Methods of PCR-Based Detection of "Ultra Short" Nucleic Acid Sequences
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.
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.