistry. The
.alpha.-amino group of the lysine derivative can then be
deprotected and the nucleobase sequence assembly initiated by
condensation of the first PNA synthon with the .alpha.-amino group
of the lysine amino acid. As discussed above, a spacer moiety may
optionally be inserted between the lysine amino acid and the first
PNA synthon by condensing a suitable spacer (e.g.
Fmoc-8-amino-3,6-dioxaoctanoic acid) with the lysine amino acid
prior to condensation of the first PNA synthon.
[0079] Alternatively, a functional group on the assembled, or
partially assembled, polymer can be introduced while the oligomer
is still support bound. The functional group will then be available
for any purpose, including being used to either attached the
oligomer to a support or otherwise be reacted with a reporter
moiety, including being reacted post-ligation (by post-ligation we
mean at a point after the oligomer has been fully formed by the
performing of one or more condensation/ligation reactions). This
method, however, requires that an appropriately protected
functional group be incorporated into the oligomer during assembly
so that after assembly is completed, a reactive functional can be
generated. Accordingly, the protected functional group can be
attached to any position within the oligomer or block, including,
at the oligomer termini, at a position internal to the
oligomer.
[0080] For example, the .epsilon.-amino group of a lysine could be
protected with a 4-methyl-triphenylmethyl (Mtt), a
4-methoxy-triphenylmethyl (MMT) or a 4,4'-dimethoxytriphenylmethyl
(DMT) protecting group. The Mtt, MMT or DMT groups can be removed
from the oligomer (assembled using commercially available Fmoc PNA
monomers and polystyrene support having a PAL linker; PerSeptive
Biosystems, Inc., Framingham, Mass.) by treatment of the synthesis
resin under mildly acidic conditions. Consequently, a donor moiety,
acceptor moiety or other reporter moiety, for example, can then be
condensed with the .epsilon.-amino group of the lysine amino acid
while the polymer is still support bound. After complete assembly
and labeling, the polymer can then cleaved from the support,
deprotected and purified using well-known methodologies.
[0081] By still another method, the reporter moiety can be attached
to the oligomer or oligomer block after it is fully assembled and
cleaved from the support. This method is preferable where the label
is incompatible with the cleavage, deprotection or purification
regimes commonly used to manufacture the oligomer. By this method,
the PNA oligomer can be labeled in solution by the reaction of a
functional group on the polymer and a functional group on the
label. Those of ordinary skill in the art will recognize that the
composition of the coupling solution will depend on the nature of
oligomer and label, such as for example a donor or acceptor moiety.
The solution may comprise organic solvent, water or any combination
thereof. Generally, the organic solvent will be a polar
non-nucleophilic solvent. Non limiting examples of suitable organic
solvents include acetonitrile (ACN), tetrahydrofuran, dioxane,
methyl sulfoxide, N,N'-dimethylformamide (DMF) and 1-methyl
pyrrolidone (NMP).
[0082] The functional group on the polymer to be labeled can be a
nucleophile (e.g. an amino group) and the functional group on the
label can be an electrophile (e.g. a carboxylic acid or activated
carboxylic acid). It is however contemplated that this can be
inverted such that the functional group on the polymer can be an
electrophile (e.g. a carboxylic acid or activated carboxylic acid)
and the functional group on the label can be a nucleophile (e.g. an
amino acid group). Non-limiting examples of activated carboxylic
acid functional groups include N-hydroxysuccinimidyl esters. In
aqueous solutions, the carboxylic acid group of either of the PNA
or label (depending on the nature of the components chosen) can be
activated with a water soluble carbodiimide. The reagent,
1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC),
is a commercially available reagent sold specifically for aqueous
amide forming condensation reactions. Such condensation reactions
can also be improved when 1-Hydroxy-7-azabenzotriazole (HOAt) or
1-hydrozybenzotriazole (HOBt) is mixed with the EDC.
[0083] The pH of aqueous solutions can be modulated with a buffer
during the condensation reaction. For example, the pH during the
condensation can be in the range of 4-10. Generally, the basicity
of non-aqueous reactions will be modulated by the addition of
non-nucleophilic organic bases. Non-limiting examples of suitable
bases include N-methylmorpholine, triethylamine and
N,N-diisopropylethylamine. Alternatively, the pH can be modulated
using biological buffers such as
(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid) (HEPES) or
4-morpholineethane-sulfonic acid (MES) or inorganic buffers such as
sodium bicarbonate.
Labeled Oligomers & Oligomer Blocks:
[0084] As discussed above, PNA oligomers can be labeled with
reporter moieties. Non-limiting examples of reporter moieties
(labels) suitable for directly labeling oligomers or oligomer
blocks include: a quantum dot, a minor groove binder, a dextran
conjugate, a branched nucleic acid detection system, a chromophore,
a fluorophore, a quencher, a spin label, a radioisotope, an enzyme,
a hapten, an acridinium ester and a chemiluminescent compound.
Quenching moieties are also considered labels. Other suitable
labeling reagents and preferred methods of attachment would be
recognized by those of ordinary skill in the art of PNA, peptide or
nucleic acid synthesis. Non-limiting examples are described or
referred to above.
[0085] Non-limiting examples of haptens include
5(6)-carboxyfluorescein, 2,4-dinitrophenyl, digoxigenin, and
biotin.
[0086] Non-limiting examples of fluorochromes (fluorophores)
include 5(6)-carboxyfluorescein (Flu),
2',4',1,4,-tetrachlorofluorescein; and
2',4',5',7',1,4-hexachlorofluorescein, other fluorescein dyes (See:
U.S. Pat. Nos. 5,188,934; 6,008,379; 6,020,481, incorporated herein
by reference), 6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic
acid (Cou), 5(and 6)-carboxy-X-rhodamine (Rox), other rhodamine
dyes (See: U.S. Pat. Nos. 5,366,860; 5,847,162; 5,936,087;
6,051,719; 6,191,278; 6,248,884, incorporated herein by reference),
benzophenoxazines (See: U.S. Pat. No. 6,140,500, incorporated
herein by reference)Cyanine 2 (Cy2) Dye, Cyanine 3 (Cy3) Dye,
Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5) Dye, Cyanine 5.5 (Cy5.5)
Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye (Cyanine dyes 2, 3,
3.5, 5 and 5.5 are available as NHS esters from Amersham, Arlington
Heights, Ill.), other cyanine dyes (Kubista, WO 97/45539),
6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE),
5(6)-carboxy-tetramethyl rhodamine (Tamara), Dye 1 (FIG. 7), Dye2
(FIG. 7) or the Alexa dye series (Molecular Probes, Eugene,
Oreg.).
[0087] Non-limiting examples of enzymes include polymerases (e.g.
Taq polymerase, Klenow PNA polymerase, T7 DNA polymerase,
Sequenase, DNA polymerase 1 and phi29 polymerase), alkaline
phosphatase (AP), horseradish peroxidase (HRP), soy bean peroxidase
(SBP)), ribonuclease and protease.
[0088] Non-limiting examples of quenching moieties include
diazo-containing moieties such as aryldiazo compounds, e.g. dabcyl
and dabsyl, homologs containing one more additional diazo and/or
aryl groups; e.g. Fast Black, (Nardone, U.S. Pat. No. 6,117,986),
and substituted compounds where Z is a substituent such Cl, F, Br,
C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.14 aryl, nitro, cyano,
sulfonate, NR.sub.2, --OR, and CO.sub.2H, where each R is
independently H, C.sub.1-C.sub.6 alkyl or C.sub.5-C.sub.14 aryl
according to the structures:
##STR00002##
cyanine dyes (Lee, U.S. Pat. No. 6,080,868), including the
exemplary structure:
##STR00003##
[0089] and other chromophores such as anthraquinone, malachite
green, nitrothiazole, and nitroimidazole compounds and the like
wherein the group X is the covalent attachment site of a bond or
linker to the oligomers of the invention.
[0090] A non-limiting example of a minor groove binder is
CDPI.sub.3, represented by the structure:
##STR00004##
where X are exemplary attachment sites to a oligomer (Dempcy, WO
01/31063).
[0091] Non-radioactive labeling methods, techniques, and reagents
are reviewed in: Non-Radioactive Labeling, A Practical
Introduction, Garman, A. J. Academic Press, San Diego, Calif.
(1997).
Detectable and Independently Detectable Moieties/Multiplex
Analysis:
[0092] In preferred embodiments of this invention, a multiplex
hybridization assay is performed. In a multiplex assay, numerous
conditions of interest are simultaneously or sequentially examined.
Multiplex analysis relies on the ability to sort sample components
or the data associated therewith, during or after the assay is
completed. In one embodiments, one or more distinct independently
detectable moieties can be used to label two or more different
detectable nucleic acid probes used in an assay. The ability to
differentiate between and/or quantitate each of the independently
detectable moieties provides the means to multiplex a hybridization
assay because the data correlates with the hybridization of each of
the distinct, independently labeled oligomer to a particular target
sequence sought to be detected in the sample. Consequently, the
multiplex assays of this invention may be used to simultaneously or
sequentially detect the presence, absence, number, position and/or
identity of two or more target sequences in the same sample and/or
in the same assay.
Spacer/Linker Moieties:
[0093] Generally, spacers are used to minimize the adverse effects
that bulky labeling reagents might have on the hybridization
properties of probes or primers. A linker is used to link two or
more segments of an oligomer or polymer. Non-limiting examples of
spacer/linker moieties used in this invention consist of:, one or
more aminoalkyl carboxylic acids (e.g. aminocaproic acid) the side
chain of an amino acid (e.g. the side chain of lysine or ornithine)
one or more natural amino acids (e.g. glycine), aminooxyalkylacids
(e.g. 8-amino-3,6-dioxaoctanoic acid), alkyl diacids (e.g. succinic
acid), alkyloxy diacids (e.g. diglycolic acid) or alkyldiamines
(e.g. 1,8-diamino-3,6-dioxaoctane). Spacer/linker moieties may also
incidentally or intentionally be constructed to improve the water
solubility of the oligomer (For example see: Gildea et al., Tett.
Lett. 39: 7255-7258 (1998)).
Hybridization Conditions/Stringency:
[0094] Those of ordinary skill in the art of hybridization will
recognize that factors commonly used to impose or control
stringency of hybridization include formamide concentration (or
other chemical denaturant reagent), salt concentration (i.e., ionic
strength), hybridization temperature, detergent concentration, pH
and the presence or absence of chaotropes. Optimal stringency for a
probe/target sequence combination is often found by the well-known
technique of fixing several of the aforementioned stringency
factors and then determining the effect of varying a single
stringency factor. The same stringency factors can be modulated to
thereby control the stringency of hybridization of a PNA to a
nucleic acid, except that the hybridization of a PNA is fairly
independent of ionic strength. Optimal stringency for an assay may
be experimentally determined by examination of each stringency
factor until the desired degree of discrimination is achieved.
Suitable Hybridization Conditions:
[0095] Generally, the more closely related the background causing
nucleic acid contaminates are to the target sequence, the more
carefully stringency must be controlled. Suitable hybridization
conditions will thus comprise conditions under which the desired
degree of discrimination is achieved such that an assay generates
an accurate (within the tolerance desired for the assay) and
reproducible result. Nevertheless, aided by no more than routine
experimentation and the disclosure provided herein, those of skill
in the art will easily be able to determine suitable hybridization
conditions for performing assays utilizing the methods and
compositions described herein.
Probing Nucleobase Sequence:
[0096] The probing nucleobase sequence of probe is the specific
sequence recognition portion of the construct. Therefore, the
probing nucleobase sequence is an aggregate nucleobase sequence of
the probe that is designed to hybridize to a specific target
sequence of interest in a sample. By aggregate nucleobase sequence,
we refer to the totality of the nucleobase subunits that bind to
the target sequence without regard to whether or not they comprise
one or more linkages atypical to the backbone of the polymer (e.g.
two segments of continuous nucleobase sequence containing subunits
separated by a linker). The target sequence can be a sequence that
identifies a gene such as for example, the CyclinD1 gene, the c-MYC
gene, the EGFR gene, the TEL gene, the E2A gene, the BCR gene, the
IGH gene, the IGL gene or the IGK gene.
Advantages of the Present Invention:
[0097] The non-nucleotide probes of this invention can be
chemically synthesized and purified in a manner that provides for
low cost and proper characterization. Consequently, the unlabeled
non-nucleotide probes (e.g. peptide nucleic acid oligomers) can be
individually prepared, characterized and quantitated before
preparing a mixture of probes for a blocking application. Hence,
the mixture of probes itself can therefore be more carefully
controlled, characterized and reproduced than are the Cot1 DNA
probes of the prior art. Since one possible application for such a
the mixture of probes is their possible use in a diagnostic assay,
the ability to more easily characterize and reproduce, in a cost
effective manner, the exact composition of the mixture from one
batch to the next is potentially advantageous.
Applications for the Present Invention:
[0098] The suppression methods described herein can be useful in
analyzing cells for the occurrence of chromosomes, chromosome
fragments, genes, or chromosome aberrations (e.g. translocations,
deletions, amplifications) associated with a condition or disease.
Any method that can detect, identify and/or quantify selected
target genomic nucleic acid in metaphase spreads, interphase
nuclei, tissue sections, and extracted DNA from these cells can
potentially take advantage of the present method as a substitute
for the conventional Cot-1 DNA blocking. These methods include, but
are not limited to, CISH (chromogen in situ hybridization), FISH,
multi-color FISH, Fiber-FISH, CGH, chromosome paints and the
analysis of BAC clones and arrays.
TABLE-US-00001 TABLE 1 Seq. Id. Nucleobase Sequence No.
GGCGGGCGGAGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCA 1
GCACTTTGGGAGGCCGAGGCGGGCGGATCACCTGAGGTCAGGA
GTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTAC
TAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCGCGCCTGTA
RTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAAC
CCGGGAGGCGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCA
CTCCAGCCTGGGCRACAAGAGCGARACTCCGTCTCAAAAAAAA
TTTTTTTTGAGACGGAGTYTCGCTCTTGTYGCCCAGGCTGGAG 2
TGCAGTGGCGCGATCTCGGCTCACTGCAACCTCCGCCTCCCGG
GTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGAY
TACAGGCGCGCGCCACCACGCCCGGCTAATTTTTGTATTTTTA
GTAGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAAC
TCCTGACCTCAGGTGATCCGCCCGCCTCGGCCTCCCAAAGTGC
TGGGATTACAGGCGTGAGCCACCGCGCCCGGCCTCCGCCCGCC GGCCGGGCGCGGTGGCT 3
GCTGGGATTACAGGCGTG 4 GGGAGGCCGAGGCGGG 5 GCCAGGCTGGTCTCGAACTCC 6
GAAACCCCGTCTCTACTAAAA 7 GCCGGGCGTGGTGGCG 8 TAGCTGGGATTACAGGCG 9
GGGAGGCTGAGGCAGGA 10 CCTCCCGGGTTCAAGCGATTC 11 TTGCAGTGAGCCGAGAT 12
TGCACTCCAGCCTGGGCGACA 13 **TT(k)TTTTT(k)TTTLysOLysOTTT(k)TTTTT(k)TT
14 AGCCACCGCGCCCGGCC 15 CACGCCTGTAATCCCAGC 16 CCCGCCTCGGCCTCCC 17
GGAGTTCGAGACCAGCCTGGC 18 TTTTAGTAGAGACGGGGTTTC 19 CGCCACCACGCCCGGC
20 CGCCTGTAATCCCAGCTA 21 TCCTGCCTCAGCCTCCC 22 GAATCGCTTGAACCCGGGAGG
23 ATCTCGGCTCACTGCAA 24 TGTCGCCCAGGCTGGAGTGCA 25
AA(k)AAAAA(k)AAA-Lys-O-Lys-O-AAA(k)AAAAA(k)AA 26 Note: k =
D-lysine; Lys = L-lysine; O = 8-amino-3,6-dioxaoctanoic acid;
II. Embodiments of the Invention:
[0099] Generally, this invention pertains to methods, kits,
non-nucleotide probes as well as other compositions for the
suppression of binding of detectable nucleic acid probes to
undesired nucleotide sequences of genomic nucleic acid in assays
designed to determine target genomic nucleic acid. In many cases,
the most problematic undesired nucleotide sequences are those known
in the art as randomly distributed repeat sequences, which include,
but are not limited to, Alu-repeats, Kpn-repeats, di-nucleotide
repeats, tri-nucleotide repeats, tetra-nucleotide repeats,
penta-nucleotide repeats, hexa-nucleotide repeats, SINEs and LINEs.
These are referred to as randomly distributed repeat sequences
since they are not prevalent in any particular section of the
genetic material, such as in a centromere or telomere region, but
rather are randomly distributed within all of the chromosomes of an
organism.
Non-Nucleotide Probes:
[0100] In one embodiment, this invention pertains to a
non-nucleotide probe of at least sixteen nucleobase containing
subunits in length having an aggregate nucleobase sequence that is
at least eighty percent homologous to a sixteen nucleotide segment
of randomly distributed repeat sequence of genomic nucleic acid.
The segment of randomly distributed repeat sequence can be a SINE
or LINE. SINEs and LINEs can be selected from the group consisting
of: Alu-repeats, Kpn-repeats, di-nucleotide repeats, tri-nucleotide
repeats, tetra-nucleotide repeats, penta-nucleotide repeats and
hexa-nucleotide repeats.
[0101] The nucleobase sequence of the non-nucleotide probe can be
chosen to be substantially, or completely, homologous to a
fraction, or part, of either: (i) a known unit repeat of a
Alu-repeat sequence; or (ii) a consensus sequence of a unit repeat
of a known Alu-repeat sequence. For example, the segment can
contain at least ten consecutive nucleobases that are at least
eighty percent homologous to the unit repeat consensus Alu-repeat
sequences selected from the group consisting of: Seq. Id. No. 1 and
Seq. Id. No. 2 (See Table 1). The ten consecutive nucleobases can
be at least ninety percent homologous to the identified Alu-repeat
consensus sequences or they can be exactly homologous to the
identified Alu-repeat consensus sequences. The non-nucleotide probe
can be from about 16 to about 50 nucleobase containing subunits in
length. The non-nucleotide probe can be a peptide nucleic acid
oligomer.
[0102] In another embodiment, this invention pertains to a
non-nucleotide probe containing an aggregate nucleobase sequence of
at least ten consecutive nucleobases that is at least eighty
percent homologous to the sequences selected from the group
consisting of: Seq. Id. No. 3, Seq. Id. No. 4, Seq. Id. No. 5, Seq.
Id. No. 6, Seq. Id. No. 7, Seq. Id. No. 8, Seq. Id. No. 9, Seq. Id.
No. 10, Seq. Id. No. 11, Seq. Id. No. 12, Seq. Id. No. 13, Seq. Id.
No. 14, Seq. Id. No. 15, Seq. Id. No. 16, Seq. Id. No. 17, Seq. Id.
No. 18, Seq. Id. No. 19, Seq. Id. No. 20, Seq. Id. No. 21, Seq. Id.
No. 22, Seq. Id. No. 23, Seq. Id. No. 24, Seq. Id. No. 25 and Seq.
Id. No. 26. Certain of these particular sequences have been
determined to be highly effective at suppressing the binding of a
detectable nucleic acid probe to undesired chromosomes in an assay
for detecting the HER-2 or MLL target nucleic acid sequences in
genomic nucleic acid (See: Examples 4 and 5). Complementary
sequences to the tested nucleobase sequences are included because
these non-nucleotide probes are directed to genomic nucleic acid
that is typically present in double stranded form. Hence, the
target sequences for these probe sequences, as well as their
complements, can be present in samples containing the complementary
strands of genomic nucleic acid.
[0103] The ten consecutive nucleobases can be either: (i) at least
ninety percent homologous to the identified sequences; or (ii)
exactly homologous to the identified sequences. The probe can be
identical in nucleobase sequence to any one of the identified
sequences. The non-nucleotide probe can be from about 10 to about
50 nucleobase containing subunits in length. The non-nucleotide
probe can be a peptide nucleic acid oligomer.
[0104] The aforementioned non-nucleotide probes will either be
labeled or unlabeled. The exact configuration of the probe will
usually depend upon its intended use. For example, if the probe is
being screened in order to determine whether or not it may be
useful in binding to undesired sequence in genomic nucleic acid, as
described in more detail below, it is likely to be labeled.
Conversely, if the probe is being used in a method to suppress the
binding of detectable nucleic acid probe to undesired sequence in
genomic nucleic acid, as described in more detail below, it is
likely to be unlabeled.
Probe Mixtures:
[0105] In still another embodiment, this invention pertains to a
mixture of two or more non-nucleotide probes wherein each probe
contains an aggregate nucleobase sequence that is at least eighty
percent homologous to a sixteen nucleotide segment of randomly
distributed repeat sequence of genomic nucleic acid. The mixture
can comprise from about 5 to about 50 probes of different
nucleobase sequence. The mixture can comprise from about 10 to
about 25 probes of different nucleobase sequence. The individual
probes of the mixture can comprise from about 10 to about 50
nucleobase containing subunits in length.
[0106] Any of the aforementioned non-nucleotide probes can be
suitable for use in the non-nucleotide probe mixture. The
non-nucleotide probes can be peptide nucleic acid oligomers. The
mixture of probes can further comprise one or more detectable
nucleic acid probes. The mixture of probes can further comprise
genomic nucleic acid of a sample to be tested.
Probes & Probe Mixtures:
[0107] The non-nucleotide probes or mixture of non-nucleotide
probes of this invention can be manufacture and purified using
conventional methods, including without limitation, those
previously described herein. They may be provided, used, handled
and/or dispensed either as a dry (lyophilized) powder, dissolved or
suspended in a solvent or mixed with a dry carrier. Mixtures of
non-nucleotide probes can be, but are not necessarily, produced by
first manufacturing and purifying the component probes followed by
a step of mixing the probes in the desired ratio to thereby produce
the mixture. Suitable solvents and carriers for the non-nucleotide
probes are known in the art and include, without limitation,
solutions of water that optionally comprise an organic modifier,
such as N,N'-dimethylformamide (DMF) or 1-Methyl-2-pyrrolidone
(NMP), and/or buffer.
Hybrid Compositions:
[0108] In yet another embodiment, this invention pertains to a
composition comprising genomic nucleic acid containing one or more
segments of randomly distributed repeat sequence selected from the
group consisting of: SINEs and LINEs. The SINEs and LINEs can be
selected from the group consisting of: Alu-repeats, Kpn-repeats,
di-nucleotide repeats, tri-nucleotide repeats, tetra-nucleotide
repeats, penta-nucleotide repeats and hexa-nucleotide repeats. The
one or more segments of randomly distributed repeat sequence can be
a fraction, or part, of a unit repeat of either: i) an Alu-repeat
sequence; or ii) a consensus sequence of a Alu-repeat sequence. The
composition further comprises two or more non-nucleotide probes of
different nucleobase sequence hybridized to at least a fraction, or
part, of the one or more segments of randomly distributed repeat
sequence of the genomic nucleic acid. Hence, the composition is the
hybrid of the segment of randomly distributed repeat sequence and
the two or more non-nucleotide probes. The non-nucleotide probes
can suppress the binding of detectable nucleic acid probe to the
randomly distributed repeat sequence of the genomic nucleic
acid.
[0109] Any of the aforementioned non-nucleotide probes, including
preferred embodiments thereof, are suitable for use in producing
the hybrid. The non-nucleotide probes can be peptide nucleic acid
oligomers.
[0110] The genomic nucleic acid of the hybrid can comprise
complementary strands of randomly distributed repeat sequence. The
genomic nucleic acid can be contained within a fixed tissue or a
cell. The genomic nucleic acid can be contained within metaphase
spreads, interphase nuclei or the nuclei of paraffin embedded
tissue material or frozen tissue sections. The nucleic acid can
also be extracted from any of the aforementioned samples.
[0111] An illustration of an exemplary construct of this hybrid is
found in FIG. 1. The design of the aforementioned construct is
intended to allow the non-nucleotide probes to hybridize to the
complementary strands of genomic nucleic acid in such a manner as
to cover as much as possible of one or the other of the
complementary strands of the genomic nucleic acid. This approach
can also serve to keep the strands separated. The ability to lock
the complementary strands of nucleic acid into an open conformation
under hybridization conditions is surprising in view of the
teachings of Perry-O'Keefe et al. (Proc. Natl. Acad. Sci. USA, 93:
14670-14675 (1996)) who specifically teach that DNA reannealing
will expel bound PNA probe that is much shorter. Because the PNA
oligomers are short, as compared with the complementary strands of
genomic nucleic acid, the PNA oligomers are expected to be expelled
and thereby should not act to block the hybridization of the longer
detectable nucleic acid probes to these undesired sequences.
[0112] Because it may be desirable to provide a mixture of
non-nucleotide probes in the same container as the detectable
nucleic acid probes, and because the detectable nucleic acid probes
can possess segments of sequence that are derived from the randomly
distributed repeat sequences, this invention is still further
directed to a composition comprising a detectable nucleic acid
probe of at least 100 by that has been derived from genomic nucleic
acid and that contains one or more segments of randomly distributed
repeat sequence selected from the group consisting of: SINEs and
LINEs. The SINEs and LINEs can be selected from the group
consisting of: Alu-repeats, Kpn-repeats, di-nucleotide repeats,
tri-nucleotide repeats, tetra-nucleotide repeats, penta-nucleotide
repeats and hexa nucleotide repeats. The composition further
comprises two or more non-nucleotide probes of different nucleobase
sequence hybridized to at least a fraction of the one or more
segments of randomly distributed repeat sequence of the detectable
nucleic acid probe. Hence, the composition is the hybrid of the
detectable nucleic acid probe hybridized to the two or more
non-nucleotide probes.
[0113] Any of the aforementioned non-nucleotide probes can be
suitable for producing the hybrid. The non-nucleotide probes can be
peptide nucleic acid oligomers.
Method for the Suppression of Undesired Detectable Probe
Binding:
[0114] In still another embodiment, this invention is directed to a
method for suppressing the binding of one or more detectable
nucleic acid probes, that are greater than 100 by and that have
been derived from genomic nucleic acid, to one or more undesired
sequences in an assay for determining target genomic nucleic acid
of a sample. The method comprises contacting the sample with a
mixture of two or more non-nucleotide probes wherein each probe
contains an aggregate nucleobase sequence that is at least eighty
percent homologous to a segment of randomly distributed repeat
sequence of genomic nucleic acid. According to the method, the
sample is also contacted with the one or more detectable nucleic
acid probes. The target genomic nucleic acid of the sample can then
be determined by determining the hybridization of the one or more
detectable nucleic acid probes to the target genomic nucleic acid
of the sample wherein the presence, absence or amount of
hybridization of the detectable nucleic acid probe to the target
genomic nucleic acid can be representative of the presence, absence
or amount of target genomic nucleic acid in the sample.
[0115] The randomly distributed repeat sequences can be selected
from the group consisting of: SINEs and LINEs. The SINEs and LINEs
can be selected from the group consisting of; Alu-repeats,
Kpn-repeats, di-nucleotide repeats, tri-nucleotide repeats,
tetra-nucleotide repeats, penta-nucleotide repeats and
hexa-nucleotide repeats. The nucleobase sequence of SINEs and LINEs
can be used as the basis for producing probes that are suitable to
suppress undesired binding since Applicants have shown that
blocking of the Alu-repeat sequences are particularly useful in
lowering the background signal that is otherwise present in such
assays (See: Example 4).
[0116] According to the method, the nucleobase sequence of the
non-nucleotide probe can be selected to be at least eighty percent
homologous to a part of a consensus sequence of a randomly
distributed repeat sequence. The nucleobase sequence of the
non-nucleotide probe can contain a segment of at least ten
consecutive nucleobases that is at least eighty percent homologous
to a fraction, or part, of the consensus unit repeat Alu-repeat
sequences selected from the group consisting of: Seq. Id. No. 1 and
Seq. Id. No. 2. The ten consecutive nucleobases can be at least
ninety percent homologous to the identified consensus sequences.
The ten consecutive nucleobases can be exactly homologous to the
identified consensus sequences.
[0117] Any of the aforementioned non-nucleotide probes can be
suitable for use in producing the hybrid that suppresses the
binding of the detectable nucleic acid probes to the undesired
sequence. The two or more non-nucleotide probes can be about 10 to
about 50 nucleobase containing subunits in length. The
non-nucleotide probes can be peptide nucleic acid oligomers.
[0118] According to the method, the genomic nucleic acid can
comprise complementary strands of randomly distributed repeat
sequence. The genomic nucleic acid can be contained in a fixed
tissue or a cell. The genomic nucleic acid can be contained in
metaphase spreads, interphase nuclei or the nuclei of paraffin
embedded tissue material or frozen tissue sections. The nucleic
acid can also be extracted from the cells or tissues.
Genomic Arrays:
[0119] (i) Array Comparative Genomic Hybridization (Array CGH)
[0120] Chromosomal comparative genomic hybridization (CGH) allows a
comprehensive analysis of multiple DNA gains and losses in entire
genomes within a single experiment. Genomic DNA from the tissue to
be investigated, such as fresh or paraffin-embedded tumor tissue
and normal reference DNA, are differentially labeled and
simultaneously hybridized in situ to normal metaphase chromosomes.
Array-based Comparative Genomic Hybridization (array CGH) provides
a higher-resolution and more quantitative alternative to chromosome
CGH for the assessment of genomic copy number abnormalities.
Instead of hybridizing to individual chromosomes (as occurs in a
ISH or FISH assay) with array CGH copy number abnormalities are
mapped onto arrays of cloned DNA sequences such as Pls, BACs or
cDNAs with the fluorescence ratios at the arrayed DNA elements
providing a locus-by-locus measure of DNA copy-number variation.
The basic assumption of a CGH experiment is that the ratio of the
binding of test and control DNA is proportional to the ratio of the
concentrations of sequences in the two samples.
[0121] (ii) Methodology of CGH
[0122] For CGH or array CGH, whole-genomic DNA is isolated from a
tumor by standard extraction protocols. Control or reference DNA is
isolated from an individual who has either a normal 46, XX
karyotype or a normal 46, XY karyotype. There is also a sample that
is to be analyzed by comparison to the control or reference DNA.
The control and sample DNA that has been extracted from the two
genomes is differentially labeled (for example fluorescein
conjugated to dUTP for the tumor genome and Cy3 conjugated to dUTP
for the normal genome). The sample and control DNA samples are
combined, and an excess of unlabelled blocking reagent (e.g. a
mixture of non-nucleic acid probes as described herein) is added
into the hybridization mixture, to suppress the repetitive
sequences that are present in both genomes. The blocking reagent is
useful because hybridization of the repetitive DNA would impair the
evaluation of the unique sequences that are either over represented
or underrepresented in the sample genome. This probe mixture is
hybridized to normal human reference metaphase chromosomes or
arrays of cloned DNA sequences in the case of array CGH. The
relative color intensities of the two fluorochromes reflect DNA
copy-number alterations in the tumor genome. In this way it is
possible to determine whether or not the sample DNA is normal
(where the color intensities of the two fluorophores is the same)
or abnormal (where the color intensities of the two samples
differs). The degree of difference in the color intensities for the
two fluorophores can also a measure of the severity or identity of
a disease state.
[0123] (iii) Array Based Embodiments of the Invention
[0124] Thus, in yet another embodiment, this invention pertains to
comparing a sample of genomic nucleic acid with that of a control
sample using a genomic nucleic acid reference array. The method
comprises providing a sample of genomic nucleic acid to be tested,
providing a control of genomic nucleic acid, wherein the control
and the sample are differentially labeled. The method further
comprises providing a genomic nucleic acid reference array, and
providing a mixture of two or more non-nucleotide probes wherein
each probe contains an aggregate nucleobase sequence that is at
least eighty percent homologous to a sixteen nucleotide segment of
randomly distributed repeat sequence of genomic nucleic acid. The
method further comprises treating the sample and control genomic
nucleic acid, the array or both the sample and control genomic
nucleic acid and the array with the mixture of non-nucleotide
probes under suitable hybridization conditions. The array is then
contacted with the treated mixture of sample and control genomic
nucleic acid under suitable hybridization conditions. The
intensities of the signals from the differential labels on the
array, caused by hybridization of the probes to genomic nucleic
acid, are then compared to thereby determine one or more variations
in copy numbers of sequences in the sample as compared with the
relative copy numbers of substantially identical sequences in the
control.
Method For Determining Non-Nucleotide Probes:
[0125] In still another embodiment, this invention is directed to a
method for determining non-nucleotide probes that hybridize to
randomly distributed repeat sequences and that are suitable for
suppressing the binding of a detectable nucleic acid probe, that is
greater than 100 by in length and that is derived from genomic
nucleic acid, to one or more undesirable sequences in an assay for
determining target genomic nucleic acid of a sample. The method
comprises designing possible nucleobase sequences of non-nucleotide
probes using sequence alignment of available sequence data for
randomly distributed repeat sequences and then preparing labeled
non-nucleotide probes having said possible nucleobase sequences.
According to the method, genomic nucleic acid of a sample that
contains the target genomic nucleic acid is treated with the
labeled non-nucleotide probes under suitable hybridization
conditions. The relative signal of the hybridized labeled probes of
the many different possible nucleobase sequences is then
determined. Based upon the signal intensity data, the probe or
probes that exhibit the strongest signal, as a result of binding to
the genomic nucleic acid, are selected and tested to thereby
determine whether or not they are suitable for suppressing the
binding of a detectable nucleic acid probe of greater than 100 by
in length that is derived from genomic nucleic acid to one or more
non-target sequences in an assay for determining target genomic
nucleic acid of a sample. In order to test the one or more selected
non-nucleotide probes, each probe can be re-synthesized in
unlabeled form and then tested using the method for suppressing the
binding of detectable probes to undesired sequences as described
above. Once tested, the best probes can be used to produce a
mixture that can be used in an assay to suppress the binding of
detectable nucleic acid probes to undesired target sequence. The
non-nucleotide probes can be peptide nucleic acid oligomers.
[0126] Those of skill in the art will appreciate that sequence
alignment is a process that can be performed using a database of
sequence information that is analyzed using a computer and software
designed to analyze the database in accordance with a particular
set of input parameters. In the context of the present invention,
the sequence of available randomly distributed repeat sequences
would be provided in the database. From this database, potential
probe sequences can be selected in accordance with the output
provided from the software program operating by computer analysis
of the database in view of the input parameters. The input
parameters can be directed toward providing a consensus sequence
where there are sequence variations known to exist among the
various randomly distributed repeats sequences. Whether using a
known randomly distributed repeat sequence, or a consensus sequence
as can be seen from the data in Example 3, one should broadly
choose all possible nucleobase sequences and then screen the
candidates since small variations of one or two nucleobases can
substantially alter the hybridization performance of the
non-nucleotide probes.
[0127] As illustrated by the Examples of this specification, this
method has been shown to be very useful in selecting probes that
can be effectively used to "block" or suppress the binding of
detectable nucleic acid probes to undesired sequences in genomic
nucleic acid. Although this method has been shown to be effective
with respect to randomly distributed repeat sequences, it is
anticipated that said method can be equally useful in the design of
probes or probe mixtures that aid in the suppression of binding of
detectable probe to other undesired sequences of genomic nucleic
acid. In order to extend the aforementioned method to other
problematic sequences it is only required that one identify a
potentially problematic sequence or sequences from which
potentially useful blocking probes can be generated for screening
purposes in accordance with the aforementioned method.
Kits:
[0128] In still another embodiment, this invention is directed to a
reagent kit comprising a mixture of two or more non-nucleotide
probes containing at least sixteen consecutive nucleobases that are
at least eighty percent homologous to a fraction of the unit repeat
Alu-repeat consensus sequence selected from the group consisting
of: Seq. Id. No. 1 or Seq. Id. No. 2. The kit further comprises one
or more other reagents, compositions and or instructions suitable
for performing an assay to thereby determine genomic nucleic acid
of a sample. For example, the reagent kit can further comprise one
or more detectable nucleic acid probes of greater than 100 by in
length and that are derived from genomic nucleic acid. The one or
more detectable nucleic acid probes can be provided in the
container that contains the mixture of two or more non-nucleotide
probes.
[0129] In yet still another embodiment, this invention is directed
to a kit comprising a mixture of two or more non-nucleotide probes
wherein at least one probe contains a segment of at least ten
consecutive nucleobases that are at least eighty percent homologous
to the Alu-repeat sequences selected from the group consisting of:
Seq. Id. No. 3, Seq. Id. No. 4, Seq. Id. No. 5, Seq. Id. No. 6,
Seq. Id. No. 7, Seq. Id. No. 8, Seq. Id. No. 9, Seq. Id. No. 10,
Seq. Id. No. 11, Seq. Id. No. 12, Seq. Id. No. 13, Seq. Id. No. 14,
Seq. Id. No. 15, Seq. Id. No. 16, Seq. Id. No. 17, Seq. Id. No. 18,
Seq. Id. No. 19, Seq. Id. No. 20, Seq. Id. No. 21, Seq. Id. No. 22,
Seq. Id. No. 23, Seq. Id. No. 24, Seq. Id. No. 25 and Seq. Id. No.
26. The kit further comprises at least one other reagent,
composition and/or set of instructions for performing a assay to
thereby determine genomic nucleic acid of a sample. For example,
the kit can further comprise one or more detectable nucleic acid
probes of greater than 100 by in length and that are derived from
genomic nucleic acid. The one or more detectable nucleic acid
probes can be provided in the container that contains the mixture
of two or more non-nucleotide probes.
[0130] Said kits are particularly useful since they provide
reagents suitable to perform a specific type of assay in convenient
packaging thereby eliminating the need to devise an assay and then
prepare the necessary reagents. The kits can provide the necessary
reagents to perform an assay for detecting the HER-2 or MLL target
sequence in a sample containing human genomic nucleic acid.
[0131] Having described the preferred embodiments of the invention,
it will now become apparent to one of skill in the art that other
embodiments incorporating the concepts described herein may be
used. It is felt, therefore, that these embodiments should not be
limited to disclosed embodiments but rather should be limited only
by the spirit and scope of the following claims.
EXAMPLES
[0132] This invention is now illustrated by the following examples
that are not intended to be limiting in any way.
General Information on PNA Oligomer Synthesis
[0133] All PNA Oligomers were prepared from commercial reagents and
instrumentation obtained from Applied Biosystems, Foster City,
Calif. using manufacturer published procedures, other well-known
procedures or those disclosed in U.S. Pat. Nos. 5,888,733,
5,985,563, 6,110,676, 6280,946, 6,287,772, 6,326,479, 6,355,421,
6,361,942 and 6,441,152 (all of which are herein incorporated by
reference). All PNA oligomers were purified by reversed-phase high
performance liquid chromatography using well-known methods. Table 2
lists PNA oligomers used in Examples 4-5, described below.
TABLE-US-00002 TABLE 2 Probe No. Label Position N-term Sequence
C-term 1 # CAGGCCGGGTGCAGTGGC 2 fluorescein # Lys(Flu)
CAGGCCGGGTGCAGTGGC 3 9-25 GGCCGGGCGCGGTGGCT 4 fluorescein 9-25
Lys(Flu) GGCCGGGCGCGGTGGCT 5 9-25 EE GGCCGGGCGCGGTGGCT EE 6
fluorescein 9-25 Flu-OEE GGCCGGGCGCGGTGGCT EE 7 fluorescein 9-25
FluOLysLys GGCCGGGCGCGGTGGCT LysLys 8 biotin 10-24 Bio-OEE
GCCGGGCGYGGTGGC EE 9 10-24 EE GCCGGGCGYGGTGGC EE 10 fluorescein
10-24 Flu-OEE GCCGGGCGYGGTGGC EE 11 26-43 GCTGGGATTACAGGCGTG 12
fluorescein 26-43 Lys(Flu) GCTGGGATTACAGGCGTG 13 26-43 Lys-Lys
GCTGGGATTACAGGCGTG Lys-Lys 14 26-43 EE GCTGGGATTACAGGCGTG EE 15
fluorescein 26-43 Flu-OEE GCTGGGATTACAGGCGTG EE 16 biotin 28-43
Bio-OEE GCTGGGAYTACAGGCG EE 17 32-48 TGTAATCCCAGCACTTT 18
fluorescein 32-48 Lys(Flu) TGTAATCCCAGCACTTT 19 fluorescein 42-56
Lys(Flu)- GCACTTTGGGAGGCC Lys(Flu) 20 fluorescein 42-56 Lys(Flu)-
GCCAGGCATGGTGAT Lys(Flu) 21 biotin 49-54 Bio-OEE CCTCCC-EOE-CCCTCC
EE 22 biotin 49-63 Bio-OEE GGGAGGCYGAGGCGG EE 23 49-64
GGGAGGCCGAGGCGGG 24 fluorescein 49-64 Flu-O GGGAGGCCGAGGCGGG 25
fluorescein 49-64 FluOLysLys GGGAGGCCGAGGCGGG LysLys 26 49-64 EE
GGGAGGCCGAGGCGGG EE 27 fluorescein 49-64 Flu-OEE GGGAGGCCGAGGCGGG
EE 28 # ACTTTGGGAGGAAGATCACC 29 fluorescein # Flu-Lys
ACTTTGGGAGGAAGATCACC 30 70-84 CACCTGAGGTCAGGA 31 fluorescein 70-84
Flu-OE CACCTGAGGTCAGGA E 32 82-102 GCCAGGATGGTCTCGATCTCC 33
fluorescein 82-102 Lys(Flu) GCCAGGATGGTCTCGATCTCC 34 82-102
GCCAGGCTGGTCTCGAACTCC 35 fluorescein 82-102 Lys(Flu)
GCCAGGCTGGTCTCGAACTCC 36 82-102 Lys-Lys GCCAGGCTGGTCTCGAACTCC
Lys-Lys 37 82-102 EE GCCAGGCTGGTCTCGAACTCC EE 38 fluorescein 82-102
Flu-OEE GCCAGGCTGGTCTCGAACTCC EE 39 98-113 TGGCCAACATGGTGA 40
fluorescein 98-113 Flu-OE TGGCCAACATGGTGA E 41 112-132
GAAACCCCGTCTCTACTAAAA 42 fluorescein ll2-132 Lys(Flu)
GAAACCCCGTCTCTACTAAAA 43 112-132 Lys GAAACCCCGTCTCTACTAAAA Lys 44
112-132 Lys-Lys GAAACCCCGTCTCTACTAAAA Lys-Lys 45 112-132 EE
GAAACCCCGTCTCTACTAAAA EE 46 fluorescein ll2-132 Flu-OEE
GAAACCCCGTCTCTACTAAAA EE 47 145-160 GCCGGGCGTGGTGGCG 48 fluorescein
l45-160 Lys(Flu) GCCGGGCGTGGTGGCG 49 145-160 EE GCCGGGCGTGGTGGCG EE
50 fluorescein l45-160 Flu-OEE GCCGGGCGTGGTGGCG EE 51 fluorescein
l45-160 FluOLysLys GCCGGGCGTGGTGGCG LysLys 52 163-180
TAGCTGGGATTACAGGCG Lys 53 fluorescein 163-180 Lys(Flu)
TAGCTGGGATTACAGGCG Lys 54 163-180 Lys-Lys TAGCTGGGATTACAGGCG
Lys-Lys 55 163-180 EE TAGCTGGGATTACAGGCG EE 56 fluorescein l63-180
Flu-OEE TAGCTGGGATTACAGGCG EE 57 184-200 GGGAGGCTGAGGCAGGA Lys 58
fluorescein l84-200 Lys(Flu) GGGAGGCTGAGGCAGGA Lys 59 184-200
Lys-Lys GGGAGGCTGAGGCAGGA Lys-Lys 60 184-200 EE GGGAGGCTGAGGCAGGA
EE 61 fluorescein l84-200 Flu-OEE GGGAGGCTGAGGCAGGA EE 62 186-200
GAGGCTGAGGCAGGA 63 fluorescein 186-200 Lys(Flu) GAGGCTGAGGCAGGA 64
201-221 CCTCCCGGGTTCACGCCATTC 65 fluorescein 201-221 Lys(Flu)
CCTCCCGGGTTCACGCCATTC 66 201-221 CCTCCCGGGTTCAAGCGATTC Lys 67
fluorescein 201-221 Lys(Flu) CCTCCCGGGTTCAAGCGATTC Lys 68 201-221
EE CCTCCCGGGTTCAAGCGATTC EE 69 fluorescein 201-221 Flu-OEE
CCTCCCGGGTTCAAGCGATTC EE 70 fluorescein 201-221 FluOLysLys
CCTCCCGGGTTCAAGCGATTC LysLys 71 228-244 Lys TTGCAGTGAGCCGAGAT 72
fluorescein 228-244 Lys(Flu) TTGCAGTGAGCCGAGAT 73 fluorescein
228-244 FluOLysLys TTGCAGTGAGCCGAGAT LysLys 74 228-244 EE
TTGCAGTGAGCCGAGAT EE 75 fluorescein 228-244 Flu-OEE
TTGCAGTGAGCCGAGAT EE 76 fluorescein 228-244 Flu-OPP
TTGCAGTGAGCCGAGAT PP 77 fluorescein 228-244 Flu-OOO
TTGCAGTGAGCCGAGAT OO 78 fluorescein 228-244 Flu-OGluGlu
TTGCAGTGAGCCGAGAT GluGlu 79 fluorescein 228-244 Lys(Flu)
DUCUCGGCUCDCUGCDD Lys 80 fluorescein 228-244 Lys(Flu)
UUGCDGUGDGCCGDGDU Lys 81 249-273 CCACTGCACTCCAGCCTGGGCGACA 82
fluorescein 249-273 Lys (Flu) CCACTGCACTCCAGCCTGGGCGACA 83 253-269
Lys TGCACTCCAGCCTGGGC 84 fluorescein 253-269 Lys (Flu)
TGCACTCCAGCCTGGGC 85 253-273 TGCACTCCAGCCTGGGCGACA 86 fluorescein
253-273 Lys (Flu) TGCACTCCAGCCTGGGCGACA 87 253-273 Lys-Lys
TGCACTCCAGCCTGGGCGACA Lys-Lys 88 253-273 EE TGCACTCCAGCCTGGGCGACA
EE 89 fluorescein 253-273 Flu-OEE TGCACTCCAGCCTGGGCGACA EE 90 #
TTTGAGACAGAGTCTCGC 91 fluorescein # Lys (Flu) TTTGAGACAGAGTCTCGC 92
275-294 TTTGAGACGGAGTCTCGCTC Lys 93 fluorescein 275-294 Lys (Flu)
TTTGAGACGGAGTCTCGCTC Lys 94 fluorescein 292-298 FluLys(Flu)OO
TTTTTTT-O-Lys-O-Lys-O- Lys TTTTTTT 95 292-298 FluLysCO
TTkTTTTT-O-Lys-O-Lys-O- Lys TTkTTTTT 96 fluorescein 292-298
FluLys(Flu)OO TTkTTTTT-O-Lys-O-Lys-O- Lys TTkTTTTT 97 PolyA Lys
TTkTTTTTkTTTLysOLysOTT Lys tail TkTTTTTkTT 98 fluorescein PolyA
FluOLys TTkTTTTTkTTTLysOLysOTT Lys tail TkTTTTTkTT 99 fluorescein
PolyA FluO TTkTTTTTkTTTOOOTTTkTTT tail TTkTT fluorescein or Flu =
5(6)-carboxyfluorescein; Lys = the amino acid L-lysine, k = the
amino acid D-lysine; O = 8-amino-3,6-dioxaoctanoic; E = the
modification resulting from use of compound 4 as described in
Gildea et al., Tett. Lett. 39: 7255-7258 (1998)), Bio = biotin; Glu
= the amino acid glutamic acid; d = the product of using
piperazine-N,N' diacetic acid-mono(2-Boc-aminoethylamide) as a
monomer; #: alu sequence, but not consensus sequence; *: score not
comparable to directly labeled oligomers
General Information on Nucleic Acid Oligomers
[0134] Those of skill in the art will appreciate that detectable
nuclei acid probes can be produced by selection of a clone covering
the desired region of interest from a public library of clones
(e.g. Resourcezemtrum in Deutchen Humangenomprojekt, RZPD). The DNA
from such a clone can then be cultured within a host organism,
extracted from the host, purified, and labeled. To amplify a
specific DNA sequence by cloning, the DNA can be inserted into a
vector and both insert and vector were amplified inside appropriate
host cells. The amplified DNA can then be extracted. Commonly used
vectors include bacterial plasmids, cosmids, PACs, BACs, and YACs,
all of which are well known to one or ordinary skill in the
art.
[0135] The purified DNA is then labeled by the Nick translation.
The Nick translation reaction employs two enzymes, Dnase I which
produces the "nicks" in the double-stranded DNA and DNA polymerase,
which incorporate labeled nucleotides along both strands of the DNA
duplex. It will be appreciated that using no more that routine
experimentation and information known to those of ordinary skill in
the art that any known labeling method can be used for labeling the
nucleic acid probes used in the embodiments and/or description of
this invention.
[0136] In Examples 4 and 5, fluorescence labeled COS or PAC based
DNA probes were produced by culturing the COS or PAC containing E.
coli and harvesting the human DNA from the cultures. The COS or PAC
DNA was purified using Qiagen large construct kit (Qiagen, Kebolab
A/S, Copenhagen, Denmark). The clones were checked by restriction
enzyme digestion. From each of the clones, the DNA was labeled by
conventional Nick translation using a monomer labeled with either
fluorescein, Cy3, or Rhodamine, as appropriate. The Nick
translation was performed using well known methods.
General Information on Preferred Method for Cytogenetic
Preparations:
[0137] Metaphase spreads and interphase nuclei were prepared from
human peripheral blood lymphocytes. A blood sample of 0.5 mL was
added to 10 mL culture medium (RPMI 1640 medium supplemented with
20% fetal calf serum, 2 mM Glutamine, 100 U/mL
Penicillin/Streptomycin, 1% Phytohemagglutine, and 50 U/mL Heparin)
and cultured for 72 hours at 37.degree. C. For metaphase arrest the
culture was incubated with 0.1 .mu.g/mL Colcemid (Gibco, BRL) for
90 min at 37.degree. C. The culture was then centrifuged at
500.times.g for 10 min. The supernatant was removed leaving 1 mL
for resuspension in 8 mL 60 mM KCl. After incubation for 30 min at
room temperature (RT), the cells were pre-fixated by adding 1 mL
freshly made Fixative (3+1 v/v methanol/acetic acid) on top of the
hypotonic suspension, and mixed carefully by turning the tube.
After 10 min at RT, the suspension was pelleted by centrifugation
at 500.times.g for 10 min. The supernatant was then removed leaving
1 mL that was resuspended in 10 mL Fixative added slowly with
gentle agitation. The fixation was repeated twice with at 10 min
incubation at RT between each fixation. After the third fixation
the cells were resuspended in 1 mL Fixative. The cells were then
dropped onto wet microscopic slides that have been cleaned in
detergent and rinsed with water. The slides are left to air dry and
stored at -20.degree. C. until hybridization.
Example 1
Design of Non-Nucleotide Probes Directed Towards Repetitive
Sequences
[0138] For this Example, the nucleobase sequences of non-target
hybridization probes directed toward Alu repeat consensus sequence
were designed. It was envisioned that the probe sequences that
would be best for suppressing the binding of detectable nucleic
acid probes, generated from nick translation of cosmid nucleic
acid, would be those nucleobase sequences possessing shared
repeated sequences of Alu repeat sequence that are most prevalent
in genomic nucleic acid. Hence, a consensus sequence of known
Alu-repeat sequences was generated by performing an alignment
analysis of five Alu consensus sequences representing the two
family branches J and S (GenBank Acc. No. U14567 and U14571-14574)
(Claverie, J-M and Makalowski, W. Science, 371; 752 (1994)), using
the Clustal W algorithm. The three identified subfamilies of the Y
family branch have not been included in the alignment shown in FIG.
2 as the relative frequency of Alu elements belonging to the Y
family is very low (presumably less than 0.5% Sherry et al.,
Genetics, 147: 1977-1982 (1997). The consensus sequence, and its
complement, that were determined using this approach are Seq. ID.
No. 1 and Seq. Id No. 2. The raw alignment data output for Seq. ID
No. 1 is also presented in FIGS. 2.
[0139] From the consensus sequence data, the nucleobase sequence of
numerous potential probes was determined. Generally however, the
nucleobase sequence design of the various probes was selected to,
as completely as possible when the probes were mixed together,
blanket the upper and lower strand of the Alu consensus sequence,
as illustrated in FIG. 1, in order to disrupt as much as possible
the hybridization between the individual strands of the repeated
(Alu) sequences of the genomic nucleic acid of a sample. Because
disruption occurs if probe is bound to one strand, the position for
hybridization of the various probes, when mixed together, was
chosen such that the probes hybridizing to one of the two strands
were substantially offset as compared with the probes hybridizing
to the other strand. In this way the entire hybrid sequence was
blanketed with as few probes as possible. This approach was taken
to minimize the number of probes need; it however is not a
limitation since additional probes or more extensive blanketing of
the randomly distributed repeat sequence is acceptable. Moreover,
it was believed that it was preferable to blanket at least fifty
percent, and more preferably at least two thirds, of the linear
double stranded molecule to thereby prevent the rehybridization of
the two strands of genomic nucleic acid. Additionally, the probe
candidates were checked for probe self dimers, probe pair dimers,
and probe hairpins to minimize the hybridization reactivity with
the probes themselves (i.e. intramolecular interactions) and with
other probes in the mixture (e.g. intermolecular interactions).
[0140] Using these design parameters, the nucleobase sequences were
chosen for the numerous probes that might, when mixed together,
suppress the binding of detectable nucleic acid probes to undesired
target genomic nucleic acid. These sequences were used to prepare
non-nucleotide (i.e. PNA oligomer) probes for further testing and
analysis.
Example 2
Evaluation of PNA Oligomer Candidates
Preferred Procedure for Performing In-Situ Hybridization Using
Directly Labeled PNAs:
[0141] Slides for in-situ hybridization using directly labeled PNAs
were prepared as described in the general information on preferred
method for cytogenetic preparations as discussed above. For
pre-treatment, the slide containing metaphase spreads and
interphase nuclei was immersed shortly in TBS (Tris-buffered
saline), followed by 3.7% formaldehyde in TBS for 2 min at RT, and
twice in TBS for 5 min each. The slide was treated with Proteinase
K (DAKO S3020, DAKO A/S, Glostrup, Denmark) diluted 1:2,000 in TBS
for 10 min at RT, and rinsed twice in TBS for 5 min each, followed
by dehydration in a cold ethanol series (70%, 85%, and 96%), 2 min
each. The slide was then air-dried. For hybridization with the
fluorescein labeled PNA probes, 10 .mu.L hybridization buffer (70%
formamide, 20 mM Tris pH 7.5, 10 mM NaCl, 10 mM phosphate buffer pH
7.5, 0.02% Ficoll, 0.02% polyvinylpyrrolidon, and 0.02% BSA (bovine
serum albumin)) with 50 nM PNA probe was added to the pre-treated
slide. An 18-mm.sup.2 coverslip was applied to cover the
hybridization mixture. The slide was denatured by incubation at
80.degree. C. for 5 min and allowed to hybridize by incubation at
RT for 30 min. The coverslip was removed in PBS (Phosphate-buffered
saline) at RT. Excess of probe was removed by washing in preheated
PBS with 1% Tween 20 at 60.degree. C. for 25 min. Finally the slide
was dehydrated in a cool ethanol series and air-dried at describe
above. The slide was mounted in 10 .mu.L anti-fade mounting medium
(Vectashield H-1000, Vector Laboratories, Inc. Burlingame)
supplemented with 0.1 .mu.g/mL 4,6-diamoni-2phenyl-indole (DAPI,
Sigma Chemicals) and sealed with a coverslip. Slides were then
analyzed using a microscope equipped with a CCD digital camera.
Digital Imaging Microscopy:
[0142] Images reproduced in FIGS. 3-7 were obtained using a Leica
fluorescent microscope equipped with a 100.times.immersion oil
objective, a 10.times.ocular (total enlargement is 1,000 fold) and
fluorescent filter cubes obtained either from Leica or Chroma
(Chroma Technology Corp., Brattleboro, Vt., US). Electronic digital
images were made of the slide using a Photometric Sensys CCD-camera
and Leica QFISH software (Leica Imaging System Ltd, Cambridge,
UK).
Experimental Design:
[0143] It is well established that the hybridization of labeled
nucleic acid, containing Alu sequences, to metaphase spreads will
produce a distinct and highly reproducible R-banding pattern
(Kornberg and Rykowski, 1988; Baldini and Ward, 1991). These
isolated nucleic acid fragments are typically greater than 100 bp
in length. Thus, it seemed reasonable to prepare labeled
non-nucleotide probes having the various chosen nucleobase
sequences and then determine whether or not they would produce
similar R-banding patterns as a way to test the binding affinity of
these probes to randomly distributed repeat sequences in genomic
nucleic acid and thereby score the result for each probe.
Particularly because it was a consensus sequence that was being
used to produce the PNA oligomers, and not one of the actual
naturally occurring Alu-repeat sequences, testing was believed to
be necessary to determine which of the nucleobase sequences
hybridized most strongly to the Alu-repeat sequences of genomic
nucleic acid.
[0144] For this purpose, a fluorescein labeled PNA oligomer was
synthesized for each nucleobase sequence candidate (chosen
candidates are listed in Table 2). The various PNA constructs were
then evaluated in a PNA-FISH assay using the procedure discussed
above. The most important of the parameters to be analyzed was the
R-banding potential but the relative intensity of the R-banding
signals was also examined. In each of the categories, the R-banding
potential and signal intensity for each of the PNA oligomers was
assigned a score from the minimum value of 0 to a maximum value of
6. To select for PNA oligomers that had the nucleobase sequences
that were most suitable for use in probe mixture for blocking of
the Alu repeats, all oligomers having an R-banding potential limit
value of less than 3 where eliminated. The process was used to
screen a total of 55 unique PNA oligomers, of which the 12
nucleobase sequences identified in Table 3 were found to be
preferred because of their superior ability to bind to genomic
nucleic acid.
TABLE-US-00003 TABLE 3 Seq. R-band Signal Id. No. Position.sup.a
Nucleobase Sequence Score Intensity 3 9-25 GGCCGGGCGCGGTGGCT 4 5 4
26-43 GCTGGGATTACAGGCGTG 5 6 5 49-64 GGGAGGCCGAGGCGGG 5 5 6 82-102
GCCAGGCTGGTCTCGAACTCC 4 4 7 112-132 GAAACCCCGTCTCTACTAAAA 3 3 8
145-160 GCCGGGCGTGGTGGCG 5 4 9 163-180 TAGCTGGGATTACAGGCG 6 5 10
184-200 GGGAGGCTGAGGCAGGA 6 6 11 201-221 CCTCCCGGGTTCAAGCGATTC 3 3
12 228-244 TTGCAGTGAGCCGAGAT 3 3 13 253-273 TGCACTCCAGCCTGGGCGACA 4
4 14 292.sup.c .sup.bTT(k)TTTTT(k)TTTLysOLysOTTT(k)TTTTT(k)TT 4 5
.sup.aRefers to the relative positions in the Alu consensus
sequence depicted in FIG 1. .sup.bTriplex maker construct. k =
D-Lysine; Lys = L-lysine; O = 8-amino-3,6-dioxaoctanoic acid.
.sup.cHybridizes to the variable polyA tail of the alu
sequence(Ullu E., TIBS: 216-219 (June, 1982)
Results:
[0145] With reference to Table 3, there is data for each identified
PNA oligomer that passed the limit value. For each entry, the
relative position of the Alu consensus sequence, depicted in FIG.
2, is identified and the recorded R-banding value and Signal
intensity score (average of 4-6 independent experiments) is
reproduced. Representative examples of fluorescein labeled PNA
oligomers that obtained a high score in the R-banding evaluation
approach (Seq. Id No. 4 and Seq. Id. No. 10) and constructs that
only just passed the limit value (Seq. Id. No. 7 and Seq. Id. No.
12) arc shown in FIGS. 3A-1, 3A-2, 3B-1 and 3B-2, respectively.
These images demonstrate that vast differences in performance exist
for the various probes examined and therefore demonstrate that
testing is a suitable way to determine which sequences were most
effective at hybridizing to the randomly distributed repeat
sequences of genomic nucleic acid.
Example 3
Effect of Sequence Variation
Experimental Design:
[0146] The Alu elements are dominated by the J and S family
branches (Britten, R. J., Proc. Natl. Acad. Sci. USA, 91: 6148-6150
(1994); Batzer M A et al., J. Mol. Evol. (1996)). Especially
noteworthy is the Sx subfamily, whereas the Y family branch
constitute less than 0.5% of the total human Alu elements. In order
to test the sensitivity by which the R-banding approach can detect
the frequency of a specific sequence within the Alu elements,
comparisons of almost identical PNA oligomer constructs were
performed.
[0147] The constructs to be compared were directed towards
identical positions within the Alu consensus sequence (FIG. 2) but
the specific nucleobase sequence design was based on either the J
and S family consensus or the Y family consensus. The images
presented in FIG. 4(A-D) were obtained using PNA oligomers that are
complementary to positions 82-102 and 201-221 of the sequence shown
in FIG. 2 (the PNA oligomer sequences are complimentary to the
depicted consensus sequence). Within positions 82-102 the J and S
consensus differ from the Y consensus by a T.fwdarw.A transversion
at position 86 and a G.fwdarw.T transversion at position 96.
Similarly, within position 201-221 a single point mutation (or
single nucleotide polymorphism) correspondingly consists of a
T.fwdarw.G transversion at position 208. The nucleobase sequence of
the various PNA oligomer probes used in the experiments, as well as
the respective R-band and Signal intensity scores of the examined
oligomer constructs (FIG. 4), are presented in Table 4. R-band and
Signal intensity scores represent average values from 8-10
independent experiments.
TABLE-US-00004 TABLE 4 Seq. Relative Id. Alu pos. in Signal No.
consensus Oligo sequence FIG. 2 R-band intensity 27 J and S family
GCCAGGCTGGTCTCGAACTCC 82-102 4 5 28 Y family GCCAGGATGGTCTCGATCTCC
82-102 2 2 29 J and S family CCTCCCGGGTTCAAGCGATTC 201-221 3 3 30 Y
family CCTCCCGGGTTCACGCGATTC 201-221 1 1
Results:
[0148] As can be seen by analysis of Table 4 and FIGS. 4(A-D),
these small changes (a single point mutation in one probe set and a
double mutation in the other probe set) in the nucleobase sequence
of the PNA oligomers significantly affects the efficiency by which
the R-bands are formed. It is not known whether the R-band signals
from hybridization experiments involving the Seq. Id. No. 28 and
Seq. Id. No. 30 constructs (Y family consensus) reflect a low
frequency of the corresponding genomic sequences, or rather that
Seq. Id. No. 28 and Seq. Id. No. 30 hybridize to the consensus
sequences of the J and S family with a reduced affinity.
Nevertheless, this data reinforces the position that actual testing
should be performed in order to determine which of all possible
nucleobase sequences will produce the most complete hybridization
to the randomly distributed repeat sequences and thereby presumably
produce the most efficient blocking probes. Moreover, the
aforementioned procedure appears to be well suited to determining
the best candidates for further testing in a representative assay
wherein there is substantial interfering cross reaction of a
detectable probe to undesired sequence.
Example 4
Suppression of Undesired Signal Using a PNA Probe Mixture
Preparation of DNA Probe/Blocking Agent Mixture:
[0149] Fluorescence labeled COS or PAC based DNA probes were
prepare made by culturing the COS or PAC containing E coli and
harvesting the human DNA from the cultures. The COS or PAC DNA was
purified using Qiagen large construct kit (Qiagen, Kebolab A/S,
Copenhagen, Denmark). The clones were checked by restriction enzyme
digestion. From each clone, the DNA was labeled by conventional
Nick translation using a monomer labeled with either fluorescein,
Cy3, or Rhodamine. For the HER2 experiments illustrated in FIG. 5,
the COSs were labeled with fluorescein; for the MLL experiments in
FIG. 6, the two PAC clones flanking the breakpoint (van der Burg et
al., Leukemia 13: 2107-2113 (1999)) were labeled in different
colors, one with fluorescein the other with Cy3; for the HER2
experiments on tissue sections (FIG. 7) the COS clones were labeled
with Rhodamine. In all experiments the labeled COS or PAC DNA
probes were mixed with a blocking agent (PNA Oligomer Mixture or
Cot-1 DNA) in DNA Hybridization Buffer (45% formamide, 300 mM NaCl,
5 mM NaPO.sub.4, 10% Dextran sulphate). Each DNA probe was present
at a final concentration of 2 ng/.mu.L. When using the PNA Oligomer
Mixture as a blocking agent, each PNA oligomer was present at a
concentration of 5 .mu.M in the hybridization buffer. With the
Cot-1 DNA as blocking agent, a 100:1 weight ratio of Cot-1
DNA:total probe DNA was used in the assay.
Preferred Procedure for Performing In-Situ Hybridization Using
Unlabeled PNAs as Blocking Agent:
[0150] Slides containing metaphase spreads and interphase nuclei
were pre-treated as described above. The slide was immersed in TBS
with 3.7% formaldehyde for 2 minutes at RT and PBS for 2 minutes at
RT followed by dehydration in chilled (5.degree. C.) ethanol series
(70%, 85%, and 96%); 2 minutes each. On each pre-treated slide, 10
.mu.L DNA of Hybridization Buffer with labeled DNA probe and
unlabeled blocking agent (PNA Oligomer Mixture or Cot-1 DNA) is
added and a 18 mm.sup.2 coverslip is applied to cover the
hybridization mixture. The edges of the coverslip were sealed with
rubber cement and air-dried until the cement had set (around 5
min). The slide was denatured by incubation at 80.degree. C. for 5
min. The slides were then hybridized O.N. at 37.degree. C. (when
Cot-1 DNA is used as blocking agent) or 45.degree. C. (when PNA
Oligomer Mixture is used as a blocking agent). After hybridization
the coverslip was removed and the slide rinsed at RT in
0.1.times.SSC followed by wash for 2.times.10 minutes in
0.1.times.SSC at 55.degree. C. (when Cot-1 DNA is used as the
blocking agent) or 60.degree. C. (when the PNA Oligomer Mixture is
used as a blocking agent). Finally the slide was dehydrated in 70%,
85%, and 96% EtOH and air-dried as describe above. Each slide was
mounted with 10 .mu.L anti-fade solution, with 0.1 .mu.g/mL
4,6-diamoni-2phenyl-indole (DAPI, Sigma Chemicals) and sealed with
a coverslip. Slides were then analyzed using a microscope equipped
with a CCD digital camera.
Experimental Design:
[0151] The Alu-banding approach (above) facilitated the
identification of nucleobase sequences, within the Alu repeats,
that seem to be present in the human genome with a high frequency.
If Alu repeats are a major reason for non-target hybridization of
large probes of genomic origin, a mixture of the identified PNA
oligomer constructs should be able to suppress this undesired
hybridization background. Therefore, unlabeled PNA oligomers having
the preferred nucleobase sequences, as identified in Table 3, were
prepared. These unlabeled and purified probes were mixed together
and used in hybridization experiments as previously described (the
"PNA Oligomer Mixture").
[0152] In addition to the unlabeled PNA probes, detectable nucleic
acid probes of genomic origin were used in an assay to detect a
genomic nucleic acid target. The detectable nucleic acid probes are
COS clones covering around 100 kb of a region which include the
HER-2 gene (17q21.1), or PAC clones covering 90 kb on each side of
MLL gene (11q23) major breakpoint region (mbr). For this
experiment, the detectable nucleic acid probes were labeled with
fluorescein and the PNA oligomers were unlabeled.
[0153] In one experiment, the fluorescein labeled HER-2 probes were
hybridized to metaphase spreads and interphase nuclei. A
representative microscope image of the resulting sample can be seen
in FIG. 5A. This result was compared with the standard art
recognized blocking reagent, Cot1 DNA (FIG. 5B: Human Cot-1 DNA
from Gibco BRL, Life Technologies). In a separate sample, neither
the PNA probe mixture nor the Cot1 DNA was added (FIG. 5C).
[0154] Translocations can be detected by a two color staining
either by the "fusion" signal principle or the "split" signal
principle. In the fusion signal principle the two genes involved in
the translocation are labeled in each a separate color. In cells
where a translocation has taken place the two colors will come
together as a "fusion" signal. In the "split" signal principle the
two differently labeled probes are localized around the breakpoint
in one of the genes participating in the translocation (van der
Burg et al., Leukemia 13: 2107-2113 (1999)). Thus, in abnormal
cells the two probes will split as a result of the translocation.
Similarly, the MLL probes labeled with fluorescein or Cy3 were
hybridized to metaphase spreads and interphase nuclei using the
same procedure as used for the HER 2 probes. A representative
microscope image of the resulting sample can be seen in FIG. 6A.
This result was compared with the standard art recognized blocking
reagent, Cot1 DNA (FIG. 6B). In a separate sample, neither the PNA
probe mixture nor the Cot1 DNA was added (FIG. 6C).
[0155] With reference to FIGS. 5A-C and 6A-C, it is apparent that
in both cases the mixture of PNA probes appears to work as well as,
if not better than, the industry standard, Cot1 DNA. It is also
noteworthy that the absence of any blocking agent (e.g. PNA probe
mixture or the Cot1 DNA) results in the formation of a
hybridization background with a R-banding pattern, thereby
indicating that non-target hybridization is caused by the presence
of Alu repeats or other randomly distributed repeat sequence. The
intensity of this background also seriously hinders the
visualization of the single locus specific signals such that it
must be removed in order to facilitate the performance of an
accurate and reproducible assay.
Example 5
Suppression of Undesired Background in Tissue Sections Using the
PNA Probe Mixture
[0156] The PNA blocking mixture was used to suppress the undesired
background staining from the HER2 probes described in Example 4
when used on tissue sections from a breast carcinoma. The DNA
probes were labeled with Rhodamine and mixed with either the PNA
Oligomer Mixture (FIG. 7A) or Cot-1 DNA (FIG. 7B). For comparison
FIG. 7C shows the same experiment without any blocking agent
added.
Preferred Procedure for Performing In-Situ Hybridization to
Paraffin Embedded Mamma Carcinoma Sections Using Unlabeled PNAs as
Blocking Agent:
[0157] Mamma carcinoma sections of 4 .mu.m were cut from paraffin
embedded tissue blocks. Slides mounted with tissue sections were
deparaffinated in Xylene and 96% EtOH according to standard
procedures (DAKO's handbook: Immunochemical Staining Methods). The
slides were pre-treated in PBS for 10 min. at RT and 10 min. in
boiling MES (2-[N-morpholino]ethanesulphonic acid) buffer, pH 6.4
followed by digestion in a 0.05% pepsin solution (0.05% pepsin in
0.02M HCl, 0.9% NaCl) for 10 min. at 37.degree. C. The slides are
washed for 3.times.2 minutes in PBS and dehydrated in chilled
(5.degree. C.) ethanol series (70%, 85%, and 96% EtOH, 2 minutes
each). The slides were allowed to air-dry. For hybridization, 10
.mu.L DNA hybridization buffer containing the labeled DNA probe and
a blocking agent was added to the pre-treated slide and a 18
mm.sup.2 coverslip was applied to cover the hybridization mixture.
The edges of the coverslip were sealed with rubber cement and
air-dried until the cement had set (around 5 min). The slide was
denatured by incubation at 90.degree. C. for 5 min. The slides were
hybridized O.N. at 45.degree. C. After hybridization the coverslip
was removed and the slide was rinsed in pre-warmed (55.degree. C.)
0.2.times.SSC with 0.1% TritonX-100 followed by a wash for 10
minutes in 0.2.times.SSC with 0.1% TritonX-100 at 55.degree. C.
Finally the slide was rinsed in PBS for 2 minutes at RT followed by
dehydration in chilled ethanol series (70%, 85%, and 96% EtOH, 2
minutes each) and air-dried as described above. Each slide was
mounted with 10 .mu.L anti-fade solution with 0.1 .mu.g/mL
4,6-diamoni-2phenyl-indole (DAPI, Sigma Chemicals) and sealed with
a coverslip. Slides were then analyzed using a microscope equipped
with a CCD digital camera.
Results:
[0158] With reference to FIGS. 7A and 7B it is clear that there is
very little difference in the performance of the PNA probe mixture
vs. the Cot-1 DNA. Moreover, the absence of any blocking agent
results in substantial background (FIG. 7C).
Example 6
More Examples of Chromosome Analysis
Synthesis and Labeling of Peptide Nucleic Acid Probes
[0159] The chromosome 17 centromere Peptide Nucleic Acids (PNAs)
were synthesized at the 2-.mu.mole scale on an Expedite 8900
nucleic acid synthesis system (Applied Biosystems, Foster City,
Calif.) using Fmoc chemistry. The Alu PNAs were synthesized at the
5 .mu.mole scale using a 433A nucleic acid synthesis system
(Applied Biosystems, Foster City, Calif.) using t-boc chemistry.
All of the PNAs were solubility enhanced using compound 4 as
described (Gildea et al 1998). Attachment of a linker group while
the oligomer was still bound to the column was accomplished by
condensation of the expedite PNA linker,
Fmoc-8-amino-3,6-dioxaotanoic acid. For 5(6)-carboxyfluorescein
labeling the synthesis support was heated at 30.degree. C. for five
hours in 250 .mu.L of a solution containing 0.08M dye as an NHS
ester, (Molecular Probes, Eugene, Oreg.), 0.25M
diisopropylethylamine and 0.2-M lutidine. The crude oligomer
samples were then cleaved from the support by the use of standard
methods, precipitated and purified by high performance liquid
chromatography (HPLC) using 0.1% trifluoroacetic acid and a linear
acetonitrile gradient.
Preparation of Chromosome Specific Probes
[0160] Chromosome 17 specific PNA probes (18-22 base units) were
selected from published data available in public databases (e.g.
Genbank). The following seven "Chromosome 17 PNA Probes" specific
for the chromosome 17 .alpha.-satellite sequences were
selected;
TABLE-US-00005 1.
Flu-OEE-AAC-GAA-TTA-TGG-TCA-CAT-EEO-Lys(Flu)-NH.sub.2 2.
Flu-OEE-GGT-GAC-GAC-TGA-GTT-TAA-EEO-Lys(Flu)-NH.sub.2 3.
Flu-OEE-AAC-GGG-ATA-ACT-GCA-CCT-EEO-Lys(Flu)-NH.sub.2 4.
Flu-OEE-ATC-ACG-AAG-AAG-GTT-CTG-EEO-Lys(Flu)-NH.sub.2 5.
Flu-OEE-TTT-GGA-CCA-CTC-TGT-GGC-EEO-Lys(Flu)-NH.sub.2 6.
Flu-OEE-GAA-TCT-TCA-CAG-GAA-AGC-EEO-Lys(Flu)-NH.sub.2 7.
Flu-OEE-GAT-TCT-ACA-CAA-AGA-GAG-EEO-Lys(Flu)-NH.sub.2
(Abbreviations are as previously described)
[0161] GenBank accession numbers U14567, U14568, U14569, U14570,
U14571, U14572, U14573, U14574 representing the Alu consensus
sequences were used to select sequences specific for the Alu family
of interspersed repeats. The following eleven "Alu PNA Blocking
Probes" selected from the consensus sequences were as follows:
TABLE-US-00006 1. H-EE-TTG-CAG-TGA-GCC-GAG-AT-EE-NH.sub.2 2.
H-EE-GGC-CGG-GCG-CGG-TGG-CT-EE-NH.sub.2 3.
H-EE-GCT-GGG-ATT-ACA-GGC-GTG-EE-NH.sub.2 4.
H-EE-GGG-AGG-CCG-AGG-CGG-G-EE-NH.sub.2 5.
H-EE-GCC-AGG-CTG-GTC-TCG-AAC-TCC--EE-NH.sub.2 6.
H-EE-GAA-ACC-CCG-TCT-CTA-CTA-AAA--EE-NH.sub.2 7.
H-EE-GCC-GGG-CGT-GGT-GGC-G--EE-NH.sub.2 8.
H-EE-TAG-CTG-GGA-TTA-CAG-GCG--EE-NH.sub.2 9.
H-EE-GGG-AGG-CTG-AGG-CAG-GA--EE-NH.sub.2 10.
H-EE-CCT-CCC-GGG-TTC-AAG-CGA-TTC-EE-NH.sub.2 11.
H-EE-TGC-ACT-CCA-GCC-TGG-GCG-ACA-EE-NH.sub.2
(Abbreviations are as previously described)
In-Situ Hybridization
[0162] (i) Slide Preparation
[0163] Slides containing metaphase spreads were prepared with
standard cytogenetic techniques, essentially as previously
described herein, and aged overnight then stored at -20.degree. C.
Prior to hybridization, the slides were removed from the freezer
and allowed to warm to room temperature.
[0164] (ii) Hybridization and Washings
[0165] To 10 .mu.L of hybridization mix containing 45% formamide,
10% dextran sulphate, 300 mM NaCl, 5 mM Na phosphate, 100 ng of
rhodamine labeled Her2 DNA probe, and optionally as discussed below
either or both of: (a) the Chromosome 17 PNA Probes at 30 nM each
and/or (b) Alu PNA Blocking Probes at 5 .mu.M each, were added to
the slide. The slides were denatured at 70.degree. C. for six
minutes followed by hybridization overnight at 37.degree. C. in a
humidified chamber. After removal of the coverslip the slides are
washed in a stringent wash solution (0.2.times.SSC with 0.1% Triton
X-100) at 65.degree. C. for 10 minutes. The slides were then rinsed
in TBS buffer for 2 minutes. Cells were counterstained with DAPI
and mounted in Vectashield anti-fade medium.
[0166] (iii) Digital Imaging Microscopy
[0167] Digital images of metaphase cells after FISH with
fluorescein labeled Chromosome 17 PNA Probes and/or Rhodamine
labeled Her2 DNA probes were acquired with a cool snap FX 12 bit
CCD camera (Roper Scientific, Tucson, Ariz.) attached to an Olympus
AX 70 microscope using Openlab software (Improvision Inc.,
Lexington, Mass.). The microscope was equipped with SP100, 41001,
SP102, SP102, SP104 and SP105 filter sets for multicolor FISH
(Chroma Technology, Brattleboro, Vt.). Images of each fluorescent
dye were acquired, avoiding over or under exposure and stored for
further analysis. After thresholding and contrast enhancement using
Openlab software (Improvision Inc., Lexington, Mass.), pixels above
a selected threshold from each fluorescent dye were projected on to
the DAPI image.
Results:
[0168] As described above, a series of experiments were designed to
establish the usefulness of a mixture of Alu PNA Blocking Probes in
the analysis of genomic nucleic acid in FISH experiments. First,
the affect of hybridizing a rhodamine labeled genomic DNA probe
(rhodamine labeled Her2 DNA) in the absence of Alu PNA Blocking
Probes was used as a control. With respect to FIG. 8A, the
interphase and metaphase cells were stained bright red with
non-specific staining clearly visible. There was little or no
differentiation between the specific Her2 signal and the
non-specific background staining. In addition, the morphology of
the chromosomes was extremely poor.
[0169] By comparison, the affect of hybridizing the same labeled
genomic probe (rhodamine labeled Her2 DNA), wherein Alu PNA
Blocking Probes were present, was performed. With respect to FIG.
8B, the interphase and metaphase cells contain low non-specific
staining and bright specific red Her2 signal on the long arm of
chromosome 17. This demonstrates that the presence of the Alu PNA
Blocking Probes significantly reduce non-specific
hybridization.
[0170] To confirm that the observed signal was present on
chromosome 17, and not on another chromosome, in the next series of
experiments a mixture of Fluorescein Chromosome 17 PNA Probes were
included in the hybridization mix along with the rhodamine labeled
Her2 DNA probe, both with (FIG. 8C) and without Alu PNA Blocking
Probes (FIG. 8D).
[0171] With reference to FIG. 8C, hybridization of the rhodamine
labeled Her2 DNA probe and the Fluorescein Chromosome 17 PNA Probe
mix, in the absence of Alu PNA Blocking Probes, produces results
that are similar to FIG. 8A, wherein the Fluorescein Chromosome 17
PNA Probe are omitted except there was non-specific binding of the
probe to the glass slide. Specifically, there is too much
non-specific signal for the assay to be of practical utility. By
comparison, FIG. 8D shows the effect of including the Alu blocking
probes. In this Figure there is a bright red specific Her2 signal
on the long arm of chromosome 17 (as indicated by the arrows) with
low non-specific staining of the interphase cells and metaphase
chromosomes with little or no non specific hybridization on the
glass slide. Because the Fluorescein Chromosome 17 PNA Probes were
present in the mix, it is confirmed that the green signal
correlates with the red signal, thereby confirming that the DNA
probe hybridizes specifically to chromosome 17 and not another
chromosome.
Example 7
Detection of Translocation Of The CyclinD1 Gene Using Suppression
of Undesired Background with the PNA Oligomer Mixture
Preparation of DNA Probe/Blocking Agent Mixture:
[0172] Fluorescence labeled BAC based DNA probe was prepare made by
culturing the BAC containing E coli and harvesting the human DNA
from the cultures. The BAC DNA was purified using Qiagen large
construct kit (Qiagen, Kebolab A/S, Copenhagen, Denmark). The clone
was checked by restriction enzyme digestion and end sequenced. The
clone DNA was labeled by conventional Nick translation using a
monomer labeled with fluorescein. In all experiments the labeled
DNA probe was mixed with a blocking agent (with or without PNA
Oligomer Mixture or Cot-1 DNA) in DNA Hybridization Buffer (45%
formamide, 300 mM NaCl, 5 mM NaPO.sub.4, 10% Dextran sulphate). The
DNA probe was present at a final concentration of 5 ng/.mu.L. The
centromeric PNA probe was labeled with Rhodamine and present at a
final concentration of 50 nM. When using the PNA Oligomer Mixture
as a blocking agent, each PNA oligomer was present at a
concentration of 5 .mu.M in the Hybridization Buffer. With the
Cot-1 DNA as blocking agent, a 100:1 weight ratio of Cot-1
DNA:total probe DNA was used in the assay.
Preferred Procedure for Performing In-Situ Hybridization using
Unlabeled PNAs as Blocking Agent:
[0173] Slides containing metaphase spreads and interphase nuclei
were pre-treated as described above. The slides were immersed in
TBS with 3.7% formaldehyde for 2 minutes at RT and PBS for 2
minutes at RT followed by dehydration in chilled (5.degree. C.)
ethanol series (70%, 85%, and 96%); 2 minutes each. On each
pre-treated slide, 10 .mu.L DNA of Hybridization Buffer with
labeled DNA probe and unlabeled blocking agent (PNA Oligomer
Mixture or Cot-1 DNA) is added and a 18 mm.sup.2 coverslip is
applied to cover the hybridization mixture. The edges of the
coverslip were sealed with rubber cement before treatment at
80.degree. C. for 5 min. The slides were then hybridized O.N. at
45.degree. C. After hybridization, the coverslip was removed and
the slide rinsed at RT in Stringent Wash Buffer (0.2.times.SSC,
0.1% Triton X-100) followed by wash for 10 minutes in Stringent
Wash Buffer at 65.degree. C. Finally the slide was dehydrated in
70%, 85%, and 96% EtOH and air-dried as describe above. Each slide
was mounted with 10 .mu.L anti-fade solution, with 0.1 .mu.g/mL
4,6-diamoni-2phenyl-indole (DAPI, Sigma Chemicals) and sealed with
a coverslip. Slides were then analyzed using a microscope equipped
with a CCD digital camera.
Experimental Design:
[0174] The Alu-banding approach (above) facilitated the
identification of nucleobase sequences, within the Alu repeats,
that seem to be present in the human genome with a high frequency.
If Alu repeats are a major reason for non-target hybridization of
large probes of genomic origin, a mixture of the identified PNA
oligomer constructs should be able to suppress this undesired
hybridization background. Therefore, unlabeled PNA oligomers having
the preferred nucleobase sequences, as identified in Table 3, were
prepared. These unlabeled and purified probes were mixed together
and used in hybridization experiments as previously described.
Results:
[0175] Since the CyclinD1 gene is located on chromosome band 11q23,
a PNA mixture for the centromere of chromosome 11 (See U.S. Ser.
No. 09/520,760 or U.S. Ser. No. 09/627,796, herein incorporated by
reference) was prepared and added to the assay to thereby identify
the chromosome to which the CyclinD1 probe hybridizes. The PNA
oligomers for the centromere of chromosome 11 were labeled with
Rhodamine. FIG. 9A shows both centromeres of chromesome 11 in red,
as well as the green CyclinD1 probes. FIG. 9B shows the same probes
wherein Cot-1 DNA is used as compared with the PNA Oligomer
Mixture. The red centromere signals are not visible as Cot-1 also
contains centromeric sequences and thus compete with the PNA in
binding to the centromere. FIG. 9C show the same probes without any
blocking agent added. It is not possible to identify either of the
red or the green signals in the interphase nuclei. In the metaphase
spread, only the centromere signals (in red) can be seen.
[0176] The CyclinD1 probe and the Centromere 11 PNA was also
hybridized to metaphase spreads of the cell line Granta 519
((Drexler, H. G. (2001) The Leukemia-Lymphoma Cell Line Facts Book.
Braunschweig, Germany)) that harbors a translocation of the
CyclinD1 gene. The probe was labeled with fluorescein and mixed
with either the PNA Oligomer Mixture (FIG. 9D) or Cot-1 DNA (FIG.
9E). For comparison FIG. 9C shows the same experiment without any
blocking agent added.
Example 8
Detection of Amplification of the c-MYC Gene Using Suppression of
Undesired Background with the PNA Oligomer Mixture
[0177] The c-MYC DNA probe was prepared as described in example 7.
Furthermore, a DNA based centromere probe was used instead of a PNA
based centromere probe. The DNA centromere DNA probe was prepare by
culturing the centromere DNA plasmid containing E coli and
harvesting the human DNA from the cultures. The plasmid DNA was
purified using Qiagen plasmid kit (Qiagen, Kebolab A/S, Copenhagen,
Denmark). The clone DNA was labeled by conventional Nick
translation using a monomer labeled with fluorescein. The DNA based
centromere probe was used at a final concentration of 2
.mu.g/.mu.L. The procedure for performing in-situ hybridization is
described in Example 7.
Results:
[0178] The PNA Oligomer Mixture was used to suppress the undesired
background staining from a c-MYC probe when used on normal
metaphase spreads and interphase nuclei. The c-MYC probe was
labeled with Texas Red and mixed with either the PNA Oligomer
Mixture (FIG. 10A) or Cot-1 DNA (FIG. 10B). For comparison FIG. 10C
shows the same experiment without any blocking agent added. Since
the c-MYC gene is located on chromosome band 8q24, a DNA probe for
the centromere of chromosome 8 was added to identify the chromosome
to which the c-MYC probe hybridizes (See discussion above with
respect to the preparation of the DNA centromere probe for
chromosome 8). The DNA for centromere 8 was labeled with
fluorescein. FIG. 10A shows both centromeres clearly and both genes
localized on chromesome 8. In the same Figure an interphase nuclei
shows two separate red and two separate green signals thereby
confirming the determination of the c-MYC gene on chromosome 8.
FIG. 10B shows the result when using Cot-1 DNA instead of the PNA
Oligomer Mixture. The green centromere signals are not visible as
Cot-1 also contains centromeric sequences and thus compete with the
DNA in binding to the centromere. FIG. 10C show the same experiment
without any blocking agent added (i.e. no PNA Oligomer Mixture and
no Cot1-PNA). Only the centromere signals (in green) can be clearly
seen. The red signals from the cMYC gene can not be seen due to the
high background staining.
[0179] The c-MYC probe and the centromere 8 DNA probe were also
hybridized to metaphase spreads of the cell line HMT3522 (Nielsen
et al., 1997) that has amplification of the c-MYC gene. The c-MYC
probe was labeled with Texas Red, the DNA probe for the centromere
of chromosome 8 was labeled with fluorescein. These probes were
mixed with either the PNA Oligomer Mixture (FIG. 10D) or Cot-1 DNA
(FIG. 10E). For comparison, FIG. 10F shows the result of the same
experiment performed without any blocking agent added. Using the
PNA Oligomer Mixture both the green centromere 8 signals and the
amplified c-MYC signals (in red) can be seen. Using Cot-1 DNA only
the red c-MYC signals can bee seen. Omitting the blocking agent
makes it impossible to discriminate between red signals and
background. [0180] Ref: Nielsen K V, Niebuhr E, Ejlertsen B,
Holstebroe S, Madsen M W , Briand P, Mouridsen H T, Bolund L.
Molecular cytogenetic analysis of a nontumorigenic human breast
epithelial cell line that eventually turns tumorigenic: Validation
of an analytical approach combining karyotyping, comparative
genomic hybridization, chromosome painting, and single-locus
fluorescence in situ hybridization. Genes Chromosomes Cancer. 1997;
20:30-37.
Example 9
Detection of Amplification of the EGFR Gene Using Suppression of
Undesired Background with the PNA Oligomer Mixture
[0181] The PNA blocking mixture was used to suppress the undesired
background staining from a EGFR probe when used on normal metaphase
spreads and interphase nuclei. The EGFR probe was prepared
essentially as described in Example 8. The procedure for in-situ
hybridization on tissue was performed essentially as described in
Example 5. The procedure for in-situ hybridization on metaphase
spreads was performed essentially described in Example 7.
Results:
[0182] The EGFR gene is located on the short arm of chromosome 7.
Therefore a DNA probe for the centromere of chromosome 7 was added
to identify the chromosome to which the EGFR probe hybridizes. The
DNA for centromere 7 was labeled with fluorescein. The EGFR probe
was labeled with Texas Red and mixed with either the PNA Oligomer
Mixture (FIG. 11A) or Cot-1 DNA (FIG. 11B). For comparison FIG. 11C
shows the result of the same experiment performed without any
blocking agent added.
[0183] FIG. 11A shows both centromeres clearly and both genes
localized on chromesome 7. FIG. 11B shows the same probes except
that Cot-1 DNA was substituted for the PNA Oligomer Mixture. The
green centromere signals are nearly invisible as Cot-1 also
contains centromeric sequences and thus compete with the DNA in
binding to the centromere. FIG. 11C shows the result of performing
the assay in the absence of any blocking agent. Only the centromere
signals (in green) can be seen clearly. The red signals from the
EGFR gene are difficult to distinguish from the high background
staining.
[0184] The EGFR probe and the centromere 7 DNA probe were also
hybridized to human lung tissue that has amplification of the EGFR
gene. The EGFR probe and the DNA probe for the centromere of
chromosome 7 were mixed with either the PNA Oligomer Mixture (FIG.
11D) or Cot-1 DNA (FIG. 11E). For comparison, FIG. 11F shows the
result of the same experiment performed without any blocking agent
added. Using the PNA Oligomer Mixture both the green centromere 7
signals and the amplified EGFR signals (in red) can be seen. Using
Cot-1 DNA only the red EGFR signals can bee seen. Omitting blocking
agent interferes marginally with signal recognition, since only a
slight increase in background staining is seen.
Example 10
Suppression of Undesired Background Using the PNA Oligomer Mixture
in Combination with a TOP2A DNA Probe and a CEN-17 PNA Probe
[0185] The PNA Oligomer Mixture was used to suppress the undesired
background staining from the TOP2A DNA probe and CEN-17 PNA probe.
This assay has been launched as a kit by DakoCytomation with the
product code no. K5333. The TOP2A DNA probe, labeled with Texas Red
and the CEN-17 PNA probe labeled with fluorescein were prepared
essentially as described in Example 4. A mixture of the PNA and DNA
probes was tested on normal metaphase spreads (FIGS. 12A, 12B and
12C), essentially as described in Example 7 and on formalin fixed,
paraffin embedded cells from the cell line MDA-361 (breast cancer
cell line with TOP2A deletion (Jarvinen T A, Tanner M, Rantanen V,
Barlund M, Borg A, Grenman S, et al. Amplification and deletion of
topoisomerase IIalpha associate with ErbB-2 amplification and
affect sensitivity to topoisomerase II inhibitor doxorubicin in
breast cancer. Am J Pathol 2000; 156: 839-4; FIGS. 12D, 12E and
12F) and a mama carcinoma with borderline amplification (FIGS. 12G,
12H and 12I) essentially as described in Example 5. The TOP2A DNA
probe and CEN-17 PNA probe (See U.S. Ser. No. 09/520,760 or U.S.
Ser. No. 09/627,796, herein incorporated by reference) were mixed
with either the PNA Oligomer Mixture (FIGS. 12A, 12D and 12G) or
Cot-1 DNA (FIGS. 12B, 12E and 12H). For comparison FIGS. 12C, 12F
and 12I show the result of performing the same assay in the absence
of any blocking agent.
Results:
[0186] FIGS. 12A, 12B and 12C demonstrates the ability for the PNA
Oligomer Mixture to block unspecific background when detecting the
TOP2A gene on normal metaphase spreads using the green CEN-17 PNA
probe as a reference. Green and red signal are clearly visible
using the PNA mixture as a blocking reagent (12A), whereas the
green signals are somewhat dimmer when using Cot-1 DNA as a
blocking reagent (12B). Specific signals cannot be distinguished
from background in the absence of any blocking reagent (12C).
[0187] FIGS. 12D, 12E and 12F demonstrates the ability of the PNA
Oligomer Mixture to block unspecific background when detecting a
TOP2A gene deletion in MDA-361 cells, using CEN-17 as a reference.
Green and red signal are clearly visible using either the PNA
Oligomer Mixture (12D) or Cot-1 DNA as a blocking reagent (12E).
Specific signals cannot be distinguished from background in the
absence of any blocking reagent (12F).
[0188] FIGS. 12G, 12H and 12I demonstrate the ability for the PNA
Oligomer Mixture to block unspecific background when determining
the ratio between the TOP2A gene and CEN-17 in a human mama
carcinoma. Green and red signal are clearly visible using either
the PNA Oligomer Mixture (12G) or Cot-1 DNA (12H) as a blocking
reagent. Specific signals cannot be distinguished from background
in the absence of any blocking reagent (12I).
Example 11
Detection of Translocation of the TEL Gene Using Suppression of
Undesired Background with the PNA Oligomer Mixture
[0189] The PNA Oligomer Mixture was used to suppress the undesired
background staining from TEL probes when used on normal metaphase
spreads and interphase nuclei. The TEL probes was prepared
essentially as described in Example 7, provided however that BAC
DNA and PAC DNA were used. The upstream clone DNA was labeled with
Texas Red and the downstream clone DNA was labeled with fluorescein
by conventional Nick translation using a monomer labeled with Texas
Red or fluorescein, as appropriate. The DNA based probes were used
at a final concentration of 50 ng/.mu.L. The procedure for
performing in-situ hybridization is essentially as described in
Example 7. The DNA probes were mixed with either the PNA Oligomer
Mixture (FIG. 13A) or Cot-1 DNA (FIG. 13B). For comparison FIG. 13C
shows the same experiment without any blocking agent added.
[0190] The TEL gene is located on chromosome band 12p13. FIG. 13A
shows normal TEL configurations on metaphases with a yellow signal
located on both chromosome 12. Two yellow signals in interphase
nuclei indicate two normal TEL loci. FIG. 13B shows the result
using Cot-1 DNA instead of the PNA Oligomer Mixture. FIG. 13C shows
the result of the same assay except that no blocking agent added.
It is not possible to identify either the red or the green signals
in the interphase nuclei and metaphase spread in the absence of a
blocking reagent.
[0191] The TEL probes were also hybridized to metaphase spreads of
the cell line REH (Drexler, H. G. (2001) The Leukemia-Lymphoma Cell
Line Facts Book. Braunschweig, Germany.) that harbors a
translocation t(12;21) (p13,q22). The assay was performed with
either the PNA Oligomer Mixture (FIG. 13D) or Cot-1 DNA (FIG. 13E).
FIG. 13D and FIG. 13E shows one green signals located on der(12)
and a red signal on der(21), indicating a split of the upstream TEL
and downstream TEL probes. This is indicative of a translocation. A
green signal on the other allele of chromosome 12 would indicate a
deletion of the upstream TEL. For comparison, FIG. 13F shows the
result of performing the same assay in the absence of any blocking
agent.
Example 12
Detection of Translocation of the E2A Gene Using Suppression of
Undesired Background with the PNA Oligomer Mixture
[0192] The PNA Oligomer Mixture was used to suppress the undesired
background staining from E2A probes when used on normal metaphase
spreads and interphase nuclei. The BCR probes was prepared
essentially as described in Example 11. In-situ hybridization was
performed essentially as described in Example 7.
[0193] The upstream E2A probe was labeled with fluorescein and the
downstream E2A probe with Texas Red. The E2A gene is located on
chromosome band 19p13. FIG. 14A show normal E2A configurations on
metaphases with a yellow signal (mixture of red and green signals)
located on both chromosome 19. Two yellow signals in interphase
nuclei indicate two normal E2A loci. FIG. 14B show the result when
the assay is performed with Cot-1 DNA instead of the PNA Oligomer
Mixture. FIG. 14C show the result when the assay is performed
without any blocking agent. It is not possible to identify neither
the red nor the green signals in the interphase nuclei and
metaphase spread.
[0194] The E2A probes were also hybridized to metaphase spreads of
the cell line 697 (Drexler, H. G. (2001) The Leukemia-Lymphoma Cell
Line Facts Book. Braunschweig, Germany) that harbors a
translocation t(1;19) (q23,p13). The E2ADNA probes were labeled as
above and mixed with either the PNA Oligomer Mixture (FIG. 14D) or
Cot-1 DNA (FIG. 14E). FIG. 14D and FIG. 14E shows one yellow signal
indicating a normal E2A allele and a red signal on der(1) while the
der(19) is lost, indicating a split of the upstream BCR and
downstream BCR probes. This result is indicative of a
translocation. For comparison FIG. 14F shows the result of
performing the assay in the absence of any blocking agent.
Example 13
Detection of Translocation of the BCR Gene Using Suppression of
Undesired Background with the PNA Oligomer Mixture
[0195] The PNA Oligomer Mixture was used to suppress the undesired
background staining from BCR probes in normal metaphase spreads and
interphase nuclei. The upstream BCR DNA probe was labeled with
fluorescein and the downstream BCR DNA probe with Texas Red. The
BCR probes were prepared essentially as described in Example 11.
However, COS DNA was also included as probe. The COS DNA was
labeled with Texas Red by conventional Nick translation using a
monomer labeled with Texas Red. The procedure for performing
in-situ hybridization was performed essentially as described in
Example 7.
[0196] These DNA probe were mixed with either the PNA Oligomer
Mixture (FIG. 15A) or Cot-1 DNA (FIG. 15B). For comparison FIG. 15C
shows the result of performing the same assay in the absence of
blocking agent.
[0197] The BCR gene is located on chromosome band 22q11. FIG. 15A
show normal BCR configurations on metaphases with a yellow signal
located on both chromosome 22. Two yellow signals in interphase
nuclei indicate two normal BCR loci. FIG. 15B show the same probes
suppressed with Cot-1 DNA. FIG. 15C show the same probes without
any blocking agent added. It is not possible to identify the red or
the green signals in the interphase nuclei and metaphase spread in
the absence of a blocking reagent.
[0198] The BCR probes were also hybridized to metaphase spreads of
the cell line BV173 (Drexler, H. G. (2001) The Leukemia-Lymphoma
Cell Line Facts Book. Braunschweig, Germany) that harbors a
translocation t(9;22) (q34,q11). The BCR probes were labeled as
above and mixed with either the PNA Oligomer Mixture (FIG. 15D) or
Cot-1 DNA (FIG. 15E). FIG. 15D and FIG. 15E shows one yellow
signal, indicating a normal BCR allele, and one green signals
located der(9) and a red signal on der(22); indicating a split of
the upstream BCR and downstream BCR probes. This result is
indicative of a translocation. An additional red signal is
indicative of an additional der(22). For comparison, FIG. 15F shows
the result of performing the same assay in the absence of blocking
agent.
Example 14
Detection of Translocation of the IGH Gene Using Suppression of
Undesired Background with the PNA Oligomer Mixture
[0199] The PNA Oligomer Mixture was used to suppress the undesired
background staining from IGH probes when used on normal metaphase
spreads and interphase nuclei. The probes were labeled with
fluorescein for the IGHV genes and with Texas Red for the IGHC
genes (Poulsen T S, Silahtaroglu A N, Gisselo C G, Gaarsdal E,
Rasmussen T, Tommerup N, Johnsen H E. Detection of illegitimate
rearrangement within the immunoglobulin locus on 14q32.3 in B-cell
malignancies using end-sequenced probes. (2001) Genes Chromosomes
Cancer, 32: 265-74), mixed with either the PNA Oligomer Mixture
(FIG. 16A) or Cot-1 DNA (FIG. 16B). The IGH probes were prepared as
described in Example 11. In-situ hybridization was performed
essentially as described in Example 11. However, in this Example
14, the pre-treatment was omitted and hybridization time was
lowered to 4 hours, thus showing the robustness of the assay.
[0200] The DNA probes were mixed with either the PNA Oligomer
Mixture (FIG. 16A) or Cot-1 DNA (FIG. 16B). For comparison FIG. 16C
shows the result of performing the same assay in the absence of
blocking agent.
[0201] The IGH genes are located on chromosome band 14q32. FIG. 16A
shows normal IGH configurations on metaphases with a yellow signal
located on both chromosome 14, band q32. FIG. 16B shows the result
when background is suppressed using Cot-1 DNA. FIG. 16C shows the
result of performing the assay in the absence of a blocking agent.
It is not possible to identify the red or the green signals in the
interphase nuclei and metaphase spread in the absence of a blocking
agent.
[0202] The IGH probes were also hybridized to metaphase spreads of
the cell line Granta 519 (Drexler, H. G. (2001) The
Leukemia-Lymphoma Cell Line Facts Book. Braunschweig, Germany) that
harbors a translocation t(11;14) (q23,q32). The IGH probes were
labeled as above and mixed with either the PNA Oligomer Mixture
(FIG. 16D) or Cot-1 DNA (FIG. 16E). FIG. 16D and FIG. 16E shows one
yellow signal located on 14q32, indicating a normal IGH
configuration on one allele and a split of the IGHV and IGHC probes
on the other allele. This result is indicative of a translocation.
For comparison FIG. 16F shows the same experiment without any
blocking agent added.
Example 15
Detection of Translocation of the IGL Gene Using Suppression of
Undesired Background with the PNA Oligomer Mixture
[0203] The PNA Oligomer Mixture was used to suppress the undesired
background staining from IGL probes when used on normal metaphase
spreads and interphase nuclei. The probes were labeled with
fluorescein for the IGLV genes and with Texas Red for the IGLC
genes (Poulsen T S, Silahtaroglu A N, Gisselo C G, Gaarsdal E,
Tommerup N, Johnsen H E. Detection of illegitimate rearrangements
within the immunoglobulin light chain loci in B-cell malignancies
using end sequenced probes. (2002) Leukemia, 16: 2148-2158), mixed
with either the PNA Oligomer Mixture (FIG. 17A) or Cot-1 DNA (FIG.
17B). The IGL probes were prepared essentially as described in
Example 11. The procedure for performing in-situ hybridization was
performed essentially as described in Example 11. However, the
hybridization time was lowered to 4 hours, thus showing the
robustness of the assay.
[0204] The DNA probes were mixed with either the PNA Oligomer
Mixture (FIG. 17A) or Cot-1 DNA (FIG. 17B). For comparison FIG. 17C
shows the result of performing the same assay in the absence of
blocking agent.
[0205] The IGL genes are located on chromosome band 22q11. FIG. 17A
show normal IGL configurations on metaphases with a yellow signal
located on both chromosome 22, band q11. FIG. 17B shows the result
of the assay when Cot-1 DNA is used. FIG. 17C shows the result of
performing the assay in the absence of a blocking agent. It is not
possible to identify the red or the green signals in the interphase
nuclei and metaphase spread in the absence of a blocking agent.
Example 16
Detection of Translocation of the IGK Gene Using Suppression of
Undesired Background with the PNA Oligomer Mixture
[0206] The PNA Oligomer Mixture was used to suppress the undesired
background staining from IGL probes when used on normal metaphase
spreads and interphase nuclei. The probes were labeled with
fluorescein for the IGKV genes and with Texas Red for the IGKC
genes (Poulsen T S, Silahtaroglu A N, Gisselo C G, Gaarsdal E,
Tommerup N, Johnsen HE. Detection of illegitimate rearrangements
within the immunoglobulin light chain loci in B-cell malignancies
using end sequenced probes. (2002) Leukemia, 16: 2148-2158). The
IGK genes are located on chromosome band 2p11. The IGK DNA probes
were prepared essentially as described in Example 11. In-situ
hybridization was performed essentially as described in Example 11,
provided however, that the hybridization time was lowered to 4
hours, thus showing the robustness of the assay.
[0207] The DNA probes were mixed with either the PNA Oligomer
Mixture (FIG. 18A) or Cot-1 DNA (FIG. 18B). For comparison, FIG.
18C shows the result of performing the assay in the absence of any
blocking agent. FIG. 18A show normal IGK configurations on
metaphases with a yellow signal located on both chromosome 2, band
p11. FIG. 18B show the same probes suppressed with Cot-1 DNA. FIG.
18C show the same probes without any blocking agent added. It is
not possible to identify neither the red nor the green signals in
the interphase nuclei and metaphase spread.
Summary of Experimental Section
[0208] The aforementioned Examples 1-16, when taken together,
demonstrate the utility of both the method for production of the
PNA Oligomer Mixture as well as for use in suppressing the binding
of detectable nucleic acid probes to randomly distributed repeat
sequence in genomic nucleic acid.
Equivalents
[0209] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those of ordinary skill in the art that various
changes in form and details may be made therein without departing
from the spirit and scope of the invention as defined by the
appended claims. Those skilled in the art will be able to
ascertain, using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed in the
scope of the claims.
REFERENCES
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Hellmann-Blumberg U, Jurka J, Labuda D, Rubin C M, Schmid C W,
Zi.cndot.tkiewicz E, Zuckerkandl E. 1996. Standardized Nomenclature
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M, Allfrey V G. 1995, Isolation of active genes containing CAG
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Evidence that most human Alu sequences were inserted in a process
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Sequence CWU 1
1
261301DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Alu-repeat consensus sequence 1ggcgggcgga ggccgggcgc
ggtggctcac gcctgtaatc ccagcacttt gggaggccga 60ggcgggcgga tcacctgagg
tcaggagttc gagaccagcc tggccaacat ggtgaaaccc 120cgtctctact
aaaaatacaa aaattagccg ggcgtggtgg cgcgcgcctg tartcccagc
180tactcgggag gctgaggcag gagaatcgct tgaacccggg aggcggaggt
tgcagtgagc 240cgagatcgcg ccactgcact ccagcctggg cracaagagc
garactccgt ctcaaaaaaa 300a 3012301DNAArtificial SequenceDescription
of Combined DNA/RNA Molecule Alu-repeat consensus sequence
2ttttttttga gacggagtyt cgctcttgty gcccaggctg gagtgcagtg gcgcgatctc
60ggctcactgc aacctccgcc tcccgggttc aagcgattct cctgcctcag cctcccgagt
120agctgggayt acaggcgcgc gccaccacgc ccggctaatt tttgtatttt
tagtagagac 180ggggtttcac catgttggcc aggctggtct gcaactcctg
acctcaggta gtccgcccgc 240ctcggcctcc caaagtgctg ggattacagg
cgtgagccac cgcgcccggc ctccgcccgc 300c 301317DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
sequence 3ggccgggcgc ggtggct 17418DNAArtificial SequenceDescription
of Combined DNA/RNA Molecule Synthetic Oligomer Sequence
4gctgggatta caggcgtg 18516DNAArtificial SequenceDescription of
Combined DNA/RNA Molecule Synthetic Oligomer Sequence 5gggaggccga
ggcggg 16621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic Oligomer Sequence 6gccaggctgg tctcgaactc c
21721DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic Oligomer Sequence 7gaaaccccgt ctctactaaa a
21816DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic Oligomer Sequence 8gccgggcgtg gtggcg 16918DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 9tagctgggat tacaggcg 181017DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 10gggaggctga ggcagga 171121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 11cctcccgggt tcaagcgatt c 211217DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 12ttgcagtgag ccgagat 171321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 13tcgactccag cctgggcgac a 211420DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 14tttttttttt tttttttttt 201517DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 15acgcaccgcg cccggcc 171618DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 16cacgcctgta atcccagc 181716DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 17cccgcctcgg cctccc 161821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 18ggagttcgag accagcctgg c 211921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 19ttttagtaga gacggggttt c 212016DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 20cgccaccacg cccggc 162118DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 21cgcctgtaat cccagcta 182217DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 22tcctgcctca gcctccc 172321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 23gaatcgcttg aacccgggag g 212417DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 24atctcggctc actgcaa 172521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 25tgtcgcccag gctggagtgc a 212620DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic Oligomer
Sequence 26aaaaaaaaaa aaaaaaaaaa 20
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