METHOD FOR ISOLATING AND ANALYZING CELL FREE DNA
The invention provides methods of detecting substantially all types of cell free DNA (cfDNA) in biological samples, including nucleosome-bound cfDNA, exosome-bound cfDNA and unbound cfDNA (including double stranded DNA (dsDNA), single stranded DNA (ssDNA) and oligonucleotides), for diagnosis, monitoring and treatment of diseases caused by, or correlated with, increased levels of cfDNA.
1 . A method for isolating a cell free DNA (cfDNA) from a biological sample comprising the cfDNA, the method comprising:
(i) contacting the biological sample with a linker histone, wherein the linker histone forms a complex with the cfDNA;
(ii) separating the complex obtained in step (i) from the biological sample, and
(iii) releasing the cfDNA from the complex separated in step (ii).
2 - 5 . (canceled)
6 . The method of claim 1 , wherein the linker histone is immobilized on a solid support.
7 . (canceled)
8 . The method of claim 1 , wherein the linker histone is bound to a magnetic particle.
9 . (canceled)
10 . The method of claim 1 , wherein the biological sample is a blood sample, a serum sample, a plasma sample, a cerebrospinal fluid (CSF) sample, an endometrial fluid sample, a urine sample, a saliva sample, a lymph sample, a tear fluid sample, a synovial fluid sample, or a sputum sample.
11 . The method of claim 10 , wherein the biological sample is a blood sample, a plasma sample, or a serum sample.
12 - 16 . (canceled)
17 . The method of claim 1 , wherein the linker histone is a mammalian somatic linker histone.
18 . The method of claim 1 , wherein the linker histone is a linker histone H1 or a linker histone H5.
19 . The method of claim 18 , wherein the linker histone H1 is selected from an H1.0 linker histone, an H1.1 linker histone, an H1.2 linker histone, an H1.3 linker histone, an H1.4 linker histone, and an H1.5 linker histone.
20 . The method of claim 19 , wherein the linker histone H1 is a human H1.3 linker histone.
21 . The method of claim 19 , wherein the linker histone H1 is a human H1.0 linker histone.
22 . The method of claim 1 , wherein the linker histone comprises an amino acid sequence which is at least 70% identical to the sequence
(SEQ ID NO: 1)
MSETAPLAPTIPAPAEKTPVKKKAKKAGATAGKRKASGPP
VSELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSR
IKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEGKPK
AKKAGAAKPRKPAGAAKKPKKVAGAATPKKSIKKTPKKVK
KPATAAGTKKVAKSAKKVKTPQPKKAAKSPAKAKAPKPKA
AKPKSGKPKVTKAKKAAPKKK
or to the sequence
(SEQ ID NO: 2)
TENSTSAPAAKPKRAKASKKSTDHPKYSDMIVAAIQAEKN
RAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTGVL
KQTKGVGASGSFRLAKSDEPKKSVAFKKTKKEIKKVATPK
KASKPKKAASKAPTKKPKATPVKKAKKKLAATPKKAKKPK
TVKAKPVKASKPKKAKPVKPKAKSSAKRAGKKK.
23 . The method of claim 1 , wherein the linker histone comprises an amino acid sequence which is at least 70% identical to the sequence
(SEQ ID NO: 3)
TDSPIPAPAPAAKPKRARAPRKPASHPTYSEMIAAAIRAD
KSRGGSSRQSIQKYVKSHYKVGQHADLQIKLAIRRLLTTG
VLKQTKGVGASGSFRLAKGDKAKRSPAGRKKKKKAARKST
SPKKAARPRKARSPAKKPKAAARKARKKSRASPKKAKKPK
TVKAKSLKTSKPKKARRSKPRAKSGARKSPKKK
or to the sequence
(SEQ ID NO: 4)
MTESLVLSPAPAKPKRVKASRRSASHPTYSEMIAAAIRAE
KSRGGSSRQSIQKYIKSHYKVGHNADLQIKLSIRRLLAAG
VLKQTKGVGASGSFRLAKSDKAKRSPGKKKKAVRRSTSPK
KAARPRKARSPAKKPKATARKARKKSRASPKKAKKPKTVK
AKSRKASKAKKVKRSKPRAKSGARKSPKKK.
24 . The method of claim 1 , wherein releasing the cfDNA comprises contacting the complex with a protease.
25 - 34 . (canceled)
35 . The methods of claim 1 , wherein the separating step (ii) comprises one or more of centrifugation, sedimentation or filtration.
36 - 56 . (canceled)