Analytical methods for cell free nucleic acids and applications

Abstract

The present invention is directed to an in vitro method of detecting cell free nucleic acids, preferably cell free DNA (cfDNA) in a body fluid sample from an individual or a patient, wherein the method comprises the step of accurately and sensitively determining the concentration of cell free nucleic acid in the sample and/or determining the concentration or amount of said cell free nucleic acid of a size range and/or the index of integrity or size fraction ratio (SFR) of said cell free nucleic acid and/or the determination of the presence of genetic polymorphisms (such as known Single Nucleotide Polymorphisms (SNPs) or mutations). The invention encompasses also a method to discriminate body fluid individuals where cfDNA are highly released by comparing the size profile obtained for at least one of three size ranges of cfDNA. The invention also encompasses a method for analysing cell free nucleic acids in individuals for the diagnosis, prognosis or for assessing the evolution of a physiological state, such as the progression of a tumor or metastatic cancer, for monitoring the efficacy of a cancer treatment in a patient or for theragnostic purposes implementing the analysis of these biomarkers.

We claim: 
     
         1 . A kit comprising two sets of nucleic acid primers amplifying distinct sequences of a same targeted nucleic acid region, wherein:
 a) a first set of primers comprises a forward primer designated A1 and a reverse primer designated A2, said primers A1 and A2:   have a minimal size of 15 nucleotides and a maximal size of 30 nucleotides in length, and   have a minimal spacing of at least 5 bp between the two primers, between the 3′ extremities of both primers, and   allow amplification of a first sequence of the targeted nucleic acid region having a length comprised between 35 and 100 bp;   b) a second set of primers comprises a forward primer designated B1 and a reverse primer designated B2, said primers B1 and B2:   have a minimal size of 15 nucleotides and a maximal size of 30 nucleotides in length, and   have a minimal spacing of at least 5 bp between the two primers, between the 3′ extremities of both primers, and   allow amplification of a second sequence of the targeted nucleic acid region having a length comprised between 35 and 100 bp;   c) wherein primers A1 and B2 allow amplification of a sequence of the targeted nucleic acid region having a length comprised between 250-450 bp; and   d) wherein the first sequence of the targeted nucleic acid region amplified by primers A1 and A2 and the second sequence of the targeted nucleic acid region amplified by primers B1 and B2 differ in length from each other by at most 20%.   
     
     
         2 . The kit of  claim 1 , wherein the amplified first sequence of the targeted nucleic acid region has a length comprised between 40 and 99 bp, preferably between 55 and 65 bp. 
     
     
         3 . The kit of  claim 1 , wherein the amplified second sequence of the targeted nucleic acid region has a length comprised between 40 and 99 bp, preferably between 55 and 65 bp. 
     
     
         4 . The kit of  claim 1 , wherein primers A1 and B2 allow amplification of a sequence of the targeted nucleic acid region having a length comprised between 250 and 350 bp. 
     
     
         5 . The kit of  claim 1 , wherein the second set of primers amplifies a sequence of the targeted nucleic acid region carrying a mutation. 
     
     
         6 . The kit of  claim 1 , wherein the first set of primers amplifies a sequence of the targeted nucleic acid region carrying a mutation. 
     
     
         7 . The kit of  claim 1 , wherein the targeted nucleic acid region is a gene mutated in a cancer. 
     
     
         8 . The kit of  claim 7 , wherein the targeted gene is KRAS. 
     
     
         9 . The kit of  claim 8 , wherein A1 is selected from SEQ ID NOs: 163 to 196; A2 is selected from SEQ ID NOs: 250 to 256; B1 is selected from SEQ ID NOs: 244 to 249 and/or B2 is selected from SEQ ID NOs: 83 to 120. 
     
     
         10 . The kit of  claim 8 , wherein A1, A2, B1 and B2 are selected from SEQ ID NOs: 1 to 15, preferably from SEQ ID NOs: 1 to 8. 
     
     
         11 . The kit of  claim 8 , wherein A1, A2, B1 and B2 are selected from SEQ ID NOs: 28 to 36. 
     
     
         12 . The kit of  claim 7 , wherein the targeted gene is BRAF. 
     
     
         13 . The kit of  claim 12 , wherein A1 is selected from SEQ ID NOs: 65 to 81; A2 is SEQ ID NO: 19; B1 is SEQ ID NO: 38 and/or B2 is selected from SEQ ID NOs: 40 to 63. 
     
     
         14 . The kit of  claim 12 , wherein A1, A2, B1 and B2 are selected from SEQ ID NOs: 16 to 21. 
     
     
         15 . The kit of  claim 12 , wherein A1, A2, B1 and B2 are selected from SEQ ID NOs: 23 to 26. 
     
     
         16 . A composition comprising two sets of nucleic acid primers as defined in  claim 1 . 
     
     
         17 . An oligonucleotide consisting of a nucleic acid sequence selected from SEQ ID NOs: 1 to 256. 
     
     
         18 . A method for detecting a targeted nucleic acid region in a sample, comprising:
 (i) contacting the sample with two sets of nucleic acid primers, wherein:   a) a first set of primers comprises a forward primer designated A1 and a reverse primer designated A2, said primers A1 and A2:   have a minimal size of 15 nucleotides and a maximal size of 30 nucleotides in length, and   have a minimal spacing of at least 5 bp between the two primers, between the 3′ extremities of both primers, and   allow amplification of a first sequence of the targeted nucleic acid region having a length comprised between 35 and 100 bp;   b) a second set of primers comprises a forward primer designated B1 and a reverse primer designated B2, said primers B1 and B2:   have a minimal size of 15 nucleotides and a maximal size of 30 nucleotides in length, and   have a minimal spacing of at least 5 bp between the two primers, between the 3′ extremities of both primers, and   allow amplification of a second sequence of the targeted nucleic acid region having a length comprised between 35 and 100 bp;   c) wherein primers A1 and B2 allow amplification of a sequence of the targeted nucleic acid having a length comprised between 250-450 bp; and   d) wherein the first sequence of the targeted nucleic acid amplified by primers A1 and A2 and the second sequence of the targeted nucleic acid amplified by primers B1 and B2 differ in length from each other by at most 20%;   (ii) performing an amplification reaction, and   (iii) detecting an amplified nucleic acid.   
     
     
         19 . The method of  claim 18 , wherein the amplified first sequence of the targeted nucleic acid has a length comprised between 40 and 99 bp, preferably between 55 and 65 bp. 
     
     
         20 . The method of  claim 18 , wherein the amplified second sequence of the targeted nucleic acid has a length comprised between 40 and 99 bp, preferably between 55 and 65 bp. 
     
     
         21 . The method of  claim 18 , wherein primers A1 and B2 allow amplification of a sequence of the targeted nucleic acid having a length comprised between 250 and 350 bp. 
     
     
         22 . The method of  claim 18 , wherein the first or second set of primers amplifies a sequence of the targeted nucleic acid carrying a mutation. 
     
     
         23 . The method of  claim 18 , wherein the targeted nucleic acid region is a gene mutated in a cancer. 
     
     
         24 . The method of  claim 23 , wherein the targeted gene is KRAS. 
     
     
         25 . The method of  claim 24 , wherein A1 is selected from SEQ ID NOs: 163 to 196; A2 is selected from SEQ ID NOs: 250 to 256; B1 is selected from SEQ ID NOs: 244 to 249 and/or B2 is selected from SEQ ID NOs: 83 to 120. 
     
     
         26 . The method of  claim 24 , wherein A1, A2, B1 and B2 are selected from SEQ ID NOs: 1 to 15, preferably from SEQ ID NOs: 1 to 8. 
     
     
         27 . The method of  claim 24 , wherein A1, A2, B1 and B2 are selected from SEQ ID NOs: 28 to 36. 
     
     
         28 . The method of  claim 23 , wherein the targeted gene is BRAF. 
     
     
         29 . The method of  claim 28 , wherein A1 is selected from SEQ ID NOs: 65 to 81;A2 is SEQ ID NO: 19; B1 is SEQ ID NO: 38 and/or B2 is selected from SEQ ID NOs: 40 to 63. 
     
     
         30 . The method of  claim 28 , wherein A1, A2, B1 and B2 are selected from SEQ ID NOs: 16 to 21. 
     
     
         31 . The method of  claim 28 , wherein A1, A2, B1 and B2 are selected from SEQ ID NOs: 23 to 26.