Amplification and sequencing of transrenal nucleic acids
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 comprising
(a) obtaining a urine sample from a patient;
(b) isolating DNA smaller than 1000 base pairs from the urine sample; and
(c) analyzing said DNA for one or more gene sequence, wherein the gene sequence is specifically amplified using polymerase chain reaction (PCR) to produce an amplicon less than 50 nucleotides in length that comprises a portion of the DNA isolated in step (b),
wherein said PCR uses a primer pair comprising two primers that are each sufficiently complementary to hybridize with a target binding sequence of the mutant gene sequence or its complement,
wherein both primers of said primer pair are sufficiently complementary to the mutant gene sequence or its complement at said primers' 3′ portion to hybridize thereto, and said both primers further comprise nucleotide fragments at the 5′ end of both primers that are not sufficiently complementary to the mutant gene sequence or its complement to hybridize thereto,
wherein
the nucleotide fragment at the 5′ end of one primer of said primer pair comprises a donor fluorophore;
the nucleotide fragment at the 5′ end of the other primer of said primer pair comprises an acceptor fluorophore; and
the nucleotide fragments at the 5′ ends of both primers of said primer pair are complementary to each other,
such that the acceptor and donor are in proximity to cause acceptor fluorescence by Förster resonance energy transfer (FRET) in the amplicon, and
wherein the gene sequence is part of a cell-free genomic DNA fragment that has crossed the kidney barrier.
2. The method of claim 1 , wherein DNA smaller than 300 base pairs is isolated from the urine samples.
3. The method of claim 1 , wherein the amplicon is less than 45 nucleotides.
4. The method of claim 1 , wherein the amplicon is less than 40 nucleotides.
5. The method of claim 1 , wherein the amplicon is less than 35 nucleotides.
6. The method of claim 1 , wherein the DNA smaller than 1000 base pairs is isolated using an anion exchange resin.
7. The method of claim 6 , wherein the anion exchange resin is Q-Sepharose.
8. A method comprising
(a) obtaining a urine sample from a patient;
(b) isolating DNA smaller than 1000 base pairs from the urine sample; and
(c) analyzing said DNA for one or more gene sequence, wherein the gene sequence is specifically amplified using polymerase chain reaction (PCR) to produce an amplicon less than 50 nucleotides in length that comprises a portion of the DNA isolated in step (b),
wherein said PCR uses a primer pair comprising two primers that are each sufficiently complementary to hybridize with a target binding sequence of the mutant gene sequence or its complement,
wherein both primers of said primer pair are sufficiently complementary to the mutant gene sequence or its complement at said primers' 3′ portion to hybridize thereto, and said both primers further comprise nucleotide fragments at the 5′ end of both primers that are not sufficiently complementary to the mutant gene sequence or its complement to hybridize thereto,
wherein
said nucleotide fragment at the 5′ end of said at least one primer is internally complementary such that it forms a stem-loop configuration, wherein the loop part of said stem-loop comprises a replication-blocking base and forms an intermediate product IP1 when it binds to the mutant gene sequence and is extended by a polymerase;
the other primer of said primer pair forms an intermediate product IP2 when it binds to Ip1 and is extended by a polymerase to the replication blocking base; and
a labeled TaqMan probe Pr that is complementary to IP2 and comprises a fluorophore and quencher, is fragmented, allowing fluorescence from the fluorophore, when a third primer complementary to a region of IP2 that binds to IP2 in a region of IP2 that is 3′ from Pr, is extended by a polymerase, and
wherein the gene sequence is part of a cell-free genomic DNA fragment that has crossed the kidney barrier.
9. The method of claim 8 , wherein DNA smaller than 300 base pairs is isolated from the urine samples.
10. The method of claim 8 , wherein the amplicon is less than 45 nucleotides.
11. The method of claim 8 , wherein the amplicon is less than 40 nucleotides.
12. The method of claim 8 , wherein the amplicon is less than 35 nucleotides.
13. The method of claim 8 , wherein the amplicon is less than 30 nucleotides.
14. The method of claim 8 , wherein the DNA smaller than 1000 base pairs is isolated using an anion exchange resin.
15. The method of claim 14 , wherein the anion exchange resin is Q-Sepharose.
16. The method of claim 8 , wherein the gene sequence is chromosome Y-specific.
17. The method of claim 15 , wherein the chromosome Y-specific gene sequence is an SRY sequence.
18. The method of claim 16 , wherein the SRY sequence is 25 base pairs.