U.S. patent application number 12/505183 was filed with the patent office on 2010-03-18 for methods of pcr-based detection of "ultra short" nucleic acid sequences.
This patent application is currently assigned to Xenomics, Inc.. Invention is credited to Hovsep S. Melkonyan, Natalya Ossina, Eugene M. Shekhtman, Samuil R. Umansky.
Application Number | 20100068711 12/505183 |
Document ID | / |
Family ID | 41171077 |
Filed Date | 2010-03-18 |
United States Patent
Application |
20100068711 |
Kind Code |
A1 |
Umansky; Samuil R. ; et
al. |
March 18, 2010 |
Methods of PCR-Based Detection of "Ultra Short" Nucleic Acid
Sequences
Abstract
The present invention provides highly sensitive methods used for
diagnosing and monitoring various diseases and disorders by
detecting and analyzing "ultra short" (20-50 base pair) nucleic
acids obtained from bodily fluids.
Inventors: |
Umansky; Samuil R.;
(Princeton, NJ) ; Melkonyan; Hovsep S.;
(Princeton, NJ) ; Ossina; Natalya; (Princeton,
NJ) ; Shekhtman; Eugene M.; (Rockville, MD) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Assignee: |
Xenomics, Inc.
Monmouth Junction
NJ
|
Family ID: |
41171077 |
Appl. No.: |
12/505183 |
Filed: |
July 17, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61135364 |
Jul 18, 2008 |
|
|
|
Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 1/6846 20130101; C12Q 1/6851 20130101; C12Q 1/6806 20130101;
C12Q 2600/106 20130101; C12Q 1/6846 20130101; C12Q 2600/156
20130101; C12Q 1/6846 20130101; C12Q 1/705 20130101; C12Q 1/6806
20130101; C12Q 1/6851 20130101; C12Q 2600/158 20130101; C12Q 1/6806
20130101; C12Q 2525/186 20130101; C12Q 2565/101 20130101; C12Q
2525/204 20130101; C12Q 2525/186 20130101; C12Q 2525/204 20130101;
C12Q 2525/186 20130101; C12Q 2565/101 20130101; C12Q 2525/207
20130101; C12Q 2523/308 20130101; C12Q 2525/161 20130101; C12Q
2525/161 20130101; C12Q 2565/101 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of detecting non-host nucleic acids originating in
areas other than the urinary tract in a patient, comprising: (a)
obtaining an urine sample from said patient; and (b) analyzing said
urine sample for one or more specific sequences of non-patient
nucleic acids that are different from sequences of nucleic acids of
the patient and have crossed the kidney barrier wherein said
analyzing comprises the step of detecting said one or more specific
sequences in the nucleic acids of 20-50 nucleotides in length from
said urine sample.
2. The method of claim 1, wherein said nucleic acids are DNA.
3. The method of claim 1, wherein said nucleic acids are RNA.
4. The method of claim 1, wherein said step of analyzing said urine
sample includes a technique selected from the group consisting of
hybridization, cycling probe reaction, polymerase chain reaction,
nested polymerase chain reaction, PCR to analyze single strand
conformation polymorphisms, ligase chain reaction, strand
displacement amplification and PCR to analyze restriction fragments
length polymorphisms.
5. The method of claim 1, wherein said step of analyzing said urine
sample includes a polymerase chain reaction that uses primer pairs
sufficiently complementary to hybridize with a target sequence of
said nucleic acids.
6. The method of claim 5, wherein the target binding sequences for
said primer pairs are overlapping or immediately adjacent to each
other.
7. The method of claim 1, wherein nucleic acid degradation in said
urine sample is reduced.
8. The method of claim 7, wherein reducing nucleic acid degradation
comprises inhibiting nuclease activity by increased pH, increased
salt concentration, heat inactivation, or by treating said urine
sample with a compound selected from the group consisting of:
ethylenediaminetetraacetic acid, guanidine-HCl guanidine
isothiocyanate, N-lauroylsarcosine, and sodium dodecylsulphate.
9. The method of claim 1, wherein said urine sample has been held
in the bladder less than 12 hours.
10. The method of claim 1, wherein step (b) comprises substantially
isolating said nucleic acids in said urine sample.
11. The method of claim 10, wherein said isolation is by
precipitation or using a solid adsorbent material.
12. The method of claim 1, further comprising filtering said urine
sample to remove contaminants.
13. The method of claim 12, wherein said filtering removes nucleic
acids comprising more than about 1000 nucleotides.
14. The method of claim 12, wherein said filtering removes nucleic
acids comprising more than about 300 nucleotides.
15. The method of claim 1, wherein said analyzing comprises
quantifying said nucleic acids.
16. A method of detecting nucleic acids of a pathogen, wherein said
nucleic acids originate in areas other than the urinary tract in a
patient, comprising: (a) obtaining an urine sample from said
patient; and (b) analyzing said urine sample for one or more
specific sequences of pathogen nucleic acids that are different
from sequences of nucleic acids of the patient and are from
pathogen nucleic acids that are 20-50 nucleotides in length and
that have crossed the kidney barrier wherein said analyzing
comprises the step of detecting said one or more specific sequences
from the pathogen.
17. The method of claim 16 wherein said pathogen nucleic acids are
DNA.
18. The method of claim 16 wherein said pathogen nucleic acids are
RNA.
19. The method of claim 16 wherein the pathogen is selected from
the group consisting of a virus, a bacterium, a fungus, a
mycoplasma, and a protozoan.
20. The method of claim 16, wherein said step of analyzing said
urine sample includes a technique selected from the group
consisting of hybridization, cycling probe reaction, polymerase
chain reaction, nested polymerase chain reaction, PCR to analyze
single strand conformation polymorphisms, ligase chain reaction,
strand displacement amplification and PCR to analyze restriction
fragments length polymorphisms.
21. The method of claim 16, wherein said step of analyzing said
urine sample includes a polymerase chain reaction that uses primer
pairs sufficiently complementary to hybridize with a target
sequence of said pathogen nucleic acids of said pathogen.
22. The method of claim 21, wherein the target binding sequences
for said primer pairs are overlapping or immediately adjacent to
each other.
23. The method of claim 16, wherein nucleic acid degradation in
said urine sample is reduced.
24. The method of claim 23, wherein reducing nucleic acid
degradation comprises inhibiting nuclease activity by increased pH,
increased salt concentration, heat inactivation, or by treating
said urine sample with a compound selected from the group
consisting of: ethylenediaminetetraacetic acid, guanidine-HCl
guanidine isothiocyanate, N-lauroylsarcosine, and sodium
dodecylsulphate.
25. The method of claim 16, wherein said urine sample has been held
in the bladder less than 12 hours.
26. The method of claim 16, wherein step (b) comprises
substantially isolating said pathogen nucleic acids in said urine
sample.
27. The method of claim 26, wherein said isolation is by
precipitation or using a solid adsorbent material.
28. The method of claim 16, further comprising filtering said urine
sample to remove contaminants.
29. The method of claim 28, wherein said filtering removes nucleic
acids comprising more than about 1000 nucleotides.
30. The method of claim 28, wherein said filtering removes nucleic
acids comprising more than about 300 nucleotides.
31. The method of claim 16, wherein said analyzing comprises
quantifying said pathogen nucleic acids.
32. A method of detecting cancer in a patient comprising: (a)
obtaining an urine sample from said patient; and (b) analyzing said
urine sample for one or more specific nucleic acids of 20-50
nucleotides in length, that are indicative of cancer, and that have
crossed the kidney barrier, wherein said analyzing comprises the
step of detecting said one or more specific nucleic acids
indicative of cancer.
33. The method of claim 32, wherein said nucleic acids are DNA.
34. The method of claim 32, wherein said nucleic acids are RNA.
35. The method of claim 32, wherein said step of analyzing said
urine sample includes a technique selected from the group
consisting of hybridization, cycling probe reaction, polymerase
chain reaction, nested polymerase chain reaction, PCR to analyze
single strand conformation polymorphisms, ligase chain reaction,
strand displacement amplification and PCR to analyze restriction
fragments length polymorphisms.
36. The method of claim 32, wherein said step of analyzing said
urine sample includes a polymerase chain reaction that uses primer
pairs sufficiently complementary to hybridize with a target
sequence of said nucleic acids indicative of cancer.
37. The method of claim 36, wherein the target binding sequences
for said primer pairs are overlapping or immediately adjacent to
each other.
38. The method of claim 32, wherein nucleic acid degradation in
said urine sample is reduced.
39. The method of claim 38, wherein reducing nucleic acid
degradation comprises inhibiting nuclease activity by increased pH,
increased salt concentration, heat inactivation, or by treating
said urine sample with a compound selected from the group
consisting of: ethylenediaminetetraacetic acid, guanidine-HCl
guanidine isothiocyanate, N-lauroylsarcosine, and sodium
dodecylsulphate.
40. The method of claim 32, wherein said urine sample has been held
in the bladder less than 12 hours.
41. The method of claim 32, wherein step (b) comprises
substantially isolating said nucleic acids, indicative of cancer,
in said urine sample.
42. The method of claim 41, wherein said isolation is by
precipitation or using a solid adsorbent material.
43. The method of claim 32, further comprising filtering said urine
sample to remove contaminants.
44. The method of claim 43, wherein said filtering removes nucleic
acids comprising more than about 1000 nucleotides.
45. The method of claim 43, wherein said filtering removes nucleic
acids comprising more than about 300 nucleotides.
46. The method of claim 32, wherein said analyzing comprises
quantifying said nucleic acids, indicative of cancer.
47. A method of detecting a genetic disease or disorder in a fetus,
comprising: (a) obtaining an urine sample from a pregnant female;
and (b) analyzing said urine sample for one or more specific fetal
nucleic acids of 20-50 nucleotides in length, that have crossed the
placental and kidney barriers, wherein said analyzing comprises the
step of detecting said one or more specific fetal nucleic acids
indicative of a genetic disease.
48. The method of claim 47, wherein said nucleic acids are DNA.
49. The method of claim 47, wherein said nucleic acids are RNA.
50. The method of claim 47, wherein said step of analyzing said
urine sample includes a technique selected from the group
consisting of hybridization, cycling probe reaction, polymerase
chain reaction, nested polymerase chain reaction, PCR to analyze
single strand conformation polymorphisms, ligase chain reaction,
strand displacement amplification and PCR to analyze restriction
fragments length polymorphisms.
51. The method of claim 47, wherein said step of analyzing said
urine sample includes a polymerase chain reaction that uses primer
pairs sufficiently complementary to hybridize with a target
sequence of said nucleic acids indicative of a genetic disease or
disorder.
52. The method of claim 51, wherein the target binding sequences
for said primer pairs are overlapping or immediately adjacent to
each other.
53. The method of claim 47, wherein nucleic acid degradation in
said urine sample is reduced.
54. The method of claim 53, wherein reducing nucleic acid
degradation comprises inhibiting nuclease activity by increased pH,
increased salt concentration, heat inactivation, or by treating
said urine sample with a compound selected from the group
consisting of: ethylenediaminetetraacetic acid, guanidine-HCl
guanidine isothiocyanate, N-lauroylsarcosine, and sodium
dodecylsulphate.
55. The method of claim 47, wherein said urine sample has been held
in the bladder less than 12 hours.
56. The method of claim 47, wherein step (b) comprises
substantially isolating said nucleic acids, indicative of a genetic
disease or disorder, in said urine sample.
57. The method of claim 56, wherein said isolation is by
precipitation or using a solid adsorbent material.
58. The method of claim 47, further comprising filtering said urine
sample to remove contaminants.
59. The method of claim 58, wherein said filtering removes nucleic
acids comprising more than about 1000 nucleotides.
60. The method of claim 58, wherein said filtering removes nucleic
acids comprising more than about 300 nucleotides.
61. The method of claim 47, wherein said analyzing comprises
quantifying said nucleic acids, indicative of a genetic disease or
disorder.
62. A method of monitoring cells, tissues or organs transplanted in
areas other than the urinary tract in a patient comprising: (a)
obtaining a urine sample from said patient; and (b) analyzing said
urine sample for one or more specific sequences of non-patient
nucleic acids of 20-50 nucleotides in length from the transplanted
cells, tissues or organs that are different from sequences of
nucleic acids of the patient and have crossed the kidney barrier to
monitor the cells, tissues or organs transplanted in areas other
than the urinary tract in the patient.
63. The method of claim 62, wherein said analyzing step further
comprises quantitatively analyzing said urine sample for one or
more specific sequences of the cell-free, transrenal nucleic acids
from dying cells in the transplanted cells, tissues or organs that
are different from sequences of nucleic acids of the patient
wherein said analyzing comprises the step of detecting said one or
more specific sequences of nucleic acids from the transplanted
cells, tissues or organs in the nucleic acids that are 20-50
nucleotides in length from urine samples and have crossed the
kidney barrier to monitor rejection or acceptance of the
transplanted cells, tissues or organs.
64. The method of claim 62, wherein said nucleic acids are DNA.
65. The method of claim 62, wherein said nucleic acids are RNA.
66. The method of claim 62, wherein said step of analyzing said
urine sample includes a technique selected from the group
consisting of hybridization, cycling probe reaction, polymerase
chain reaction, nested polymerase chain reaction, PCR to analyze
single strand conformation polymorphisms, ligase chain reaction,
strand displacement amplification and PCR to analyze restriction
fragments length polymorphisms.
67. The method of claim 62, wherein said step of analyzing said
urine sample includes a polymerase chain reaction that uses primer
pairs sufficiently complementary to hybridize with a target
sequence of said nucleic acids.
68. The method of claim 67, wherein the target binding sequences
for said primer pairs are overlapping or immediately adjacent to
each other.
69. The method of claim 62, wherein nucleic acid degradation in
said urine sample is reduced.
70. The method of claim 69, wherein reducing nucleic acid
degradation comprises inhibiting nuclease activity by increased pH,
increased salt concentration, heat inactivation, or by treating
said urine sample with a compound selected from the group
consisting of: ethylenediaminetetraacetic acid, guanidine-HCl
guanidine isothiocyanate, N-lauroylsarcosine, and sodium
dodecylsulphate.
71. The method of claim 62, wherein said urine sample has been held
in the bladder less than 12 hours.
72. The method of claim 62, wherein step (b) comprises
substantially isolating said nucleic acids in said urine
sample.
73. The method of claim 72, wherein said isolation is by
precipitation or using a solid adsorbent material.
74. The method of claim 62, further comprising filtering said urine
sample to remove contaminants.
75. The method of claim 74, wherein said filtering removes nucleic
acids comprising more than about 1000 nucleotides.
76. The method of claim 74, wherein said filtering removes nucleic
acids comprising more than about 300 nucleotides.
77. The method of claim 62, wherein said analyzing comprises
quantifying said nucleic acids.
Description
RELATED APPLICATIONS
[0001] The present application claims priority to, and the benefit
of, U.S. Provisional Application No. 61/135,364, filed Jul. 18,
2008. The contents of this application is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention provides highly sensitive methods used
for diagnosing and monitoring various diseases and disorders by
detecting and analyzing "ultra short" (20-50 base pair) nucleic
acids obtained from bodily fluids.
BACKGROUND OF THE INVENTION
[0003] Cell death is an essential event in the development and
functioning of multicellular organisms. In adult organisms, cell
death plays a complementary role to mitosis in the regulation of
cell populations. The pathogenesis of numerous diseases involves
failure of tissue homeostasis which is presumed to be linked with
cytotoxic injury or loss of normal control of cell death.
[0004] There exist two major types of cell death, necrosis and
apoptosis, marked by different morphological and molecular
characteristics (Kerr et al., Br. J. Cancer. 26: 239-257, 1972;
Umansky, J. Theor. Biol. 97: 591-602, 1982; Umansky and Tomei, Adv
Pharmacol. 41: 383-407, 1997; Ameisen, Cell Death Differ. 11: 4-10,
2004; Lockshin and Zakeri, Int J Biochem Cell Biol. 36: 2405-19,
2004; and Kroemer, et al., Cell Death and Differentiation 12:
1463-1467, 2005). Necrosis is considered to be catastrophic
metabolic failure resulting directly from severe molecular and/or
structural damage and leads to inflammation and secondary damage to
surrounding cells. Apoptosis, also termed programmed cell death, is
a much more prevalent biological phenomenon than necrosis and can
be induced by specific signals such as hormones, cytokines, by
absence of specific signal such as growth or adhesion factors, or
by molecular damage that does not cause catastrophic loss of
integrity. Apoptosis is a result of an active cellular response
involving initiation of an orderly and specific cascade of
molecular events. Apoptosis leads to the appearance of distinctive
chromatin condensation and margination, nuclear fragmentation, cell
shrinkage, and membrane blebbing. Enzymatic internucleosomal
fragmentation of nuclear DNA is a hallmark of apoptosis, although
some cells die by apoptosis without internucleosomal DNA cleavage
(Umansky et al., Biochim Biophys Acta. 655: 9-17, 1981; Arends et
al., Am J. Pathol. 136: 593-608, 1990; and Zimmermann et al.,
Pharmacol Ther. 92: 57-70, 2001). Other, more rare forms of cell
death, characterized by specific morphology, for example the
so-called autophagic cell death, have also been described.
[0005] It is well known that apoptosis, or programmed cell death,
which is a major form of cell death in multicellular organisms, is
accompanied by internucleosomal fragmentation of nuclear DNA. This
DNA originates from all cells undergoing apoptosis and thus from
all tissues throughout the body. Many laboratories have
demonstrated that in humans a portion of this DNA appears in blood
(Lo et al., Ann N Y Acad. Sci. 945: 1-7, 2001; Lichtenstein et al.,
Ann N Y Acad. Sci. 945: 239-249, 2001; Taback and Hoon, Curr. Opin
Mol. Ther. 6: 273-278, 2004; and Bischoff et al., Hum Reprod
Update. 8: 493-500, 2002). It has also been shown that this
fragmented DNA crosses the kidney barrier (Transrenal DNA or
Tr-DNA) and can be detected in the urine (Botezatu et al., Clin
Chem. 46:1078-1084, 2000; Su et al., J Mol. Diagn. 6:101-107, 2004;
and Su et al., Ann N Y Acad. Sci. 1022: 81-89, 2004).
[0006] Both cell-free plasma DNA and Transrenal-DNA (Tr-DNA) have
been used as diagnostic tools when the diagnostic marker is the
presence of specific, known sequences different from bulk genomic
DNA. For example, detection of tumor-specific DNA that results from
characteristic mutations can be used for tumor diagnostics,
detection of male Y chromosome-specific sequences in urine or blood
of a pregnant woman can be used to determine the male gender of the
fetus and detection of mutations characteristic of inherited
disease can provide a tool for prenatal genetic testing (Chan and
Lo, Semin Cancer Biol. 12: 489-496, 2002; Goessl, Expert Rev Mol.
Diagn. 3: 431-442, 2003; Su et al., J Mol. Diagn. 6: 101-107, 2004;
Wataganara and Bianchi, Ann N Y Acad Sci. 1022: 90-99, 2004;
Botezatu et al., Clin Chem. 46: 1078-1084, 2000; and Ding et al.,
Proc Natl Acad Sci USA. 101: 10762-10767, 2004).
[0007] The fate of RNA from dying cells, in particular the
mechanisms of its degradation, is much less investigated. However,
it is known that fetal RNA can be detected in plasma of pregnant
women and RNA with tumor-specific mutations is detectable in plasma
of patients with different types of cancer (Tsui et al., Ann N Y
Acad. Sci. 2006; 1075:96-102; Lo and Chiu, Nat Rev Genet. 8: 71-77,
2007; and Tsang and Lo, Pathology 39: 197-207, 2007).
[0008] These specific nucleic acid biomarkers are often very short
and their concentration in body fluids is usually low, especially
if a test addresses an early stage of pregnancy or a disease. Thus,
new methods are needed to detect these sensitive biomarkers. The
present invention addresses this need in the art.
SUMMARY OF THE INVENTION
[0009] The present invention provides a method of detecting
non-host nucleic acids originating in areas other than the urinary
tract in a patient, including obtaining an urine sample from the
patient; and analyzing the urine sample for one or more specific
sequences of non-patient nucleic acids that are different from
sequences of nucleic acids of the patient and have crossed the
kidney barrier wherein the analyzing comprises the step of
detecting said one or more specific sequences in the nucleic acids
of 20-50 nucleotides in length from the urine sample
[0010] The present invention also provides a method of detecting
nucleic acids of a pathogen, where the nucleic acids originate in
areas other than the urinary tract in a patient, including
obtaining an urine sample from the patient; and analyzing the urine
sample for one or more specific sequences of pathogen nucleic acids
that are different from sequences of nucleic acids of the patient
and are from pathogen nucleic acids that are 20-50 nucleotides in
length and that have crossed the kidney barrier where the analyzing
includes the step of detecting the one or more specific sequences
from the pathogen.
[0011] The present invention also provides a method of detecting
cancer in a patient including obtaining an urine sample from the
patient; and analyzing the urine sample for one or more specific
nucleic acids of 20-50 nucleotides in length, that are indicative
of cancer, and that have crossed the kidney barrier, where the
analyzing includes the step of detecting the one or more specific
nucleic acids indicative of cancer.
[0012] The present invention also provides a method of detecting a
genetic disease or disorder in a fetus, including obtaining an
urine sample from a pregnant female; and analyzing the urine sample
for one or more specific fetal nucleic acids of 20-50 nucleotides
in length, that have crossed the placental and kidney barriers,
where the analyzing includes the step of detecting the one or more
specific fetal nucleic acids indicative of a genetic disease.
[0013] The present invention also provides a method of monitoring
cells, tissues or organs transplanted in areas other than the
urinary tract in a patient including obtaining a urine sample from
the patient; and analyzing the urine sample for one or more
specific sequences of non-patient nucleic acids of 20-50
nucleotides in length from the transplanted cells, tissues or
organs that are different from sequences of nucleic acids of the
patient and have crossed the kidney barrier to monitor the cells,
tissues or organs transplanted in areas other than the urinary
tract in the patient. The analyzing step can further include
quantitatively analyzing the urine sample for one or more specific
sequences of the cell-free, transrenal nucleic acids from dying
cells in the transplanted cells, tissues or organs that are
different from sequences of nucleic acids of the patient wherein
the analyzing comprises the step of detecting said one or more
specific sequences of nucleic acids from the transplanted cells,
tissues or organs in the nucleic acids that are 20-50 nucleotides
in length from urine samples and have crossed the kidney barrier to
monitor rejection or acceptance of the transplanted cells, tissues
or organs.
[0014] The nucleic acids can be DNA or RNA. The pathogen can be a
virus, a bacterium, a fungus, a mycoplasma, or a protozoan.
[0015] The step of analyzing the urine sample can include
hybridization, cycling probe reaction, polymerase chain reaction,
nested polymerase chain reaction, PCR to analyze single strand
conformation polymorphisms, ligase chain reaction, strand
displacement amplification or PCR to analyze restriction fragments
length polymorphisms.
[0016] The step of analyzing the urine sample can include a
polymerase chain reaction that uses primer pairs sufficiently
complementary to hybridize with a target sequence of the nucleic
acids of interest. Preferably, the target binding sequences for
said primer pairs are overlapping or immediately adjacent to each
other.
[0017] The nucleic acid degradation in the urine sample can be
reduced. Reducing the nucleic acid degradation can include
inhibiting nuclease activity by increased pH, increased salt
concentration, heat inactivation, or by treating said urine sample
with a compound selected from the group consisting of:
ethylenediaminetetraacetic acid, guanidine-HCl guanidine
isothiocyanate, N-lauroylsarcosine, or sodium dodecylsulphate.
Preferably, the urine sample has been held in the bladder less than
12 hours.
[0018] The step of analyzing the urine sample can further include
substantially isolating the nucleic acids of interest in said urine
sample. Preferably, the isolation can be by precipitation or using
a solid adsorbent material. Preferably, the isolation is by
adsorption of the nucleic acids on a resin.
[0019] In some embodiments, the methods further comprise filtering
the urine sample to remove contaminants. Preferably, the filtering
removes nucleic acids comprising more than about 1000 nucleotides
or more than about 300 nucleotides.
[0020] In some embodiments, the analyzing comprises quantifying
said nucleic acids of interest
[0021] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0022] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a schematic showing the dependence of PCR
sensitivity on amplicon size.
[0024] FIG. 2 is a schematic showing FRET-based PCR with primers
labeled with fluorophores near the 3'-end.
[0025] FIG. 3 is a graph showing dependence of fluorescence signal
on the distance between the donor and acceptor fluorophores.
[0026] FIG. 4, Panel A, is a graph showing detection of
EBV-specific sequences by FRET-based real-time PCR. Panel B shows a
calibration curve.
[0027] FIG. 5 is a graph showing amplification of a 36-bp SRY
target in labeled-primer FRET real-time PCR assay.
[0028] FIG. 6 is a graph showing fetal gender detection by
labeled-primer FRET real-time PCR assay of DNA extracted from urine
of pregnant women.
[0029] FIG. 7 is a schematic showing FRET-based PCR with primers
having tails and labeled with fluorophores near the 5'-end.
[0030] FIG. 8 is a schematic showing one version of a the two-stage
single-tube real-time PCR assay.
[0031] FIG. 9, Panel A, is a graph showing detection of M
tuberculosis IS6110 sequences (39-bp target) by the two-stage
single-tube real-time PCR assay of FIG. 8. Panel B shows detection
of M tuberculosis Tr-DNA in the urine samples from patients with
pulmonary tuberculosis and non-infected controls.
[0032] FIG. 10 is a schematic showing a second version of a the
two-stage single-tube real-time PCR assay.
[0033] FIG. 11 is a graph showing amplification of SRY standards
(25-bp target) by the two-stage single-tube real-time PCR assay of
FIG. 10.
[0034] FIG. 12 is a graph showing fetal gender detection of DNA
extracted from urine of pregnant women by the two-stage single-tube
real-time PCR assay of FIG. 10.
[0035] FIG. 13 is a graph showing a detection comparison of Y
chromosome-specific TSPY sequences (43-bp target) in urinary DNA
purified by Q-Sepharose and silica methods by the two-stage
single-tube real-time PCR assay of FIG. 8.
[0036] FIG. 14 is a photograph of a silica gel showing separation
of high/medium and low molecular weight urinary nucleic acids based
on their differential retention by silica in the presence of
ethanol.
[0037] FIG. 15 is a photograph of a silica gel showing comparing
abundance of MTB-specific templates in fractionated nucleic acids
purified from the urine of a patient with active pulmonary
tuberculosis.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention provides various methods providing a
significant increase in sensitivity for analyzing of cell-free,
ultra short (20-50 base pairs), nucleic acids obtained from a
bodily fluid.
[0039] Currently, the most sensitive methods for detection of
specific DNA or RNA sequences are based on PCR or other
amplification techniques. Analysis of cell-free DNA isolated from
plasma, urine, and stool by conventional silica-based methods
demonstrated that DNA fragments originated from dying fetal or
tumor cells are relatively small, around 150 nucleotides (Chan et
al., Cancer Res. 63: 2028-2032, 2003; Botezatu et al., Clin Chem.
46:1078-1084, 2000; Su et al., J Mol. Diagn. 6: 101-107, 2004; and
Diehl et al., Gastroenterology 135: 489-98, 2008). However, it is
not always recognized that apoptotic DNA fragmentation is random,
and that, consequently, a target sequence of interest is located in
DNA fragments that have been cleaved out in a variety of ways. In
fact, in any given population of DNA fragments produced by random
cleavage the probability of any given target sequence surviving
intact to be available for use as PCR template is inversely
proportional to the length of such target sequence, as illustrated
in FIG. 1. Different lines in FIG. 1 represent short DNA fragments
(the 157 base pairs fragments are given as an example) generated by
random cleavage of K-RAS in the area of codon 12. Bold solid line
represents the only fragment that is amplified by primers designed
for the 157-bp amplicon. Thin solid lines represent a subset of DNA
fragments amplified by the pair of primers targeting the 87-bp
amplicon. Dashed lines are DNA fragments that are not amplified by
either set of primers. For example, Su et al., utilize two sets of
primers designed for detection of mutant K-RAS (Su, et al., Ann.
New York Acad. Sci. 1022: 81-89, 2004).
[0040] Most longer targets are out of frame determined by the
respective primers. Thus, the advantage of a shorter target size is
very significant, especially when target sizes are close to the
average fragment length. DNA isolated from urine with a standard
silica-based method consists of two fractions, high molecular
weight DNA, which originates from shed cells and low molecular
weight (150-250 base pair) fraction of Tr-DNA (Botezatu et al.,
Clin Chem. 46: 1078-1084, 2000; and Su et al., J Mol. Diagn. 6:
101-107, 2004).
[0041] Furthermore, the application of newly developed technique
for isolation of cell-free nucleic acids from body fluids to the
isolation of transrenal nucleic acids has revealed the presence in
urine of DNA and RNA fragments much shorter than 150 base pairs
(U.S. Patent Application Publication No. 20080139801). If these
fragments also represent transrenal nucleic acids, amplification of
"ultra short" PCR targets or other techniques capable to detect
very short nucleic acid sequences with sufficient specificity can
significantly increase sensitivity of tests based on analysis of
cell-free nucleic acids in urine and other body fluids.
[0042] Another reason to aim for "ultra short" targets is the
possible presence of single-strand breaks (nicks) in cell-free DNA
fragments. In the PCR reaction nucleic acid fragments are used as
templates in their single-stranded form, thus further reducing
their effective length if cell-free DNA fragments in plasma and
Tr-DNA are nicked. These considerations necessitate a PCR assay
design capable of detecting exceptionally short target sequences.
All considerations discussed above are also applicable any
situation when randomly degraded short DNA fragments must be
analyzed, e.g. DNA from paraffin-embedded tissues, forensic, or
paleontology samples.
[0043] Several approaches were used to design primers and probes
for detection of ultra short DNA targets by conventional and real
time PCR and are described in detail herein. Data obtained with
primers/probe sets, designed for detection of ultra short DNA
targets (20-50 base pairs), demonstrate: (i) the presence in the
urine of DNA and RNA fragments, which are much shorter than those
described earlier; (ii) the presence in this low molecular weight
nucleic acid fragments of sequences that originated in tissues
located outside the urinary system, which means that they have
crossed the kidney barrier transrenal NA (Tr-NA); (iii) much higher
sensitivity of Tr-DNA-based tests when ultra short DNA targets are
detected compared to conventional PCR target size.
[0044] Detection and analysis of ultra short (20-50 base pairs) DNA
targets in bodily fluids can significantly increase sensitivity of
tests based on analysis of Tr-DNA and cell-free DNA fragments from
other bodily fluids. The most commonly used quantitative PCR (qPCR)
approach is the Real-Time TaqMan PCR system, which involves the use
of 3 target sequence-specific components: 2 primers and 1 labeled
TaqMan probe. This standard assay is suitable for amplicons no
shorter than about 50 bases, the minimum size being limited by the
combined footprint length of the 3 sequence-specific components.
There exist a number of alternative qPCR approaches that eliminate
the need for a separate TaqMan probe by using various forms of
labeled primers, thus allowing for correspondingly shorter targets.
However, the elimination of 1 of the 3 sequence-specific components
is likely to reduce target specificity of the assay. The present
invention addresses target specificity vs. minimum target length by
developing novel labeled-primer qPCR assays and modified TaqMan
assays. Several new approaches for designing primers or
primers/probe sets for detection and quantitative analysis of ultra
short sequences by Real-Time PCR are provided. Using these
techniques, it has been demonstrated that: (i) analysis of ultra
short DNA targets significantly increases sensitivity of the
Tr-DNA-based tests; (ii) short DNA fragments (30-150 base pairs)
contain human and pathogen Tr-DNA sequences; (iii) larger Tr-DNA
fragments are also more effectively detected by primers for ultra
short DNA targets, most likely due to the presence of single-strand
breaks in those DNA fragments.
[0045] The invention relates to a significant increase in
sensitivity of tests based on analysis of cell-free nucleic acids
obtained from a bodily fluid selected from, but not limited to, the
group comprising blood, blood plasma, serum, lymph, interstitial
fluid, urine, saliva, sweat, cerebrospinal fluid and others.
Preferably, the bodily fluid is obtained non-invasively.
[0046] The present invention can be used for many applications,
including, but not limited to, analyzing for the presence of
pathogen nucleic acids, detecting the presence of nucleic acids
indicative of cancer, detecting the presence of nucleic acids
indicative of a genetic disease in a fetus, analyzing for the
presence of fetal nucleic acids, analyzing for the presence of
specific host and specific non-host nucleic acid sequences, for
analyzing the form and degree of methylation of a target nucleic
acid, detection of single nucleotide polymorphisms and forensic
analysis.
[0047] The present invention provides methods of detecting nucleic
acids of a pathogen, wherein said nucleic acids originate in areas
other than the urinary tract in a patient, comprising: (a)
obtaining an urine sample from said patient; and (b) analyzing said
urine sample for one or more specific sequences of pathogen nucleic
acids that are different from sequences of nucleic acids of the
patient and are from pathogen nucleic acids that are 20-50
nucleotides in length and that have crossed the kidney barrier,
wherein said analyzing comprises the step of detecting said one or
more specific sequences from the pathogen.
[0048] A pathogen is a biological agent that can cause disease to
its host. A synonym of pathogen is "infectious agent". The term
"pathogen" is most often used for agents that disrupt the normal
physiology of a multicellular organism.
[0049] Infection is the invasion and multiplication of
microorganisms in body tissues, which may be clinically unapparent
or result in local cellular injury due to competitive metabolism,
toxins, intracellular replication or antigen antibody response.
[0050] The pathogen nucleic acids can be DNA or RNA. The pathogen
is selected from the group consisting of a virus, a bacterium, a
fungus, a mycoplasma, and a protozoan.
[0051] The methods of the invention are applicable to all viral
pathogenic agents, including RNA, DNA, episomal, and integrative
viruses. They also apply to recombinant viruses, such as the
adenoviruses or lentiviruses utilized in gene therapy. Examples of
infectious virus include: Retroviridae (e.g., human
immunodeficiency viruses, such as HIV-1, also referred to as
HTLV-III, LAV or HTLV-III/LAV, or HIV-111; and other isolates, such
as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus;
enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses);
Calciviridae (e.g., strains that cause gastroenteritis);
Togaviridae (e.g., equine encephalitis viruses, rubella viruses);
Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow
fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae
(e.g., vesicular stomatitis viruses, rabies viruses); Fidoviridae
(e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza
viruses, mumps virus, measles virus, respiratory syncytial virus);
Orthomyxoviridae (e.g., influenza viruses); Buiigaviridae (e.g.,
Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses);
Arenaviridae (hemorrhagic fever virus); Reoviridae (e.g.,
reoviruses, orbiviruses and rotaviruses); Birnaviridae;
Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses);
Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae
(most adenoviruses); Herperviridae (herpes simplex virus (HSV) 1
and 2, varicella zoster virus, cytomegalovirus (CMV), herpes
viruses); Poxyiridae (variola virsues, vaccina viruses, pox
viruses); andlridoviridae (e.g., African swine fever virus); and
unclassified viruses (e.g., the etiological agents of Spongiform
encephalopathies, the agent of delta hepatitides (thought to be a
defective satellite of hepatitis B virus), the agents of non-A,
non-B hepatitis (class 1-internally transmitted; class
2-parenterally transmitted (i.e., Hepatitis C); Norwalk and related
viruses, and astroviruses).
[0052] The methods of the invention are applicable to all bacterial
pathogenic agents. Examples of infectious bacteria include:
Helicobacter pyloris, Borrelia (e.g., Borrelia afzelii, Borrelia
anserine, Borrelia burgdorferi, Borrelia garinii, Borrelia hermsii,
Borrelia recurrentis, Borrelia valaisiana, and Borrelia vincentii);
Rickettsia (e.g., Rickettsia felis, Rickettsia prowazekii,
Rickettsia rickettsii, Rickettsia typhi, Rickettsia conorii,
Rickettsia africae, or Rickettsia akari); Legionella pneumophilia,
Mycobacteria sps (e.g., M. tuberculosis, M. avium, M.
Intracellulare, M. kansaii M. gordonae), Staphylococcus aureus,
Neisseria gonorrhoeae, Neisseria meningitidis,
Listeriamonocytogenes, Streptococcuspyogenes (Group A
Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.),
Streptococcuspneumoniae, pathogenic Campylobacter sp.,
Enterococcussp., Haemophilus influenzae, Bacillus antracis,
corynebacterium diphtheriae, corynebacteium sp., Erysipelothrix
rhusiopathiae, Clostridium penfiingers, Clostridium tetani,
Enterobacter erogenes, Klebsiellapneuomiae, Pasturella multicoda,
Bacteroides sp., Fusobacterium nucleatum,
Sreptobacillusmoniliformis, Treponema pallidium, Treponemapertenue,
Leptospira, and Actinomeycesisraelli.
[0053] Examples of infectious fungi include: Cryptococcus
neoformans, Histoplasmacapsulatum, Coccidioides immitis,
Blastomyces dermatitidis, Chlamydia trachomatis, Candidaalbicans.
Other infectious organisms (i.e., protists) include: Plasmodium
falciparum and Toxoplasma gondii.
[0054] In some preferred embodiments, the non-viral pathogen can be
Helicobacter pylori, Bacillus anthracis, Plasmodium species or
Leishmania species.
[0055] The step of analyzing for the presence of said pathogen
nucleic acid sequence of 20-50 nucleotides in length can be
performed using one or more of a variety of techniques, including,
but not limited to, hybridization, cycling probe reaction,
polymerase chain reaction, nested polymerase chain reaction,
polymerase chain reaction-single strand conformation polymorphism,
ligase chain reaction, strand displacement amplification and
restriction fragments length polymorphism. Preferably, the analysis
step also includes FRET-dependent fluorescence detection.
Preferably, the step of performing the polymerase chain reaction
can comprise using primers with binding sites which are either
immediately adjacent to each other on the target sequence or
slightly overlapping (having no intervening sequences between the
primer binding sites).
[0056] The present invention further encompasses methods having the
step of reducing DNA degradation in said urine sample, which in one
embodiment encompasses treatment with a compound selected from the
group comprising: ethylenediaminetetraacetic acid, guanidine-HCl,
Guanidine isothiocyanate, N-lauroylsarcosine, and
Na-dodecylsulphate. DNA degradation can further be reduced by
taking a urine sample that has been held in the bladder less than
12 hours.
[0057] In one embodiment, it is beneficial to substantially isolate
said nucleic acid sequence prior to assaying the urine for the
presence of a pathogen nucleic acid sequence, that has crossed the
kidney barrier. In alternate embodiments, the nucleic acid sequence
is substantially isolated by precipitation or by treatment with a
solid adsorbent material. In another embodiment, the urine sample
is filtered to remove contaminants, and, in a specific embodiment,
the filtering removes DNA comprising more than about 1000
nucleotides. Preferably, the filtering removes DNA comprising more
than about 300 nucleotides.
[0058] Further encompassed by the present invention is a diagnostic
kit for detecting pathogen in the urine, comprising: reagents to
facilitate the isolation of DNA of 20-50 nucleotides in length from
urine; reagents to facilitate amplification of DNA of 20-50
nucleotides in length by the polymerase chain reaction; a heat
stable DNA polymerase; and an oligodeoxynucleotide specific for a
pathogen nucleic acid sequence.
[0059] The present invention provides methods of detecting cancer
in a patient, comprising: providing a urine sample from a patient;
and analyzing said urine sample for a nucleic acid sequence of
20-50 nucleotides in length, indicative of cancer, that has crossed
the kidney barrier.
[0060] In one embodiment, analyzing for the presence of said
nucleic acid sequence comprises amplifying said nucleic acid
sequence indicative of cancer. In another specific embodiment, said
analyzing comprises quantifying the number of copies of said
nucleic acid sequence. In one embodiment said nucleic acid sequence
contains an anomaly indicative of colon cancer. In another
embodiment, said nucleic acid sequence contains mutant K-ras
DNA.
[0061] Cancer includes solid tumors, as well as, hematologic tumors
and/or malignancies. Various cancers to be treated include but are
not limited to breast cancer, lung cancer, colorectal cancer,
pancreatic cancer, ovarian cancer, prostate cancer, renal
carcinoma, hepatoma, brain cancer, melanoma, multiple myeloma,
lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood
lymphomas, and lymphomas of lymphocytic and cutaneous origin,
leukemia, childhood leukemia, hairy-cell leukemia, acute
lymphocytic leukemia, acute myelocytic leukemia, chronic
lymphocytic leukemia, chronic myelocytic leukemia, chronic
myelogenous leukemia, and mast cell leukemia, myeloid neoplasms,
mast cell neoplasms, hematologic tumor, and lymphoid tumor,
including metastatic lesions in other tissues or organs distant
from the primary tumor site. Cancers to be treated include but are
not limited to sarcoma, carcinoma, and adenocarcinoma.
[0062] The step of analyzing for the presence of said nucleic acid
sequence of 20-50 nucleotides in length can be performed using one
or more of a variety of techniques, including, but not limited to,
hybridization, cycling probe reaction, polymerase chain reaction,
nested polymerase chain reaction, polymerase chain reaction-single
strand conformation polymorphism, ligase chain reaction, strand
displacement amplification and restriction fragments length
polymorphism. Preferably, the analysis step also includes
FRET-dependent fluorescence detection. Preferably, the step of
performing the polymerase chain reaction can comprise using primers
with binding sites which are either immediately adjacent to each
other on the target sequence or slightly overlapping (having no
intervening sequences between the primer binding sites).
[0063] The present invention further encompasses methods having the
step of reducing DNA degradation in said urine sample, which in one
embodiment encompasses treatment with a compound selected from the
group comprising: ethylenediaminetetraacetic acid, guanidine-HCl,
Guanidine isothiocyanate, N-lauroylsarcosine, and
Na-dodecylsulphate. DNA degradation can further be reduced by
taking a urine sample that has been held in the bladder less than
12 hours.
[0064] In one embodiment, it is beneficial to substantially isolate
said nucleic acid sequence prior to assaying the urine for the
presence of a nucleic acid sequence, indicative of cancer, that has
crossed the kidney barrier. In alternate embodiments, the nucleic
acid sequence is substantially isolated by precipitation or by
treatment with a solid adsorbent material. In another embodiment,
the urine sample is filtered to remove contaminants, and, in a
specific embodiment, the filtering removes DNA comprising more than
about 1000 nucleotides. Preferably, the filtering removes DNA
comprising more than about 300 nucleotides.
[0065] In yet another embodiment, a method of monitoring cancer
treatment in a patient is encompassed, comprising: providing a
urine sample from a patient; and analyzing said urine sample for
the quantity of a nucleic acid sequence of 20-50 nucleotides in
length, indicative of cancer, that has crossed the kidney
barrier.
[0066] Further encompassed by the present invention is a diagnostic
kit for detecting a genetic mutation indicative of cancer in the
DNA of a patient, comprising: reagents to facilitate the isolation
of DNA of 20-50 nucleotides in length from urine; reagents to
facilitate amplification of DNA of 20-50 nucleotides in length by
the polymerase chain reaction; a heat stable DNA polymerase; and an
oligodeoxynucleotide specific for a sequence only occurring in a
genetic mutation characteristic of cancer.
[0067] The present invention provides methods of analyzing a
fragment of fetal DNA of 20-50 nucleotides in length that has
crossed the placental and kidney barriers, comprising: obtaining a
urine sample, suspected of containing fetal polymeric transrenal
nucleic acids, from a pregnant female; and assaying for the
presence of said fetal polymeric DNA of 20-50 nucleotides in length
in said urine sample.
[0068] In one embodiment of the present invention, the presence of
the particular unique fetal DNA of 20-50 nucleotides in length
sequence is indicative of a genetic disease.
[0069] The target fetal DNA sequence can be, for example, a
sequence that is present only on the Y chromosome. The step of
assaying for the presence of unique fetal DNA sequence of 20-50
nucleotides in length can be performed using one or more of a
variety of techniques, including, but not limited to,
hybridization, cycling probe reaction, cleavage product detection,
polymerase chain reaction, nested polymerase chain reaction,
polymerase chain reaction-single strand conformation polymorphism,
ligase chain reaction, strand displacement amplification and
restriction fragments length polymorphism. Preferably, the analysis
step also includes FRET-dependent fluorescence detection. The step
of performing the polymerase chain reaction can comprise using
primers substantially complementary to a portion of the unique
fetal DNA sequence, and the unique fetal DNA sequence can be a
sequence that is present in the paternal genome and not present in
the maternal genome. Preferably, the step of performing the
polymerase chain reaction can comprise using primers with binding
sites which are either immediately adjacent to each other on the
target sequence or slightly overlapping (having no intervening
sequences between the primer binding sites).
[0070] The present invention further encompasses methods having the
step of reducing DNA degradation in the urine sample. Reducing DNA
degradation can be by treatment with compounds selected from the
group consisting of: ethylenediaminetetraacetic acid,
guanidine-HCl, Guanidine isothiocyanate, N-lauroylsarcosine, and
Na-dodecylsulphate. DNA degradation can further be reduced by
taking a urine sample that has been held in the bladder less than
12 hours.
[0071] The present invention encompasses methods where DNA in the
urine sample is substantially isolated prior to assaying for the
presence of a unique fetal DNA sequence in the urine sample.
Substantial isolation can be by, but is not limited to,
precipitation and adsorption on a resin.
[0072] In some cases, it can be desirable to filter the urine
sample to remove contaminating nucleic acids before assaying. In a
specific embodiment, the filtering removes DNA comprising more than
about 1000 nucleotides. Preferably, the filtering removes DNA
comprising more than about 300 nucleotides.
[0073] The present invention further encompasses a method of
determining the sex of a fetus, comprising: obtaining a urine
sample, suspected of containing fetal male DNA of 20-50 nucleotides
in length, from a pregnant female; amplifying a portion of the male
DNA of 20-50 nucleotides in length present in the urine sample by
the polymerase chain reaction, using an oligodeoxynucleotide primer
having sequences specific to a portion of the Y chromosome, to
produce amplified DNA; and detecting the presence of the amplified
DNA.
[0074] The present invention also encompasses a diagnostic kit for
detecting the presence of human male fetal DNA of 20-50 nucleotides
in length in maternal urine, comprising: reagents to facilitate the
isolation of DNA from urine; reagents to facilitate amplification
of DNA by the polymerase chain reaction; a heat stable DNA
polymerase; and an oligodeoxynucleotide specific for a sequence
only occurring on the Y chromosome.
[0075] The present invention also encompasses methods of analyzing
a target nucleic acid sequence of 20-50 nucleotides in length in
urine, comprising: providing a urine sample; and assaying the urine
sample for the presence of a target nucleic acid sequence of 20-50
nucleotides in length that has crossed the kidney barrier.
[0076] In one embodiment, the target nucleic acid sequence of 20-50
nucleotides in length comprises an altered gene sequence, and that
altered gene sequence can comprise a modification occurring in
tumor cells in specific. The target nucleic acid sequence of 20-50
nucleotides in length can be a host nucleic acid or a non-host
nucleic acid.
[0077] The target nucleic acid can also be a single nucleotide
polymorphism (SNP), which is a DNA sequence variation occurring
when a single nucleotide in the genome (or other shared sequence)
differs between members of a species (or between paired chromosomes
in an individual). The nucleic acid sequence comprising the SNP can
be any length. Preferably, the nucleic acid sequence comprising the
SNP is less than 50 nucleotides. More preferably, the nucleic acid
sequence comprising the SNP is between 20-50 nucleotides in length.
The nucleic acid sequence comprising the SNP can be a host nucleic
acid or a non-host nucleic acid.
[0078] The present invention also encompasses methods of analyzing
a target nucleic acid sequence of 20-50 nucleotides in length in
any bodily fluid or tissue to perform forensic analysis,
comprising: providing a fluid or tissue sample; and assaying the
fluid or tissue sample for the presence of a target nucleic acid
sequence of 20-50 nucleotides in length that has crossed the kidney
barrier. The method can further comprise quantitative analysis of
said target nucleic acid sequence. Preferably, the method can
further comprise the sequencing of said target nucleic acid
sequence using any method known in the art.
[0079] The step of assaying for the presence of a target DNA
sequence can be selected from the group consisting of
hybridization, cycling probe reaction, polymerase chain reaction,
nested polymerase chain reaction, polymerase chain reaction-single
strand conformation polymorphism, ligase chain reaction, strand
displacement amplification and restriction fragments length
polymorphism. The step of assaying for the presence of a target DNA
sequence can comprise techniques for amplifying the target DNA.
Preferably, the analysis step also includes FRET-dependent
fluorescence detection. Preferably, the step of performing the
polymerase chain reaction can comprise using primers with binding
sites which are either immediately adjacent to each other on the
target sequence or slightly overlapping (having no intervening
sequences between the primer binding sites).
[0080] The present invention further encompasses methods having the
step of reducing DNA degradation in the urine sample prior to
assaying the urine sample for the presence of a target DNA sequence
that has crossed the kidney barrier. Reducing DNA degradation can
be by treatment with compounds selected from the group consisting
of: ethylenediaminetetraacetic acid, guanidine-HCl, Guanidine
isothiocyanate, N-lauroylsarcosine, and Na-dodecylsulphate. DNA
degradation can further be reduced by taking a urine sample that
has been held in the bladder less than 12 hours.
[0081] The present invention encompasses methods where DNA in the
urine sample is substantially isolated prior to assaying for the
presence of a target DNA sequence that has crossed the kidney
barrier. Substantial isolation can be by, but is not limited to,
precipitation and adsorption on a resin. In some cases, it is
desirable to filter the urine sample to remove contaminating
nucleic acids before assaying for the presence of a target DNA
sequence that has crossed the kidney barrier. In a specific
embodiment, the filtering removes DNA comprising more than about
1000 nucleotides. Preferably, the filtering removes DNA comprising
more than about 300 nucleotides.
[0082] The present invention also encompasses methods of analyzing
a target nucleic acid sequence in urine, comprising: providing a
urine sample, suspected of containing DNA that has crossed the
kidney barrier, from a patient; amplifying a target DNA sequence in
the DNA that has crossed the kidney barrier, comprising using a
primer substantially complementary to a portion of the target DNA
sequence that does not occur in cells of the urinary tract of the
patient, to make amplified target DNA; and detecting the presence
of the amplified target DNA. Amplification can comprise performing
a polymerase chain reaction. The target DNA sequence can comprise
an altered gene sequence, such as a modification occurring in tumor
cells.
[0083] The present invention provides methods of monitoring
transplanted material in a patient, comprising: providing a urine
sample suspected of containing nucleic acid from transplanted
material; and analyzing said urine sample for a nucleic acid
sequence of 20-50 nucleotides in length that has crossed the kidney
barrier and that was not present in the patient prior to
transplantation. In a specific embodiment, the nucleic acid
sequence of 20-50 nucleotides in length is not present in cells of
the urinary tract of said patient.
[0084] In a specific embodiment, the analyzing comprises amplifying
said nucleic acid sequence of 20-50 nucleotides in length with a
primer substantially complementary to a part of said nucleic acid
sequence that does not occur in cells of the urinary tract of the
patient, to make amplified target DNA, and detecting the presence
of said amplified target DNA. More specifically, the amplifying can
comprise performing a polymerase chain reaction. Preferably, the
analysis step also includes FRET-dependent fluorescence detection.
Preferably, the step of performing the polymerase chain reaction
can comprise using primers with binding sites which are either
immediately adjacent to each other on the target sequence or
slightly overlapping (having no intervening sequences between the
primer binding sites).
[0085] In another specific embodiment is included the additional
step of reducing DNA degradation in said urine sample, which can be
performed in any way known, but, without limitation, includes
situations wherein reducing DNA degradation is by treatment with a
compound selected from the group consisting of:
ethylenediaminetetraacetic acid, guanidine-HCl, Guanidine
isothiocyanate, N-lauroylsarcosine, and Na-dodecylsulphate. DNA
degradation can further be reduced by taking a urine sample that
has been held in the bladder less than 12 hours.
[0086] It is desirable in some embodiments to substantially isolate
said nucleic acid sequence. In alternate embodiments, the nucleic
acid sequence is substantially isolated by precipitation, and/or by
adsorption on a resin. Additionally, one can filter the urine
sample to remove contaminants. In a specific embodiment, this
filtering removes DNA comprising more than about 1000
nucleotides.
[0087] Further encompassed by the present invention is a diagnostic
kit for detecting DNA from a transplanted material in the urine of
a patient, comprising: reagents to facilitate the isolation of DNA
of 20-50 nucleotides in length from urine; reagents to facilitate
amplification of DNA of 20-50 nucleotides in length by the
polymerase chain reaction; a heat stable DNA polymerase; and an
oligodeoxynucleotide specific for a sequence that occurs in the
transplanted material, and did not occur in the patient prior to
transplantation.
[0088] The present invention provides methods of analyzing for the
presence of specific fetal ultra short nucleic acid sequences or
ultra short nucleic acid modifications by detecting specific fetal
nucleic acid sequences in bodily fluids. Preferably, the nucleic
acid sequences have crossed the placental and kidney barriers and
are present in maternal urine. The methods generally involve the
steps of obtaining a urine sample from a pregnant woman and
subjecting the material to a method of detecting a specific fetal
ultra short nucleic acid sequence or modification of interest. In
one embodiment, the method further encompasses substantially
purifying nucleic acids present in the urine sample prior to
detecting the specific ultra short nucleic acid sequence or
modification of interest. These methods have a variety of
diagnostic applications, including the determination of fetus sex
and the identification of fetal genetic diseases, such as those
inherited from the father for various purposes, including
determinations of paternity.
[0089] The inventions described herein can be used, for example, to
diagnose any of the more than 3000 genetic diseases currently known
or to be identified (e.g. hemophilias, thalassemias, Duchenne
muscular dystrophy, Huntington's disease, Alzheimer's disease and
cystic fibrosis). Any genetic disease for which the mutation(s) or
other modification(s) and the surrounding nucleotide sequence is
known can be identified by methods of the present invention. Some
diseases may be linked to known variations in methylation of
nucleic acids, that can also be identified by methods of the
present invention.
[0090] Further, there is growing evidence that some DNA sequences
can predispose an individual to any of a number of diseases such as
diabetes, arteriosclerosis, obesity, various autoimmune diseases
and cancer (e.g. colorectal, breast, ovarian, lung), or chromosomal
abnormality (either prenatally or postnatally). The diagnosis for a
genetic disease, chromosomal aneuploidy or genetic predisposition
can be performed prenatally by collecting an appropriate bodily
fluid, such as, urine from the pregnant mother.
[0091] Ultra short DNA analysis obtained non-invasively from a
bodily fluid provides an easier and safer way to perform prenatal
testing. Preferably, the bodily fluid is urine. A fetus receives
equal amount of genetic information from both parents. The loss of
a large number of fetal cells during development is a major part of
the genetic program for embryonic differentiations and formation of
a normal body. DNA from these dying embryonic cells not only
escapes into the bloodstream of the mother, but also crosses the
kidney barriers where it appears in the mother's urine. Pieces of
the male-specific Y chromosome have been found in the urine of
women pregnant with male fetuses. Fetal genetic information was
found in the mother's urine as early as the 7.sup.th to 8.sup.th
week of pregnancy, that is, at least 6-8 weeks earlier than can be
obtained by either amniocentesis or chorionic villus sampling. See,
for Example U.S. Pat. No. RE39,920.
[0092] In one embodiment of this invention, a simple noninvasive
test can be used for early determination of the fetal gender.
However, there are more far-reaching consequences of these findings
with regard to development of modern safe diagnostic techniques.
The discovery that ultra short DNA from the developing embryo
appears in the mother's urine presents the opportunity to quickly
develop products for analysis of genes inherited from the father.
These include genes that contain disease-related mutations or can
cause problems on different genetic backgrounds. As an example, if
a pregnant woman is Rh-(negative Rhesus factor) and produces
anti-RhD antibodies and a father is Rh.sup.+, amniocentesis is
currently recommended for early diagnostics of Rh incompatibility,
which often causes life threatening hemolytic anemia in the newborn
baby. Detection of the RhD gene-specific sequence in the mother's
urine will be an excellent alternative to amniocentesis, which is
considered hazardous by a growing number of physicians worldwide.
This test is also less expensive and more cost-effective, because
it avoids the necessity of a surgical step in obtaining samples for
analysis.
[0093] With the advent of broad-based genetic mapping initiatives
such as the Human Genome Project, there is an expanding list of
targets and applications for genetic analysis of urine ultra short
DNA. Many diseases inherited by the fetus will be easily detectable
by analysis of the mother's urine DNA. These include Marfan
Syndrome, Sickle Cell Anemia, Tay Sachs Disease, and a group of
neurodegenerative disorders, including Huntington's Disease,
Spinocerebellar Ataxia 1, Machado-Joseph Disease,
Dentatorubraopallidoluysian Atrophy, and others that affect the
fetus and newborn. Urine DNA analysis can detect the presence of
the mutant gene inherited from the father. Also, if the mother's
genome bears a mutation, the test can help determine whether a
normal version of the gene has been inherited from the father.
[0094] In addition to providing answers to commonly asked questions
from expectant couples, determination of fetal sex can also be very
helpful if there is a risk of X chromosome-linked inherited
disease, e.g. Hemophilia or Duchenne Muscular Dystrophy. Again
prenatal testing for inherited diseases is currently performed with
specimens obtained by amniocentesis. There are two major
disadvantages of this technology: First, amniocentesis can only be
performed after the 14.sup.th week of pregnancy. Second, in some
instances, the risk associated with an inherited disorder is
comparable to the risk associated with the surgical procedure of
amniocentesis. Urine DNA based technology can present the
information while avoiding both problems.
[0095] The methods of the present invention provide a significant
increase in sensitivity for analyzing fetal, cell-free, ultra short
(20-50 base pairs), nucleic acids
[0096] Another important factor contributing to the success of any
new diagnostic test is the necessity that patients and doctors
express a preference for the new test. Invasive prenatal testing is
often declined by the patient because of the attendant risks to the
fetus and mother. If the same information can be obtained from a
safe and simple urine test, it is likely that the test will be
given widespread acceptance by the public and medical
community.
[0097] The present invention further provides methods enabling the
detection of specific ultra short nucleic acid sequences
originating from the patient's own endogenous nucleic acid. These
ultra short nucleic acid sequences are obtained non-invasively from
a bodily fluid. Preferably, the nucleic acids sequences must cross
the kidney barrier to appear in the urine. The method generally
involves the steps of obtaining a urine sample from a patient and
subjecting the material to a method of detecting a target nucleic
acid sequence. In one embodiment, the method further encompasses
substantially purifying nucleic acids present in the urine sample
prior to detecting the target nucleic acid. This method has a
variety of diagnostic applications, including, but not limited to,
tumor diagnosis and the diagnosis of diseases caused by clonal
expansion of cells containing DNA modifications accompanied by
death of at least a subset of the cells bearing DNA
modifications.
[0098] Success of tumor treatment is currently dependent on tumor
type and method of treatment. However, the most important factor
determining the success of cancer therapy is detection of the tumor
at the earliest possible stage of development. The earlier a tumor
is detected the better is the prognosis. In many per-neoplastic
conditions, such as inherited predisposition to a specific tumor
type or a disease promoting neoplastic transformation, (e.g.
chronic hepatitis and cirrhosis), significant efforts for early
tumor detection are currently being applied but existing techniques
are usually invasive and expensive. The oncologist's arsenal now
includes tests that are not only invasive, often hazardous, but
also less reliable than expected.
[0099] From the patient's point of view, the invasive tests are
expensive and sufficiently unpleasant to warrant decisions to forgo
needed tests such as rectocolonoscopy for diagnostics of colorectal
cancer. The problem of compliance is of critical importance when
high-risk patients are encouraged to submit to procedures that are
clearly uncomfortable and unpleasant. Dramatic improvement of
high-risk patient compliance is an absolute necessity for the
future. Thus, development of new methods for early tumor detection
is absolutely necessary for a substantial progress in this area of
medicine. It is also clear that such methods should be based not
only on more sensitive techniques for detection of clinical
symptoms of neoplastic growth, but rather on revealing tumor
cell-specific markers.
[0100] The methods of the present invention provide a significant
increase in sensitivity for analyzing cell-free, ultra short (20-50
base pairs), nucleic acids originating from the patient's or
subject's own endogenous nucleic acid.
[0101] The earliest cellular changes that can be used as a marker
of neoplastic transformation are changes that cause the
transformation, i.e. genetic and epigenetic DNA modification.
Various changes in DNA sequences and/or in the methylation status
of CpG islands (especially of those located in promoter regions of
tumor suppressor genes) are currently used as tumor markers. As
more such markers are discovered, it has become evident that some
are characteristic of early tumor stages, or even of pre-neoplastic
conditions. Other DNA modifications can indicate relatively late
phases of neoplastic transformation. Also there are expectations
that some changes in DNA sequences and its methylation pattern will
help predict metastatic potential and tumor cell sensitivity to
different chemotherapeutic agents. Cell death occurs at all stages
of tumor growth and detection of tumor-specific changes in the
urine DNA can be an excellent marker for tumor diagnosis and
monitoring of anti-tumor therapy. A tumor-specific mutation of the
K-ras gene can be detected in the urine of patients with colorectal
tumors that bear this mutation.
[0102] One of the greatest clinical challenges for tumor
chemotherapy is the variable sensitivity of different tumors to
anti-tumor drugs, and the absence of a simple test for the quick
early stage evaluation of anti-tumor therapy. Normally, the
oncologist can observe the results of treatment only after several
months. Meanwhile, the tumor can continue to grow and possibly
metastasize if the chemotherapeutic regimen is ineffective. One
embodiment of the present invention, useful for the immediate
monitoring of the effectiveness of tumor therapy, is the
quantitative analysis of tumor-specific mutations in the patient's
urine DNA. If the treatment is effective, then more tumor cells
die, and the ratio of the mutant sequence to any normal reference
sequence contained in the urine will increase. Eventually, if
chemotherapy is effective the mutant tumor-specific sequence will
disappear. Periodic analysis of a patient's urine DNA can be used
for monitoring of possible tumor re-growth. Early indication of
chemotherapeutic ineffectiveness would allow time to try other
chemotherapeutics and anti-tumor treatments. This approach is
similarly effective for the evaluation of the effectiveness of
radiation therapy and other cancer therapies and for monitoring
after surgical treatment of cancerous growths.
[0103] The present invention also provides methods enabling the
detection of specific ultra short nucleic acid sequences that do
not originate from the patient's endogenous nucleic acid sequences.
These ultra short nucleic acid sequences are obtained
non-invasively from a bodily fluid. Preferably, the nucleic acid
sequences must cross the kidney barrier to appear in the urine. The
steps are the same as for the detection of host originated nucleic
acids, except that the detection method selects for non-host
nucleic acid sequences. This method has a variety of diagnostic
applications, including, but not limited to, diagnosis of infection
by nucleic acid containing pathogens that infect areas other than
the urinary tract, and do not shed nucleic acids directly into the
urinary tract.
[0104] The methods of the present invention provide a significant
increase in sensitivity for analyzing cell-free, ultra short (20-50
base pairs), nucleic acids that do not originate from the patient's
or subject's own endogenous nucleic acid.
[0105] In one embodiment, the present invention has important
applications in organ and tissue transplanting science.
Transplantation of different organs, tissues, and cells or other
material that contains nucleic acids (referred to as "transplanted
material") is now widely used in clinical practice. The most
important problem faced by the transplant patient and the
healthcare delivery system is the requirement to carefully control
the normal immune response of the recipient that leads to
transplant rejection and failure. In spite of intensive therapy
designed to suppress the recipient's immune response, rejection
episodes often occur during the post-transplantation period and
their early detection can be very useful, if not critical for
effective clinical management.
[0106] Each person has a distinct and unique pattern of genes that
are encoded by DNA. Since the donor's DNA is genetically different
from the recipient's DNA, the present invention can be used to
"monitor transplanted material" which is defined as detecting
and/or measuring the rejection or acceptance of transplanted
organs, tissues and cells by the recipient. This will reduce and
even eliminate in some instances the necessity of taking tissue
biopsies from already debilitated patients. A test for the
appearance of Y chromosome-specific DNA sequences in the urine of
female recipients who had received blood transfusions with blood
from males has been described. See, for Example U.S. Pat. No.
RE39,920. These experiments showed that due to the death of white
blood cells from the male donor, Y chromosome-specific sequences
appeared in the urine of the female recipient. These blood cells
die in such the same manner as the cells of a transplanted organ
that has been attacked by the recipient's immune system. Methods of
the present invention can be used to track the progress of
recipients of cell, tissue and organ transplants.
[0107] Changes in DNA methylation of specific genomic areas affect
chromatin structure and DNA transcription, and consequently, are
being investigated for their involvement in various pathological
processes. As such, analysis of ultra short transrenal DNA
methylation is a useful diagnostic tool.
[0108] Mutations and changes in DNA methylation status that happen
during tumor progression can be used as the tumor markers (Esteller
et al., Cancer Res 59:67-70, 1999; Wong et al., Cancer Res 59:
71-3, 1999). Various changes in DNA sequences and/or in the
methylation status of CpG islands (especially of those located in
promoter regions of tumor suppressor genes) are currently used as
tumor markers (Baylin et al., Adv Canc Res 72:141-96, 1998). Also
there are expectations that some changes in DNA sequences and its
methylation pattern will help to predict metastatic potential and
tumor cell sensitivity to different chemotherapeutic agents.
methylation in CpG islands of some genes, e.g. MYF-3 gene, can be
bound to different stages of carcinogenesis. Hypermethylation of
this gene in comparison with normal mucosa was observed in 88% of
adenomas and 99% of carcinomas (Shannon et al., Int J Cancer
84:109-13, 1999).
[0109] There are no reliable markers based on DNA mutations for
HCC. However, in this case there is a growing group of markers that
are based on CpG island methylation in a gene promoter region, e.g.
the p16 or GSTPI promoter. p16 methylation was found in more than
70% of HCC tissues and among HCC cases with aberrant methylation
similar changes were also detected in about 80% of the plasma
samples (Wong et al., Cancer Res 59:71-3, 1999; Matsuda et al.,
Gastroenterology 116:394-400, 1999). Somatic hypermethylation of
GSTP1 CpG islands was observed in DNA from more than 80% of HCC
cases (Tchou et al., Int J Oncol 16:663-76, 2000)
[0110] Methylation of CpG islands in promoters of tumor suppressor
genes lead to their inactivation and are involved in pre-neoplastic
conditions and carcinogenesis, and, as such, can be used for
diagnostics of those pathological processes. Methylation of
estrogen-receptor gene has been linked to heart disease (Fricker
J., Mol. Med. Today, 5, 505-506, 1999). Fragile X chromosome
syndrome is associated not only with the expansion of the number of
CGG trinucleotide tandem repeats at the 5' untranslated region of
the FMR1 gene but also with hypermethylation in the CGG repeats and
the adjacent CpG islands (Panagopoulos et al., Hum. Mutat., 14,
71-79, 1999). Analysis of the methylation status at the CpG islands
of the small nuclear ribonucleoprotein associated polypeptide
N(SNRPN) gene using amniotic fluid cell cultures or cultivated
chorionic vilus samples has been recommended for prenatal diagnosis
of Prader-Willi and Angelman syndromes (Kubota et al., J. Med.
Genet., 33, 1011-1014, 1996). DNA hypermethylation of the promoter
region of the E-cadherin gene is characteristic of chronic
hepatitis and liver cirrhosis (Kanai et al., Cancer Lett., 148,
73-80, 2000). Of course many more modifications in DNA methylation
status will be linked to various disease in the future. Detection
of those modifications in transrenal DNA will be a useful marker in
prenatal testing as well as for diagnosis of pathological processes
in adult organisms.
[0111] It is also known that aging is accompanied by specific
changes in the genome methylation status, hypermethylation of some
CpG islands and demethylation in coding regions of genome (Toyota
and Issa, Seminars in Cancer Biol., 9, 349-357, 1998). Detection of
these changes in transrenal DNA, that contains DNA fragments from
various cell types, can be used as a marker of normal and
pathological aging processes.
[0112] Techniques for nucleic acid manipulation useful for the
practice of the present invention are described in a variety of
references, including but not limited to, Molecular Cloning: A
Laboratory Manual, 2nd ed., Vol. 1-3, eds. Sambrook et al. Cold
Spring Harbor Laboratory Press (1989); and Current Protocols in
Molecular Biology, eds. Ausubel et al., Greene Publishing and
Wiley-Interscience: New York (1987) and periodic updates. Specific
descriptions, while not intended to limit the scope of the present
invention, provide guidance in practicing certain aspects of the
present invention.
[0113] DNA is subject to degradation by DNases present in bodily
fluids, such as urine. The present invention encompasses several
methods for preventing or reducing the degradation of DNA while in
urine so that sufficiently large sequences are available for
detection by known methods of DNA detection such as those described
below. In one embodiment, samples of urine are taken when the urine
has been held in the bladder for less than 12 hours, in a specific
embodiment the urine is held in the bladder for less than 5 hours,
more preferable for less than 2 hours. Collecting and analyzing a
urine sample before it has been held in the bladder for a long
period of time reduces the exposure of DNA to the any DNase present
in the urine.
[0114] In another embodiment of the present invention, after
collection, the urine sample is treated using one or more methods
of inhibiting DNase activity. Methods of inhibiting DNase activity
include, but are not limited to, the use of
ethylenediaminetetraacetic acid (EDTA), guanidine-HCl, GITC
(Guanidine isothiocyanate), N-lauroylsarcosine, Na-dodecylsulphate
(SDS), high salt concentration and heat inactivation of DNase.
[0115] In yet another embodiment, after collection, the urine
sample is treated with an adsorbent that traps DNA, after which the
adsorbent is removed from the sample, rinsed and treated to release
the trapped DNA for detection and analysis. This method not only
isolates DNA from the urine sample, but, when used with some
adsorbents, including, but not limited to Hybond N membranes
(Amersham Pharmacia Biotech Ltd., Piscataway, N.J.) protects the
DNA from degradation by DNase activity.
[0116] In some cases, the amount of DNA in a urine sample is
limited. Therefore, for certain applications, the present invention
encompasses embodiments wherein sensitivity of detection is
increased by any method(s) known in the art, including, without
limitation, one or more of the following methods.
[0117] Where DNA is present in minute amounts in the urine, larger
urine samples can be collected and thereafter concentrated by any
means that does not effect the detection of DNA present in the
sample. Some examples include, without limiting the breadth of the
invention, reducing liquid present in the sample by butanol
concentration or concentration using Sephadex G-25 (Pharmacia
Biotech, Inc., Piscataway N.J.).
[0118] Nested PCR can be used to improve sensitivity by several
orders of magnitude. Because of the vulnerability of nested PCR to
inaccurate results due to DNA contamination, in one embodiment of
the present invention, precautions are taken to avoid DNA
contamination of the sample. For example, without limiting the
present invention, one can treat PCR reagents with restriction
endonuclease(s) that cleave within the target sequence, prior to
adding them to the test DNA sample.
[0119] In one embodiment, the present invention encompasses
substantially purifying or isolating nucleic acids from a sample
prior to detection. Nucleic acid molecules can be isolated from
urine using any of a number of procedures, which are well-known in
the art. Any method for isolation that facilitates the detection of
target nucleic acid is acceptable. For example, DNA can be isolated
by precipitation, as described by Ishizawa et al., Nucleic Acids
Res. 19, 5972 (1991). Where a large volume sample contains a low
concentration of DNA, as with urine, a preferred method of
isolating DNA is encompassed. In this method, a sample is treated
with an adsorbent that acts to concentrate the DNA. For example, a
sample can be treated with a solid material that will adsorb DNA,
such as, without limitation, DEAE Sephadex A-25 (Pharmacia Biotech,
Inc., Piscataway N.J.), a DNA filter, and/or glass milk. Sample DNA
is eluted from the adsorbent after other compositions are washed
away.
[0120] In consideration of the sensitivity of various nucleic acid
analyzing techniques, such as PCR, the present invention also
encompasses methods of reducing the presence of contaminating
nucleic acids in the urine sample. Contamination of urine samples
by nucleic acid sequences that have not crossed the kidney barrier
can be introduced by cells shedding from the urinary tract lining,
by sexual intercourse, or during processing of the urine sample
prior to detection of the DNA sequence of interest. Without
intending to limit the present invention to any mechanism, it is
believed that DNA passing the kidney barrier and appearing in urine
is likely to have on average a shorter length than DNA introduced
from contaminating sources because of the fragmentation that occurs
in apoptotic cells and necrotic cells in the body, combined with
the action of DNase in the blood and urine.
[0121] Filtration can be used to reduce the level of contaminating
DNA in a urine sample prior to detection, by selecting for shorter
sequences of DNA. In one embodiment of the present invention
nucleic acids containing more than about 1000 base pairs, or 1000
nucleotides when denatured, are removed from the sample prior to
detection. In a specific embodiment of the present invention, urine
samples are filtered prior to amplification by PCR to remove
substantially all DNA comprising greater than 300 base pairs, or
300 nucleotides when denatured. Without limiting the invention to a
specific mechanism, it is proposed that such a filtration removes
contaminating DNA from cells shed from the urethral/bladder wall or
introduced into the urethra during sexual intercourse. The majority
of DNA from such contaminating sources are likely to comprise more
than 300 nucleotides as the DNA is not for the most part a product
of fragmentation of nucleic acids as a result of apoptotic cell
death.
[0122] Nucleic acid molecules can also be isolated by gel
electrophoresis, whereby fragments of nucleic acid are separated
according to molecular weight. The technique of restriction
fragments length polymorphisms (RFLP), applies the methods of
electrophoresis separation, followed by nucleic acid detection
enabling comparison by molecular weight of fragments from two or
more alleles of a specific gene sequence.
[0123] The above-mentioned methods of purification are meant to
describe, but not limit, the methods suitable for use in the
invention. The methods of isolating nucleic acids are within the
ability of one skilled in the art and are not described in detail
here.
[0124] The expression "assaying for the presence of a nucleic acid
sequence" refers to the use of any method to determine whether or
not a nucleic acid sequence is present in a sample. Methods
include, but are not limited to, techniques for hybridization,
amplification and detection of nucleic acids. One skilled in the
art has access to a multitude of these methods, including, but not
limited to, those set forth in Current Protocols in Molecular
Biology, eds. Ausubel et al., Greene Publishing and
Wiley-Interscience: New York (1987) and periodic updates. It is
contemplated that two or more methods can be used in combination to
confirm the results or improve the sensitivity of the assay. An
example of analyzing by the combination of methods to determine
whether or not a nucleic acid sequence is present is the technique
of restriction fragment length polymorphism based PCR ("PCR-RFLP"),
where nucleic acid sequences are amplified, treated with
restriction enzymes, and separated by electrophoresis, allowing for
the detection of nucleic acids containing small modifications, such
as point mutations.
[0125] The terms "detect" and "analyze" in relation to a nucleic
acid sequence, refer to the use of any method of observing,
ascertaining or quantifying signals indicating the presence of the
target nucleic acid sequence in a sample or the absolute or
relative quantity of that target nucleic acid sequence in a sample.
Methods can be combined with nucleic acid labeling methods to
provide a signal by, for example: fluorescence, radioactivity,
colorimetry, gravimetry, X-ray diffraction or adsorption,
magnetism, enzymatic activity and the like. The signal can then be
detected and/or quantified, by methods appropriate to the type of
signal, to determine the presence or absence, of the specific DNA
sequence of interest.
[0126] To "quantify" in relation to a nucleic acid sequence, refers
to the use of any method to study the amount of a particular
nucleic acid sequence, including, without limitation, methods to
determine the number of copies of a nucleic acid sequence or to
determine the change in quantity of copies of the nucleic acid
sequence over time, or to determine the relative concentration of a
sequence when compared to another sequence.
[0127] To assist in detection and analysis, specific DNA sequences
can be "amplified" in a number of ways, including, but not limited
to cycling probe reaction (Bekkaoui, F. et al, BioTechniques 20,
240-248 (1996), polymerase chain reaction (PCR), nested PCR,
PCR-SSCP (single strand conformation polymorphism), ligase chain
reaction (LCR) (F. Barany Proc. Natl. Acad. Sci. USA 88:189-93
(1991)), cloning, strand displacement amplification (SDA) (G. K.
Terrance Walker et al., Nucleic Acids Res. 22:2670-77 (1994), and
variations such as allele-specific amplification (ASA).
[0128] An alternative to amplification of a specific DNA sequence
that can be used to indicate the presence of that sequence in
methods of the present invention is based on hybridization of a
nucleic acid cleavage structure with the specific sequence,
followed by cleavage of the cleavage structure in a site-specific
manner. This method is herein referred to as "cleavage product
detection." This method is described in detail in U.S. Pat. Nos.
5,541,331 and 5,614,402, and PCT publication Nos. WO 94/29482 and
WO 97/27214. It allows for the detection of small amounts of
specific nucleic acid sequences without amplifying the DNA sequence
of interest.
[0129] The methods of the present invention provide a significant
increase in sensitivity for analyzing cell-free, ultra short (20-50
base pairs), nucleic acids.
[0130] One method of detecting and analyzing specific ultra short
DNA targets utilizes specific primers with internally labeled
fluorophores. In one example, primer pairs were designed
specifically for the ultra short DNA target of interest such that
the primer binding sites lacking any intervening sequences in the
double stranded PCR product. That is, the primer target sequences
are immediately adjacent to each other or overlapping. The each of
the primers in the primer pair are internally labeled with a
fluorophore near 3'-end. Appropriate fluorophores are selected from
those known in the art. In some embodiments, the fluorophores are
6-carboxyfluorescein and carboxy-X-rhodamine. Preferably, the two
bases closest to the 3' end are unlabeled to ensure unhindered
initiation of the DNA polymerization reaction. The fluorophores are
spaced such that 6-11 bases are between the fluorophores on the two
primers. Preferably, the spacing between fluorophores is 6-10
bases. Following binding of the labeled primers, and inclusion of
the appropriate materials required for PCR amplification, a PCR
reaction amplifies the target sequence generating double-stranded
oligonucleotide products, trans-labeled with the two fluorophores
in close proximity. Amplified labeled product is then detected by
Forster resonance energy transfer (FRET)-dependent fluorescence. In
some embodiments, non-specific products are differentiated from
specific products by measuring melting (dissociation) temperature.
Example 1 and FIG. 2 describe these primers and the subsequent
amplification reaction.
[0131] Another method of detecting and analyzing specific ultra
short DNA targets utilizes specific primers comprising
oligonucleotide tails at the 5' ends of their target-binding
sequences. These oligonucleotide tails are labeled at their 5' ends
with appropriate fluorophores. The oligonucleotide tails have no
homology to any other sequences in the reaction, except short
sequences adjacent to the fluorophores that are designed to be
complementary to sequences on the opposite primer pair
oligonucleotide tail, such that, if the two oligonucleotide tails
are brought into close proximity, they will bind to each other.
Each primer in the primer pair contains a replication blocking base
to separate the target-binding region from the oligonucleotide tail
comprising the fluorophore. This ensures that the tails are not
replicated during PCR and remain single stranded. Any replication
blocking base known in the art may be utilized, such as, iso-dC.
Following binding of the labeled primers, and inclusion of the
appropriate materials required for PCR amplification, and a PCR
reaction amplifies the target sequence generating double-stranded
oligonucleotide products. The complementary sequences of the
oligonucleotide tails anneal, bringing the fluorophore pairs into
close proximity. The amplified labeled product is then detected by
FRET-dependent fluorescence. Fluorescence is measured at a
temperature at which sticky ends are annealed only if they are part
of the same double-stranded PCR product molecule. Example 2 and
FIG. 7 describe these primers and the subsequent amplification
reaction.
[0132] Another method of detecting and analyzing specific ultra
short DNA targets utilizes three sequence-specific components,
including a TaqMan probe. This method permits for very short
amplicons by means of a partial target recognition sequence overlap
of the TaqMan probe with the sense (same-strand) target-specific
primer. This method utilizes a two stage, single tube, qPCR scheme.
In stage 1, the target DNA is which is amplified using primers P1
and P2, which map in very close proximity to each other on the
target sequence, thus allowing for very short templates. Primer P1
carries a target-unrelated 5'-end extension sequence, which is
incorporated into the intermediate PCR products IP1/IP2 along with
the template sequence. The resulting intermediate PCR product IP2
is sufficiently long to serve as template in stage 2, which
involves primers P3 and P2 and a TaqMan probe Pr, which is labeled
with fluorophore and quencher.
[0133] The mechanics of stage 2 are largely identical to those of a
standard TaqMan qPCR assay. During this stage, as in a standard
TaqMan qPCR assay, the amount of the final PCR product is monitored
by measuring the increase in fluorescence of the PCR mixture. The
three target-specific components in the assay are primers P3 and P2
and the TaqMan probe (Pr). Determination of the annealing
temperatures (T.sub.a) of the participant oligonucleotides, their
concentrations, extension temperatures, and the number of cycles in
each stage is an important part of assay development. The resulting
assay proved to be exceptionally sensitive, highly
sequence-specific, and suitable for the detection of target
fragments as short as 20 to 50 bases ("ultra short" targets).
[0134] When choosing among potential targets, preference was given
to those located in genomic sequence regions of relatively high
T.sub.m, which allow for the design of correspondingly short
primers and probes. The T.sub.m of the probe Pr-PCR product complex
was chosen to be 68.degree. C. to 70.degree. C. The T.sub.m of the
target recognition sequences of primers P2 and P3 were chosen to be
8.degree. C. to 10.degree. C. below that of probe Pr, as they would
normally be in a standard TaqMan assay. The T.sub.m of the target
recognition sequence of primer P1 was chosen to be 8.degree. C. to
10.degree. C. below that of primers P2 and P3 to allow control of
the length of each stage by altering the annealing/extension phase
temperature. This low T.sub.m requirement also allows for very
short target recognition sequence of in primer P1, thus reducing
the minimum required overall length of the template. This method is
shown schematically in FIG. 8 and described in Example 3.
[0135] In preferred example of the method described above, the P1
primer is further modified such that it is in a folded, stem-loop
configuration and maintains that structure at the
annealing/extension phase temperature of stage 2. This achieves
better linearity of the assay, as it prevents primer P1 from
competing for template with probe Pr in stage 2. Further, the stem
loop region further comprises a replication blocking base known in
the art. The inclusion of a replication blocking base prevents the
stem loop region from being copied into the PCR product. In one
example, the replication blocking base is iso-dC. This method is
shown schematically in FIG. 10 and described in Example 4.
[0136] Without limiting the present invention to any specific
methods of detection, analysis or quantification of methylated
regions of DNA, the following techniques are useful for evaluating
DNA methylation. Methods for the mapping and quantification of
methylated regions of DNA, in general, and for analysis of
transrenal DNA, in particular, may be grouped in two classes:
methods allowing to assess overall methylation status of CpG
islands and methods for analysis of sequence specific
methylation.
[0137] Methods in the first group rely on Southern hybridization
approach, based on utilization of properties of methylation
sensitive restriction nucleases. Hatada et al., describes a genomic
scanning method for higher organisms using restriction sites as
landmarks (Proc Natl Acad Sci USA 88(21):9523-7, 1991). Issa et
al., shows that methylation of the oestrogen receptor CpG island
links ageing and neoplasia in human colon (Nat Genet (4):536-40,
1994). Pogribny and Yi, describe a sensitive new method for rapid
detection of abnormal methylation patterns in global DNA with and
within CpG islands (Biochem Biophys Res Commun 262(3):624-8,
1999).
[0138] Recently designed DNA microarray based technology can also
be included in this group. Huang et al., describes methylation
profiling of CpG islands in human breast cancer cells (Genet
(3):459-70, 1999).
[0139] The methods in the second group are based on registration of
the sequence differences between methylated and unmethylated
alleles resulting from bisulfite treatment of DNA. Registration
usually is carried out by PCR amplification using primers specific
to methylated and unmethylated DNA. Herman et al., describes
methylation-specific PCR: a novel PCR assay for methylation status
of CpG islands (Proc Natl Acad Sci USA 93(18):9821-6, 1996).
Depending on the experimental setting several approaches based on
this strategy have been developed.
[0140] There are also several options for the quantification of
methylated CpG islands in small amount of DNA (Xiong and Laird,
Nucleic Acids Res 25(12):2532-4, 1997, describing COBRA: a
sensitive and quantitative DNA methylation assay, and Olek et al.,
Nucleic Acids Res 24(24):5064-6, 1996, describing a modified and
improved method for bisulphite based cytosine methylation analysis)
and partially degraded DNA received from micro-dissected pathology
sections (Gonzalgo and Jones, Nucleic Acids Res 25(12):2529-31,
1997, describing rapid quantitation of methylation differences at
specific sites using methylation-sensitive single nucleotide primer
extension (Ms-SNuPE)). Additionally, there is a methylation
sensitive SSCP that was developed for the analysis multiple
methylation sites in CpG islands (Kinoshita et al., Anal Biochem
278(2):165-9, 2000, describing methods for screening
hypermethylated regions by methylation-sensitive single-strand
conformational polymorphism) and an extremely sensitive methylation
specific Real Time PCR (Eads et al., Nucleic Acids Res 28(8):E32,
2000, describing MethyLight: a high-throughput assay to measure DNA
methylation).
[0141] Ligation-mediated polymerase chain reaction (LMPCR) has been
used for the detection of DNA adducts at individual nucleotide
positions in mammalian genes. Adduct-specific enzymes, such as T4
endonuclease V, base excision repair enzymes, like UVTABC nuclease,
and chemical cleavage can be used to convert the adducts into DNA
strand breaks. The positions of these breaks are then detected by
LMPCR. Yoon and Lee, Mol Cells 10(1):71-5, 2000, describes the
mapping of altromycin B-DNA adduct at nucleotide resolution in the
human genomic DNA by ligation-mediated PCR. Pfeifer, et al., Proc
Natl Acad Sci USA 88(4):1374-8, 1991, describes the in vivo mapping
of a DNA adduct at nucleotide resolution: detection of pyrimidine
(6-4) pyrimidone photoproducts by ligation-mediated polymerase
chain reaction. Pfeifer and Tang, Toxicol Left 102-130:447-51,
1998, describes PCR-based approaches to adduct analysis.
[0142] Using this approach the distribution of benzo[a]pyrene diol
epoxide adducts (formed by cigarette smoke major carcinogen
benzo[a]pyrene) in the P53 gene was mapped at nucleotide
resolution. Adduct formation was observed at the nucleotide
positions that appeared to be mutational hotspots in human lung
cancers. Denissenko et al., Science 274(5286):430-2, 1996,
describes preferential formation of benzo[a]pyrene adducts at lung
cancer mutational hotspots in P53. A similar trend was observed in
the case of skin cancer. Tommasi et al., Cancer Res 57(21):4727-30,
1997, shows that sunlight induces pyrimidine dimers occur
preferentially at 5-methylcytosine bases. Thus, the distribution of
DNA adducts in the p53 gene caused by environmental carcinogens
corresponds to the mutational hotspots of certain cancers.
[0143] These data indicate that both quantitation of DNA adducts
and their gene specific nucleotide mapping in transrenal DNA can be
used for the evaluation of genotoxic effects of environmental
factors, dietary and other carcinogens as well as for prediction of
resulting predisposition to a specific type of cancer.
[0144] To facilitate understanding of the invention, a number of
terms are defined below.
[0145] The term "gene" refers to a DNA sequence that comprises
control and coding sequences necessary for the transcription of an
RNA sequence. The term "genome" refers to the complete gene
complement of an organism, contained in a set of chromosomes in
eukaryotes.
[0146] The term "ultra short" refers to a DNA or RNA sequence of
less than 50 nucleotides. Preferably, less than 45 nucleotides,
less than 40 nucleotides, less than 35 nucleotides, less than 30
nucleotides, less than 25 nucleotides, less than 20 nucleotides,
less than 15 nucleotides, less, than 10 nucleotides, less than 5
nucleotides. More preferably, the DNA or RNA sequence is between 20
and 50 nucleotides.
[0147] A "wild-type" gene or gene sequence is that which is most
frequently observed in a population and is thus arbitrarily
designed the "normal" or "wild-type" form of the gene. In contrast,
the term "modified", "mutant", "anomaly" or "altered" refers to a
gene, sequence or gene product which displays modifications in
sequence and or functional properties (i.e., altered
characteristics) when compared to the wild-type gene, sequence or
gene product. For example, an altered sequence detected in the
urine of a patient can display a modification that occurs in DNA
sequences from tumor cells and that does not occur in the patient's
normal (i.e. non cancerous) cells. It is noted that
naturally-occurring mutants can be isolated; these are identified
by the fact that they have altered characteristics when compared to
the wild-type gene or gene product. Without limiting the invention
to the detection of any specific type of anomaly, mutations can
take many forms, including addition, addition-deletion, deletion,
frame-shift, missense, point, reading frame shift, reverse,
transition and transversion mutations as well as microsatellite
alterations.
[0148] A "disease associated genetic anomaly" refers to a gene,
sequence or gene product that displays modifications in sequence
when compared to the wild-type gene and that is indicative of the
propensity to develop or the existence of a disease in the carrier
of that anomaly. A disease associated genetic anomaly encompasses,
without limitation, inherited anomalies as well as new
mutations.
[0149] The term "unique fetal DNA sequence" is defined as a
sequence of nucleic acids that is present in the genome of the
fetus, but not in the maternal genome.
[0150] The terms "oligonucleotide" and "polynucleotide" and
"polymeric" nucleic acid are interchangeable and are defined as a
molecule comprised of two or more deoxyribonucleotides or
ribonucleotides, preferably more than three, and usually more than
ten. The exact size will depend on many factors, which in turn
depends on the ultimate function or use of the oligonucleotide. The
oligonucleotide can be generated in any manner, including chemical
synthesis, DNA replication, reverse transcription, or a combination
thereof.
[0151] Because mononucleotides are reacted to make oligonucleotides
in a manner such that the 5' phosphate of one mononucleotide
pentose ring is attached to the 3' oxygen of its neighbor in one
direction via a phosphodiester linkage, an end of an
oligonucleotide is referred to as the "5' end" if its 5' phosphate
is not linked to the 3' oxygen of a mononucleotide pentose ring and
as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of
a subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide, also
can be said to have 5' and 3' ends.
[0152] When two different, non-overlapping oligonucleotides anneal
to different regions of the same linear complementary nucleic acid
sequence, and the 3' end of one oligonucleotide points towards the
5' end of the other, the former can be called the "upstream"
oligonucleotide and the latter the "downstream"
oligonucleotide.
[0153] The term "primer" refers to an oligonucleotide which is
capable of acting as a point of initiation of synthesis when placed
under conditions in which primer extension is initiated. An
oligonucleotide "primer" can occur naturally, as in a purified
restriction digest or be produced synthetically.
[0154] A primer is selected to be "substantially" complementary to
a strand of specific sequence of the template. A primer must be
sufficiently complementary to hybridize with a template strand for
primer elongation to occur. A primer sequence need not reflect the
exact sequence of the template. For example, a non-complementary
nucleotide fragment can be attached to the 5' end of the primer,
with the remainder of the primer sequence being substantially
complementary to the strand. Non-complementary bases or longer
sequences can be interspersed into the primer, provided that the
primer sequence has sufficient complementarity with the sequence of
the template to hybridize and thereby form a template primer
complex for synthesis of the extension product of the primer.
[0155] A "target" nucleic acid is a nucleic acid sequence to be
evaluated by hybridization, amplification or any other means of
analyzing a nucleic acid sequence, including a combination of
analysis methods.
[0156] "Hybridization" methods involve the annealing of a
complementary sequence to the target nucleic acid (the sequence to
be analyzed). The ability of two polymers of nucleic acid
containing complementary sequences to find each other and anneal
through base pairing interaction is a well-recognized phenomenon.
The initial observations of the "hybridization" process by Marmur
and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al.,
Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the
refinement of this process into an essential tool of modern
biology. Hybridization encompasses, but is not limited to, slot,
dot and blot hybridization techniques.
[0157] It is important for some diagnostic applications to
determine whether the hybridization represents complete or partial
complementarity. For example, where it is desired to detect simply
the presence or absence of pathogen DNA (such as from a virus,
bacterium, fungi, mycoplasma, protozoan) it is only important that
the hybridization method ensures hybridization when the relevant
sequence is present; conditions can be selected where both
partially complementary probes and completely complementary probes
will hybridize. Other diagnostic applications, however, could
require that the hybridization method distinguish between partial
and complete complementarity. It may be of interest to detect
genetic polymorphisms.
[0158] Methods that allow for the same level of hybridization in
the case of both partial as well as complete complementarity are
typically unsuited for such applications; the probe will hybridize
to both the normal and variant target sequence. The present
invention contemplates that for some diagnostic purposes,
hybridization be combined with other techniques (such as
restriction enzyme analysis). Hybridization, regardless of the
method used, requires some degree of complementarity between the
sequence being analyzed (the target sequence) and the fragment of
DNA used to perform the test (the probe). (Of course, one can
obtain binding without any complementarity but this binding is
nonspecific and to be avoided.)
[0159] The complement of a nucleic acid sequence as used herein
refers to an oligonucleotide which, when aligned with the nucleic
acid sequence such that the 5' end of one sequence is paired with
the 3' end of the other, is in "antiparallel association." Specific
bases not commonly found in natural nucleic acids can be included
in the nucleic acids of the present invention and include, for
example, inosine and 7-deazaguanine. Complementarity need not be
perfect; stable duplexes can contain mismatched base pairs or
unmatched bases. Those skilled in the art of nucleic acid
technology can determine duplex stability empirically considering a
number of variables including, for example, the length of the
oligonucleotide, base composition and sequence of the
oligonucleotide, ionic strength and incidence of mismatched base
pairs.
[0160] As used herein, the term "Tm" is used in reference to the
"melting temperature." The melting temperature is the temperature
at which a population of double-stranded nucleic acid molecules
becomes half dissociated into single strands. The equation for
calculating the Tm of nucleic acids is well known in the art. As
indicated by standard references, a simple estimate of the Tm value
can be calculated by the equation: Tm=81.5+0.41(% G+C), when a
nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson
and Young, Quantitative Filter Hybridisation, in Nucleic Acid
Hybridisation (1985). Other references include more sophisticated
computations which take structural as well as sequence
characteristics into account for the calculation of Tm.
[0161] The term "probe" as used herein refers to an oligonucleotide
(i.e., a sequence of nucleotides), whether occurring naturally as
in a purified restriction digest or produced synthetically, which
forms a duplex structure or other complex with a sequence in
another nucleic acid, due to complementarity or other means of
reproducible attractive interaction, of at least one sequence in
the probe with a sequence in the other nucleic acid. Probes are
useful in the detection, identification and isolation of particular
gene sequences. It is contemplated that any probe used in the
present invention will be labeled with any "reporter molecule," so
that it is detectable in any detection system, including, but not
limited to, enzyme (e.g., ELISA, as well as enzyme-based
histochemical assays), fluorescent, radioactive, and luminescent
systems. It is further contemplated that the oligonucleotide of
interest (i.e., to be detected) will be labeled with a reporter
molecule. It is also contemplated that both the probe and
oligonucleotide of interest will be labeled. It is not intended
that the present invention be limited to any particular detection
system or label.
[0162] The term "label" as used herein refers to any atom or
molecule which can be used to provide a detectable (preferably
quantifiable) signal, and which can be attached to a nucleic acid
or protein. Labels provide signals detectable by any number of
methods, including, but not limited to, fluorescence,
radioactivity, colorimetry, gravimetry, X-ray diffraction or
absorption, magnetism, and enzymatic activity.
[0163] The term "substantially single-stranded" when used in
reference to a nucleic acid target means that the target molecule
exists primarily as a single strand of nucleic acid in contrast to
a double-stranded target which exists as two strands of nucleic
acid which are held together by inter-strand base pairing
interactions.
[0164] The term "sequence variation" as used herein refers to
differences in nucleic acid sequence between two nucleic acid
templates. For example, a wild-type structural gene and a mutant
form of this wild-type structural gene can vary in sequence by the
presence of single base substitutions and/or deletions or
insertions of one or more nucleotides. These two forms of the
structural gene are said to vary in sequence from one another. A
second mutant form of the structural gene can exit. This second
mutant form is said to vary in sequence from both the wild-type
gene and the first mutant form of the gene.
[0165] The terms "structure probing signature," "hybridization
signature" and "hybridization profile" are used interchangeably
herein to indicate the measured level of complex formation between
a target nucleic acid and a probe or set of probes, such measured
levels being characteristic of the target nucleic acid when
compared to levels of complex formation involving reference targets
or probes.
[0166] "Oligonucleotide primers matching or complementary to a gene
sequence" refers to oligonucleotide primers capable of facilitating
the template-dependent synthesis of single or double-stranded
nucleic acids. Oligonucleotide primers matching or complementary to
a gene sequence can be used in PCRs, RT-PCRs and the like.
[0167] "Nucleic acid sequence" as used herein refers to an
oligonucleotide, nucleotide or polynucleotide, and fragments or
portions thereof, and to DNA or RNA of genomic or synthetic origin
which can be single- or double-stranded, and represent the sense or
antisense strand.
[0168] A "deletion" is defined as a change in either nucleotide or
amino acid sequence in which one or more nucleotides or amino acid
residues, respectively, are absent.
[0169] An "insertion" or "addition" is that change in a nucleotide
or amino acid sequence which has resulted in the addition of one or
more nucleotides or amino acid residues, respectively, as compared
to, naturally occurring sequences.
[0170] A "substitution" results from the replacement of one or more
nucleotides or amino acids by different nucleotides or amino acids,
respectively.
[0171] A "modification" in a nucleic acid sequence refers to any
change to a nucleic acid sequence, including, but not limited to a
deletion, an addition, an addition-deletion, a substitution, an
insertion, a reversion, a transversion, a point mutation, a
microsatellite alteration, methylation or nucleotide adduct
formation.
[0172] As used herein, the terms "purified", "decontaminated" and
"sterilized" refer to the removal of contaminant(s) from a
sample.
[0173] As used herein, the terms "substantially purified" and
"substantially isolated" refer to nucleic acid sequences that are
removed from their natural environment, isolated or separated, and
are preferably 60% free, more preferably 75% free, and most
preferably 90% free from other components with which they are
naturally associated. An "isolated polynucleotide" is therefore a
substantially purified polynucleotide. It is contemplated that to
practice the methods of the present invention polynucleotides can
be, but need not be substantially purified. A variety of methods
for the detection of nucleic acid sequences in unpurified form are
known in the art.
[0174] "Amplification" is defined as the production of additional
copies of a nucleic acid sequence and is generally carried out
using polymerase chain reaction or other technologies well known in
the art (e.g., Dieffenbach and Dveksler, PCR Primer, a Laboratory
Manual, Cold Spring Harbor Press, Plainview, N.Y. [1995]). As used
herein, the term "polymerase chain reaction" ("PCR") refers to the
method of K. B. Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202,
hereby incorporated by reference), which describe a method for
increasing the concentration of a segment of a target sequence in a
mixture of genomic DNA without cloning or purification. This
process for amplifying the target sequence consists of introducing
a large excess of two oligonucleotide primers to the DNA mixture
containing the desired target sequence, followed by a precise
sequence of thermal cycling in the presence of a DNA polymerase.
The two primers are complementary to their respective strands of
the double stranded target sequence. To effect amplification, the
mixture is denatured and the primers then annealed to their
complementary sequences within the target molecule. Following
annealing, the primers are extended with a polymerase so as to form
a new pair of complementary strands. The steps of denaturation,
primer annealing and polymerase extension can be repeated many
times (i.e., denaturation, annealing and extension constitute one
"cycle"; there can be numerous "cycles") to obtain a high
concentration of an amplified segment of the desired target
sequence. The length of the amplified segment of the desired target
sequence is determined by the relative positions of the primers
with respect to each other, and therefore, this length is a
controllable parameter. By virtue of the repeating aspect of the
process, the method is referred to as the "polymerase chain
reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified".
[0175] As used herein, the term "polymerase" refers to any enzyme
suitable for use in the amplification of nucleic acids of interest.
It is intended that the term encompass such DNA polymerases as Taq
DNA polymerase obtained from Thermus aquaticus, although other
polymerases, both thermostable and thermolabile are also
encompassed by this definition.
[0176] With PCR, it is possible to amplify a single copy of a
specific target sequence in genomic DNA to a level that can be
detected by several different methodologies (e.g., staining,
hybridization with a labeled probe; incorporation of biotinylated
primers followed by avidin-enzyme conjugate detection;
incorporation of 32P-labeled deoxynucleotide triphosphates, such as
dCTP or dATP, into the amplified segment). In addition to genomic
DNA, any oligonucleotide sequence can be amplified with the
appropriate set of primer molecules. In particular, the amplified
segments created by the PCR process itself are, themselves,
efficient templates for subsequent PCR amplifications. Amplified
target sequences can be used to obtain segments of DNA (e.g.,
genes) for insertion into recombinant vectors.
[0177] As used herein, the terms "PCR product" and "amplification
product" refer to the resultant mixture of compounds after two or
more cycles of the PCR steps of denaturation, annealing and
extension are complete. These terms encompass the case where there
has been amplification of one or more segments of one or more
target sequences.
[0178] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0179] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the base-pairing rules. For
example, for the sequence "A-G-T," is complementary to the sequence
"T-C-A." Complementarity can be "partial," in which only some of
the nucleic acids' bases are matched according to the base pairing
rules. Or, there can be "complete" or "total" complementarity
between the nucleic acids. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, as well as
detection methods that depend upon binding between nucleic
acids.
[0180] The term "homology" refers to a degree of complementarity.
There can be partial homology or complete homology (i.e.,
identity). A partially complementary sequence is one that at least
partially inhibits a completely complementary sequence from
hybridizing to a target nucleic acid is referred to using the
functional term "substantially homologous." The inhibition of
hybridization of the completely complementary sequence to the
target sequence can be examined using a hybridization assay
(Southern or Northern blot, solution hybridization and the like)
under conditions of low stringency. A substantially homologous
sequence or probe will compete for and inhibit the binding (i.e.,
the hybridization) of a completely homologous to a target under
conditions of low stringency. This is not to say that conditions of
low stringency are such that are non-specific binding is permitted;
low stringency conditions require that the binding of two sequences
to one another be a specific (i.e., selective) interaction. The
absence of non-specific binding can be tested by the use of a
second target which lacks even a partial degree of complementarity
(e.g., less than about 30% identity); in the absence of
non-specific binding the probe will not hybridize to the second
non-complementary target.
[0181] Numerous equivalent conditions can be employed to comprise
either low or high stringency conditions; factors such as the
length and nature (DNA, RNA, base composition) of the probe and
nature of the target (DNA, RNA, base composition, present in
solution or immobilized, etc.) and the concentration of the salts
and other components (e.g., the presence or absence of formamide,
dextran sulfate, polyethylene glycol) are considered and the
hybridization solution can be varied to generate conditions of
either low or high stringency hybridization different from, but
equivalent to, the above listed conditions. The term
"hybridization" as used herein includes "any process by which a
strand of nucleic acid joins with a complementary strand through
base pairing" (Coombs, Dictionary of Biotechnology, Stockton Press,
New York N.Y. [1994].
[0182] "Stringency" typically occurs in a range from about
Tm-5.degree. C. (5.degree. C. below the Tm of the probe) to about
20.degree. C. to 25.degree. C. below Tm. As will be understood by
those of skill in the art, a stringent hybridization can be used to
identify or detect identical polynucleotide sequences or to
identify or detect similar or related polynucleotide sequences.
[0183] As used herein the term "hybridization complex" refers to a
complex formed between two nucleic acid sequences by virtue of the
formation of hydrogen bonds between complementary G and C bases and
between complementary A and T bases; these hydrogen bonds can be
further stabilized by base stacking interactions. The two
complementary nucleic acid sequences hydrogen bond in an
antiparallel configuration. A hybridization complex can be formed
in solution (e.g., C0t or R0t analysis) or between one nucleic acid
sequence present in solution and another nucleic acid sequence
immobilized to a solid support (e.g., a nylon membrane or a
nitrocellulose filter as employed in Southern and Northern
blotting, dot blotting or a glass slide as employed in situ
hybridization, including FISH [fluorescent in situ
hybridization]).
[0184] As used herein, the term "antisense" is used in reference to
RNA sequences which are complementary to a specific RNA (e.g.,
mRNA) or DNA sequence. Antisense RNA can be produced by any method,
including synthesis by splicing the gene(s) of interest in a
reverse orientation to a viral promoter which permits the synthesis
of a coding strand. Once introduced into a cell, this transcribed
strand combines with natural mRNA produced by the cell to form
duplexes. These duplexes then block either further transcription of
the mRNA or its translation. In this manner, mutant phenotypes can
be generated. The term "antisense strand" is used in reference to a
nucleic acid strand that is complementary to the "sense" strand.
The designation (-) (i.e., "negative") is sometimes used in
reference to the antisense strand, with the designation (+)
sometimes used in reference to the sense (i.e., "positive")
strand.
[0185] The term "sample" as used herein is used in its broadest
sense. A biological sample suspected of containing nucleic acid can
comprise, but is not limited to, genomic DNA (in solution or bound
to a solid support such as for Southern blot analysis), cDNA (in
solution or bound to a solid support), and the like.
[0186] The term "urinary tract" as used herein refers to the organs
and ducts which participate in the secretion and elimination of
urine from the body.
[0187] The terms "transrenal DNA" and "transrenal nucleic acid" as
used herein refer to nucleic acids that have crossed the kidney
barrier. Transrenal DNA as used herein differs from miRNA.
Specifically, transrenal DNA comprises randomness in the 3' and 5'
ends, which is not present in miRNA.
[0188] The present invention encompasses a platform for the
detection and gene specific analysis of "ultra short" transrenal
DNA fragments carrying different nucleotide lesions and adducts
caused by various external and internal DNA modifying factors.
Without limiting the scope of the invention, but in the interest of
clarity, some factors that generate DNA modifications might be
grouped in three classes: (i) physical, including but not limited
to gamma and UV irradiation, temperature fluctuations; (ii)
chemical, including but not limited to environmental pollutants,
naturally occurring genotoxins, carcinogens, anticancer drugs and
(iii) reactive metabolites such as active forms of oxygen, lipid
peroxidation products and hydrolytic agents.
[0189] The invention is further described below, by way of the
following examples. The examples also illustrate useful methodology
for practicing the invention. These examples do not limit the
claimed invention.
EXAMPLES
Example 1
[0190] Example 1 shows the design of primers with internally
located fluorophores for detection of ultra short DNA targets by
PCR using fret-dependent fluorescence.
[0191] The specificity of a typical labeled-primer qPCR assay is
determined solely by the specificity of the two primers, and
therefore such an assay is not capable of distinguishing between
templates having identical primer binding sites but different
intervening sequences. To detect very short target fragments and
overcome this limitation, novel labeled-primer assays were
developed specific for targets in which the primer binding sites
are either immediately adjacent to each other or even slightly
overlapping, thus lacking any intervening sequences. Such amplicons
are characterized by the pair of primer-derived sequences
positioned in close proximity to each other in the double-stranded
PCR product. In the assay, the mutual proximity of fluorescently
labeled oligonucleotides was detected by Forster resonance energy
transfer (FRET) between them. To test the effectiveness of a FRET
assay, a model system was designed consisting of two primers
internally labeled with a widely used FRET fluorophore pair,
6-carboxyfluorescein (FAM) and carboxy-X-rhodamine (ROX).
TABLE-US-00001 SEQ ID Direction ID Template NO: Forward ES0343-FL
5'-CGTCCGTGCTGTCGACG- 1 primer: [FL-dT]-AG-3' Reverse ES0344-RX
5'-CATACCACGCCATCAGAG- 2 primer: [ROX-dT]-GC-3'
[0192] As shown in FIG. 2, each fluorophore was conjugated to a
thymine base at position 3 from the 3' end of each primer. In FIG.
2, T designates a template; P1 and P2 designate primers; D and A
designate donor and acceptor fluorophores, respectively. Labeling
the primers at internal bases and leaving at least the 2 bases
closest to the 3' end unlabeled is necessary to ensure unhindered
initiation of DNA polymerization reaction (Ahmad and Ghasemi, Anal
Bioanal Chem. 387: 2737-43, 2007). This model system was utilized
to determine the spacing between the fluorophores that yields the
highest FRET efficiency by testing it on a number of
oligonucleotide templates with a range of spacing between the 2
primer binding sites.
TABLE-US-00002 FLUOROPHORE TEMPLATE SPACING SEQ ID NO:
5'-CAGCACGTCCGTGCTGTCGACGTAGACATCAGC 11 3
ACTCTGATGGCGTGGTATGACGAC-3' 4 5'-CAGCACGTCCGTGCTGTCGACGTAGCATCAGC
10 5 ACTCTGATGGCGTGGTATGACGAC-3' 6
5'-CAGCACGTCCGTGCTGTCGACGTAGATCAGC 9 7 ACTCTGATGGCGTGGTATGACGAC-3'
8 5'-CAGCACGTCCGTGCTGTCGACGTAGATGC 7 9 ACTCTGATGGCGTGGTATGACGAC-3'
10 5'-CAGCACGTCCGTGCTGTCGACGTAGAGC 6 11 ACTCTGATGGCGTGGTATGACGAC-3'
12 5'-CAGCACGTCCGTGCTGTCGACGTAGGC 5 13 ACTCTGATGGCGTGGTATGACGAC-3'
14 5'-CAGCACGTCCGTGCTGTCGACGTAC 4 15 ACTCTGATGGCGTGGTATGACGAC-3'
16
[0193] PCR generates a double-stranded oligonucleotide product
trans-labeled with the 2 fluorophores, the spatial distance between
which is assumed to directly correlate with the spacing between the
primer binding sites on the supplied template. Although the signal
variation for different templates was not very significant, FIG. 3
shows that a peak in FRET efficiency at fluorophore spacing of 6 to
10 bases in the PCR product. Based on the results in FIG. 3, the
assay is capable of detecting very short target fragments, those
containing both of the primer binding sites in immediate proximity
to each other. The minimum length of target fragments detectable
with this system is approximated by the combined length of the 2
primer binding sites, totaling about 20 to 50 bases.
[0194] This method was then tested for its ability to detect
Epstein-Barr virus (EBV) BamHI-W region sequence. A pair of primers
were designed and internally labeled with FAM and ROX, and PCR
amplification of the target in real-time was monitored by measuring
610 nm fluorescence emission signal with excitation at 492 nm.
TABLE-US-00003 SEQ ID Direction ID Template NO Forward ES0430-FL
5'-ATCGCAGAGCCCAGGATG- 17 primer: [FL-dT]-CC-3' Reverse ES0431-RX
5'-ACGAGCTCTAGGGTCCCTTG- 18 primer: [ROX-dT]-GG-3' 39-bp
5'-CAGAGCCCAGGATGTCCCCCA 19 target GAAGGGACCCTAG-3'
[0195] To monitor the level of fluorescence of individual
fluorophores, fluorescence emission was also measured at 516 nm
with 492 nm excitation (FAM), and emission at 610 nm with 585 nm
excitation (ROX). No concurrent increase in the level of
fluorescence of the individual fluorophores was observed in the
reaction, indicating that all of the fluorescence increase (492 nm
ex/610 nm em) was attributable to FRET between the fluorophores.
FIG. 4 shows that this method permits the detection of as few as 10
copies of the target EBV sequence in a 25 .mu.l reaction.
Specifically, FIG. 4, Panel A shows amplification of EBV standards
in labeled-primer FRET real-time PCR assay and FIG. 4, Panel B
shows the calibration curve.
[0196] In another example of the application of labeled-primer FRET
Real-Time PCR, a pair of fluorophore-labeled primers were designed
for the detection of a chromosome Y-specific sequence. PCR with
these 2 primers amplifies a 36-bp region of the SRY gene, yielding
a product with the donor and acceptor fluorophores spaced 6 bases
apart.
TABLE-US-00004 Direction ID Template SEQ ID Forward ES0619-FL
5'-CCGCAGATCCCGC- 20 primer: [FL-dT]-TCG-3' Reverse ES0620-RX
5'-GCACTTCGCTGCAGAG- 21 primer: [ROX-dT]-ACC-3' 39-bp
5'-CCGCAGATCCCGCTTCGGTAC 22 target TCTGCAGCGAAGTGC-3'
[0197] FIG. 5 shows the amplification of the 36-bp SRY region as
described above.
[0198] The FRET SRY assay was applied to DNA samples purified from
the urine of pregnant women. The results in FIG. 6 show the
detection of SRY sequences in samples from women pregnant with male
fetuses, but not in a sample from a woman pregnant with a female
fetus. In FIG. 6, M designates samples from women pregnant with
male fetuses and F designates a sample from a woman pregnant with a
female fetus.
[0199] The fluorescence signal detected by the labeled-primer FRET
method indicated the presence of double-stranded products of
amplification in which (i) both labeled primers are incorporated,
and (ii) the primers are incorporated in such a manner that the
fluorophores are positioned in close proximity to each other. In
most cases, these two requirements are sufficient to ensure that
only the specific PCR products generate detectable fluorescence
signal. However, the downside of using a highly sensitive PCR
method is the occasional, uncontrolled generation of nonspecific
products, which may also give FRET-dependent fluorescence signal.
In conventional PCR, nonspecific products are typically
differentiated by size using gel electrophoresis. The
labeled-primer FRET method allows these nonspecific products to be
differentiated by measuring their melting (dissociation)
temperature. For example, a larger nonspecific product is likely to
exhibit a higher melting temperature than that of the specific one.
Since FRET takes place only when these products are in their
double-stranded state, their transition to a single-stranded state
is accompanied by a decrease in FRET fluorescence. Therefore, the
temperature of this transition can be measured immediately after
the amplification phase, with no need for any additional dye. The
amplification phase and the melting phase fluorescence signals are
based on the same FRET effect, thus ensuring that in both cases the
same product is analyzed.
Example 2
[0200] Example 2 shows the design of primers with fluorophores
located on 5'-end for detection of ultra short DNA targets by PCR
using fret-dependent fluorescence
[0201] Compared to unlabeled primers, the use of primers labeled
with fluorophores near their 3' ends may lower the efficiency of
the PCR, most likely due to reduction in polymerase processivity as
it reads fluorophore-labeled bases of the primers incorporated into
templates. To overcome this problem, another variant of the
labeled-primer FRET qPCR scheme was developed. In this example, as
shown in FIG. 7, the primers contain oligonucleotide tails at the
5' ends of their target-binding sequences. In FIG. 7, T designates
a template; P1 and P2 designate primers; D and A designate donor
and acceptor fluorophores, respectively; black circles denote the
positions of replication-blocking modifications. The tails are
labeled at their 5' ends with appropriate fluorophores, so that
each double-stranded PCR product molecule carries a FRET
fluorophore pair. On each primer, the tail is separated from
target-binding region by a replication-blocking base, such as
iso-dC, to ensure that the tails are not replicated during the PCR,
and thus remain single-stranded. The tails are chosen to have no
homology to any other sequence in the system, except that short
sequence stretches immediately adjacent to the fluorophores are
designed to be complementary to each other (sticky ends). Annealing
of the sticky ends brings the fluorophores of the FRET pair into
close proximity to each other. Due to their increased local
concentration, these sticky ends are expected to anneal more
readily when both are part of the same double-stranded PCR product.
Fluorescence is measured at a temperature at which sticky ends are
annealed only if they are part of the same double-stranded PCR
product molecule. The intensity of FRET fluorescence measured at
this temperature directly correlates to the yield of PCR product.
Nonspecific PCR products of significantly greater length should not
contribute to the FRET fluorescence signal because their sticky
ends are too far apart to anneal to each other.
[0202] A model system was designed to test this version of
labeled-primer assay. Two "primer" oligonucleotides labeled on
their 5' ends with FAM and ROX dyes, respectively, were mixed
together and added to a range of "template" oligonucleotides, and
the dissociation curves of the mixtures were measured. The
templates contained sequences complementary to each of the two
primers, and differed in the distance between those sequences. The
dissociation curves were generated for each mixture. An increased
FRET signal from the labeled oligonucleotides in the presence of
the "templates" was observed, showing that it is possible to detect
incorporation of the labeled primers into PCR products.
Example 3
[0203] Example 3 shows the design of single-tube PCR scheme for
detection of ultra short DNA targets.
[0204] A novel qPCR scheme was developed that offers high target
specificity by utilizing three sequence-specific components,
including a TaqMan probe, yet allows for very short amplicons by
means of a partial target recognition sequence overlap of the
TaqMan probe with the sense (same-strand) target-specific primer.
To accommodate the overlapping probe and primer, a novel 2-stage
single-tube qPCR scheme was devised. The flow diagram provided in
FIG. 8 shows the specific details and steps of the reaction. In
FIG. 8, T designates a template; P1, P2, and P3 designate primers;
IP1 and IP2 designate intermediate products; Pr designates
TaqMan.TM. probe, dual-labeled with fluorophore F and quencher Q.
In stage 1, the target DNA template T is amplified using primers P1
and P2, which map in very close proximity to each other on the
target sequence, thus allowing for very short templates. Primer P1
carries a target-unrelated 5'-end extension sequence, which is
incorporated into the PCR product IP1/IP2 along with the template
sequence. The resulting intermediate PCR product IP2 is
sufficiently long to serve as template in stage 2, which involves
primers P3 and P2 and a labeled TaqMan probe Pr. The mechanics of
stage 2 are largely identical to those of a standard TaqMan qPCR
assay. During this stage, as in a standard TaqMan qPCR assay, the
amount of the final PCR product is monitored by measuring the
increase in fluorescence of the PCR mixture. The three
target-specific components in the assay are primers P3 and P2 and
the TaqMan probe (Pr). Determination of the annealing temperatures
(T.sub.a) of the participant oligonucleotides, their
concentrations, extension temperatures, and the number of cycles in
each stage is an important part of assay development. The resulting
assay proved to be exceptionally sensitive, highly
sequence-specific, and suitable for the detection of target
fragments as short as 20 to 50 bases ("ultra short" targets).
[0205] The oligonucleotide components involved in the 2-stage qPCR
assay for ultra short targets were designed with the following
considerations: [0206] When choosing among potential targets,
preference was given to those located in genomic sequence regions
of relatively high T.sub.m, which allow for the design of
correspondingly short primers and probes. [0207] The T.sub.m of the
probe Pr-PCR product complex was chosen to be 68.degree. C. to
70.degree. C. [0208] The T.sub.m of the target recognition
sequences of primers P2 and P3 were chosen to be 8.degree. C. to
10.degree. C. below that of probe Pr, as they would normally be in
a standard TaqMan assay. [0209] The T.sub.m of the target
recognition sequence of primer P1 was chosen to be 8.degree. C. to
10.degree. C. below that of primers P2 and P3 to allow control of
the length of each stage by altering the annealing/extension phase
temperature. This low T.sub.m requirement also allows for very
short target recognition sequence of in primer P1, thus reducing
the minimum required overall length of the template.
[0210] A specific example of this method is an assay developed for
the purpose of detecting M. tuberculosis sequences in urine DNA
samples. The target is a 39-bp region within the IS6110 repeat of
the bacteria.
TABLE-US-00005 Direction ID Template SEQ ID 1.sup.st-stage ES0564
5'-GAACACGACCTACGACGAGTCAG 23 forward CATCTAGCTTCGGACCACCA-3'
primer (P1) reverse ES0563 5'-CTGCTACCCACAGCCGGTTA 24 primer G-3'
(P2) 2.sup.nd-stage ES0565 5'-CACGACCTACGACGAGTCAG 25 forward C-3'
primer (P3) TaqMan ES0566-M FAM-5'-TTCGGACCACCAGCAC- 26 MGB
3'-MGB-NFQ probe (Pr) MTB 5'-GCTTCGGACCACCAGCACCTAAC 27 target
CGGCTGTGGGTAGCAG-3'
[0211] FIG. 9, Panel A, demonstrates that this technique
effectively detects 5 genome-equivalents of M. tuberculosis per
reaction. FIG. 9, Panel B, illustrates application of this test for
detection of M. tuberculosis Tr-DNA in the urine samples from
patients with pulmonary tuberculosis and non-infected controls.
Specifically, Panel A shows amplification of IS6110 standards and
Panel B shows the detection of IS6110 in DNA from urine samples of
8 tuberculosis patients (TB1 through TB8) and two healthy
individuals (H1 and H2).
Example 4
[0212] Example 4 shows an alternative design of single-tube PCR
scheme for detection of ultra short DNA targets.
[0213] In performing the above-described experiments, it was
determined that to achieve better linearity of the assay it is
important to prevent primer P1 from competing for template with
probe Pr in stage 2 of the reaction. To that end, primer P1 was
modified in such a way that it preferentially exists in a folded,
stem-loop configuration at the annealing/extension phase
temperature(s) of stage 2. As shown in FIG. 10, to prevent the
stem-loop region from being copied into the PCR product, an iso-dC
replication-blocking base was introduced into the loop part of
primer P1. In FIG. 10, T designates a template; P1, P2, and P3
designate primers; IP1 and IP2 designate intermediate products; Pr
designates TaqMan.TM. probe, dual-labeled with fluorophore F and
quencher Q. designates iso-dC.
[0214] The 2-stage Real-Time PCR assay has been shown to be able to
detect a number of various targets. For each target, a custom set
of primer and probe sequences were designed, and the physical
conditions of the assay were optimized. The optimized factors
included Mg.sup.2+ concentration, primers and probe concentrations,
the temperatures of the annealing/extension phase for each stage,
and the length (i.e., the number of amplification cycles) of stage
1. The responses of the system which were optimized included assay
sensitivity, specificity, and linearity. It was found that the
2-stage qPCR assay can be used to reliably detect ultra short
targets at concentrations as low as 1 to 5 copies per reaction.
[0215] One such 2-stage Real-Time PCR system was designed to detect
25-bp SRY target. The amplification curves of the standard
concentration of positive control template are shown in FIG. 11.
FIG. 12 illustrates detection of fetal SRY sequences in DNA
isolated from urine of women pregnant with male but not with female
fetuses. In FIG. 12, M and F designate samples from women pregnant
with male and female fetuses, respectively.
Example 5
[0216] Example 5 shows the detection of fetal sequences of
different size in DNA isolated from maternal urine by two
methods.
[0217] DNA from the urine from women pregnant with male fetuses was
isolated both by the silica-based method of Botezatu et al., Clin
Chem. 46:1078-1084, 2000 and by the anion exchanger-based technique
described in U.S. Patent Application Publication No. 20080139801.
The isolated DNA was then analyzed for the presence of Y
chromosome-specific TSPY sequences by real time PCR using primers
designed for detection of 84 base pairs and ultra short 43 base
pairs targets. The silica-based method isolates 100-150 base pairs
DNA fragments and larger. The anion-exchanger-based technique
isolates DNA fragments larger than 10 base pairs. Independent of
the DNA purification method, the male-specific sequences were
successfully detected in urinary DNA using the 43 base pairs
amplicon assay, but not the 84 base pairs assay. Furthermore, FIG.
13 demonstrates that the detected amount of TSPY sequences in
Q-Sepharose preparation was twice as high as that in the silica
preparation.
[0218] The next experiments were designed to characterize fetal
Tr-DNA in more details. Using four sets of primers and probes,
which amplified 25 base pairs, 39 base pairs, 65 base pairs, and 88
base pairs sequences of the SRY gene, real time PCR was performed
with DNA isolated from the same urine specimens of women pregnant
with male fetuses by two techniques, based on the silica or Q-resin
absorption. Data presented in the following table clearly
demonstrate that both factors are very important, a method of DNA
isolation and the amplicon size. First, sensitivity is higher with
shorter amplicon size. Even the increase of a target sequence size
from 25 base pairs to 39 base pairs decreased test sensitivity, and
fetal DNA was undetectable in all samples with 88 base pairs
amplicon. Second, isolation of DNA fragments shorter than 150 base
pairs with the Q-resin-based technique significantly increased
sensitivity when 25 base pairs and 39 base pairs sequences were
amplified. Both DNA isolation methods gave similar results with the
65 base pairs amplicon.
[0219] Numbers of successfully detected pregnancies with male
fetuses depending on the amplicon size. DNA was isolated by two
methods from urine samples of ten women pregnant with male
fetuses.
TABLE-US-00006 DNA isolation Size of SRY target method 25 base
pairs 39 base pairs 65 base pairs 88 base pairs Q-resin 10 8 3 0
Silica 7 4 3 0
[0220] Data obtained provide information on properties of Tr-DNA,
in particular characteristics of fetal Tr-DNA in the maternal
urine. First, higher sensitivity of detection of fetal sequence in
DNA purified with Q-resin when compared to DNA isolated by the
silica method demonstrates that 50-<150 base pairs DNA fragments
contain fetal Tr-DNA. This difference in sensitivity is seen with
25 base pairs and 39 base pairs amplicons only, which means that
larger fragments of fetal Tr-DNA detectable with 65 base pairs
amplicon belong to DNA fractions isolated by both methods, most
likely to 150-200 base pairs DNA fragments.
[0221] Second, sensitivity of detection of fetal sequences in DNA
isolated with the silica method depends on the amplicon size in a
size range of 25-88 base pairs, although the shortest DNA fragments
isolated by this technique are about 150 base pairs long. The most
plausible explanation of these results is the presence of
single-strand breaks in 150-200 base pairs fragments of Tr-DNA,
which makes amplifiable targets significantly shorter.
[0222] Based on the foregoing results, detection of ultra short DNA
targets is essential to the successful detection of Tr-DNA
sequences.
Example 6
[0223] Example 6 shows the detection of bacterial transrenal DNA
sequences of different size in DNA isolated from urine of
tuberculosis patients by two methods.
[0224] Since bacterial DNA is covered by different proteins than
eukaryotic DNA and is not packed in nucleosomes, data obtained with
human Tr-DNA are not necessarily correct for prokaryotic DNA.
Recently, Mycobacterium tuberculosis (MTB) DNA was detected by
means of nested PCR in the urine from pulmonary tuberculosis
patients (Cannas, A. et al., Int. J. Tuberc. Lung Dis. 12: 146-151,
2008). In the first set of experiments with MTB DNA, urinary DNA
was isolated by the two methods described above and analyzed by
real time PCR using primers designed for amplicons of various
sizes. The table below demonstrates that bacterial Tr-DNA fragments
can be detected in the urine from infected patients by
amplification of short and ultra short targets, but in the latter
case a significantly higher number of MTB-specific gene copies are
detected. Still more copies of the MTB DNA sequences were detected
with primers designed for ultra short targets in DNA preps isolated
by anion exchanger-based methods.
[0225] Amplicon size dependence of MTB Tr-DNA detection (copies/ml)
in nucleic acids isolated by two methods from urine of a patient
with active pulmonary tuberculosis.
TABLE-US-00007 Target Size Silica Q-Sepharose 39 base pairs 24 50
49 base pairs 5 4 99 base pairs 0 0
Example 7
[0226] Example 7 shows the detection of prokaryotic and eukaryotic
transrenal DNA in fractionated urinary DNA.
[0227] Nucleic acids isolated from urine were further separated
into high/medium and low molecular weight fractions. Ethanol was
added to nucleic acids eluted from the Q-Sepharose to 30% v/v, and
the mixture was passed through a silica column. A flow-through
fraction was collected, and after the addition of ethanol up to 70%
the mixture was loaded onto another silica column. High/medium and
low molecular weight nucleic acids, respectively, were eluted from
the first and the second columns. FIG. 14 illustrates separation of
urinary nucleic acids of different molecular weights. In FIG. 14,
Lane 1 shows total nucleic acids; Lane 2 shows high/medium
molecular weight fraction and Lane 3 shows low molecular weight
fractions.
[0228] In the first set of experiments, the amount of Y
chromosome-specific TSPY and SRY sequences in the high/medium and
in low molecular weight fractions was compared. Data presented in
the following table demonstrate that the latter contained the
significant amount of fetus-specific sequences.
[0229] Concentrations of fetal Tr-DNA in nucleic acid fractions
from urine of a woman pregnant with a male fetus.
TABLE-US-00008 TSPY SRY DNA GE/ml of urine GE/ml of urine Total DNA
44 67 High/medium MW 3 19 Low MW 33 61
[0230] Similar experiments were performed for analysis of bacterial
Tr-DNA distribution in fractionated urinary DNA. The results of MTB
DNA analysis in fractionated DNA from the urine of a patient with
pulmonary tuberculosis are presented in FIG. 15. In FIG. 15, Lane 1
shows total DNA; Lane 2 shows high/medium molecular weight DNA;
Lane 3 shows low molecular weight DNA; Lane 5 shows no template
(control reaction); and Lane 5 shows positive control with MTB
genomic DNA (Erdman strain).
[0231] Once again, MTB-specific sequences were detected in both
high/medium and low molecular weight fraction, the latter contains
more copies of bacterial Tr-DNA.
Sequence CWU 1
1
27120DNAArtificial Sequenceprimer sequence 1cgtccgtgct gtcgacgtag
20221DNAArtificial Sequenceprimer sequence 2cataccacgc catcagagtg c
21333DNAArtificial Sequenceprimer sequence 3cagcacgtcc gtgctgtcga
cgtagacatc agc 33424DNAArtificial Sequenceprimer sequence
4actctgatgg cgtggtatga cgac 24532DNAArtificial Sequenceprimer
sequence 5cagcacgtcc gtgctgtcga cgtagcatca gc 32624DNAArtificial
Sequenceprimer sequence 6actctgatgg cgtggtatga cgac
24731DNAArtificial Sequenceprimer sequence 7cagcacgtcc gtgctgtcga
cgtagatcag c 31824DNAArtificial Sequenceprimer sequence 8actctgatgg
cgtggtatga cgac 24929DNAArtificial Sequenceprimer sequence
9cagcacgtcc gtgctgtcga cgtagatgc 291024DNAArtificial Sequenceprimer
sequence 10actctgatgg cgtggtatga cgac 241128DNAArtificial
Sequenceprimer sequence 11cagcacgtcc gtgctgtcga cgtagagc
281224DNAArtificial Sequenceprimer sequence 12actctgatgg cgtggtatga
cgac 241327DNAArtificial Sequenceprimer sequence 13cagcacgtcc
gtgctgtcga cgtaggc 271424DNAArtificial Sequenceprimer sequence
14actctgatgg cgtggtatga cgac 241525DNAArtificial Sequenceprimer
sequence 15cagcacgtcc gtgctgtcga cgtac 251624DNAArtificial
Sequenceprimer sequence 16actctgatgg cgtggtatga cgac
241721DNAArtificial Sequenceprimer sequence 17atcgcagagc ccaggatgtc
c 211823DNAArtificial Sequenceprimer sequence 18acgagctcta
gggtcccttc tgg 231934DNAArtificial Sequenceprimer sequence
19cagagcccag gatgtccccc agaagggacc ctag 342017DNAArtificial
Sequenceprimer sequence 20ccgcagatcc cgcttcg 172120DNAArtificial
Sequenceprimer sequence 21gcacttcgct gcagagtacc 202236DNAArtificial
Sequenceprimer sequence 22ccgcagatcc cgcttcggta ctctgcagcg aagtgc
362343DNAArtificial Sequenceprimer sequence 23gaacacgacc tacgacgagt
cagcatctag cttcggacca cca 432421DNAArtificial Sequenceprimer
sequence 24ctgctaccca cagccggtta g 212521DNAArtificial
Sequenceprimer sequence 25cacgacctac gacgagtcag c
212616DNAArtificial Sequenceprimer sequence 26ttcggaccac cagcac
162739DNAArtificial Sequenceprimer sequence 27gcttcggacc accagcacct
aaccggctgt gggtagcag 39
* * * * *