Method and device for separating molecular targets in a complex mixture

Abstract

The invention relates to a method of analysing molecular targets contained in a complex mixture, comprising the following steps consisting in: a) bringing the mixture of molecular targets to be analysed into contact with an array of different types of primary probes, whereby each type of primary probe forming the array can bind specifically to a type of target selected from among the molecular targets, under conditions that enable specific binding between the molecular targets and the primary probes; b) optionally eliminating the primary probes that are not bound specifically to a molecular target; c) separating the molecular targets and the primary probes which are bound specifically in a probe/target complex, such as to recover the array of primary probes representing a fingerprint of the molecular targets to be analysed; and d) quantitatively analysing the primary probes eluted in step c.

1 - 55 . (canceled) 
     
     
         56 . A method for analysing molecular targets contained in a complex mixture comprising:
 a. putting into contact in solution the mixture of molecular targets to be analysed with a set of primary probes of different types, each type of primary probe of the set being suitable to be bound by a specific bond to a single type of molecular target of said complex mixture, in conditions allowing a specific bond between said molecular targets and said primary probes to form a primary probe-target complex in solution, each primary probe being comprised of a polynucleotide or containing a portion forming a polynucleotide of which the nucleotide sequence is known and is, for each type of primary probe, different from all the nucleotide sequences of the polynucleotides of the other types of primary probe,   b. optionally eliminating the primary probes not specifically bound with a molecular target,   c. separating the molecular targets and the primary probes bound by a specific bond in said primary probe-target complex, so as to recover the primary probes representing a fingerprint of the molecular targets to be analysed, and   d. analysing of the primary probes eluted at step c, comprising identifying and quantifying the polynucleotide of each eluted primary probe.   
     
     
         57 . The method according to  claim 56 , wherein the molecular targets of the mixture are bound to particles before being put in contact with the set of primary probes. 
     
     
         58 . The method according to  claim 56 , wherein the molecular targets of the mixture are nucleic acids. 
     
     
         59 . The method according to  claim 56 , wherein the molecular targets of the mixture are polypeptides or are both nucleic acids and polypeptides. 
     
     
         60 . The method according to  claim 56 , wherein the polynucleotide of each primary probe is intended to bind by means of a specific binding and of a single site with a single type of molecular target in the mixture. 
     
     
         61 . The method according to  claim 56 , wherein the primary probes of the set of probes comprise a polypeptide portion associated with a polynucleotide, each type of primary probe being suitable, via its polypeptide portion, to recognise and bind specifically, by means of a single site, with a unique type of polypeptide molecular target, the polynucleotide portion of each type of probe (called a tag) also being specific to a unique type of molecular target. 
     
     
         62 . The method according to  claim 56 , wherein step a) is carried out by mixing an excess of primary probes with the molecular targets. 
     
     
         63 . The method according to  claim 56 , wherein step b) of eliminating the primary probes not specifically bound to the molecular targets is carried out after the step of separating the primary probes contained in the probe-target complex, prior to step d) of analysing the separated primary probes. 
     
     
         64 . The method according to  claim 56 , wherein the separation of the primary probes and the molecular targets during step c) is obtained by initially immobilising the targets on magnetic particles, and by applying a magnetic field to separate the “target-magnetic particles” entities from the probe-target complexes and recovering the primary probes from the complexes, after having separated them. 
     
     
         65 . The method according to  claim 56 , wherein the separation of the primary probes and the molecular targets during step c) is obtained by initially immobilising the molecular targets on particles, and by centrifuging to recover the primary probes, after having separated them. 
     
     
         66 . The method according to  claim 56 , wherein the polynucleotides of the set of primary probes are all of homogenous size, preferably identical, and each has a determined sequence for obtaining primary probes that are different from each other. 
     
     
         67 . The method according to  claim 66 , wherein the analysis of the polynucleotides of the primary probes is carried out using a set of polynucleotide probes called secondary, each type of secondary probe being specific to a unique type of primary probe. 
     
     
         68 . The method according to  claim 67 , wherein the analysis of the primary probes separated in step c) comprises:
 d. putting the primary probes separated at step c) into contact with secondary probes of different types, each type of secondary probe being suitable to be bound by a specific bond by hybridisation to the polynucleotide portion of the primary probes,   e. identifying the molecular targets from the detection and/or recovery and/or analysis of the polynucleotide portion of the primary probes hybridised to the secondary probes.   
     
     
         69 . The method according to  claim 68 , wherein step e) is carried out by making the primary probes circulate over the secondary probes immobilised on a substrate, by simple diffusion of the primary probes or by applying electrical potentials. 
     
     
         70 . The method according to  claim 69 , wherein the electrical potentials are applied by at least one network of electrodes. 
     
     
         71 . The method according to  claim 56 , wherein the polynucleotides of the set of primary probes are Peptide Nucleic Acids (PNAs), composite mixtures of nucleic acid / Peptide Nucleic Acid (PNA) or modified nucleic acids. 
     
     
         72 . The method according to  claim 68 , wherein the specific bond between the secondary probes and the primary probes is a hybridisation between the complementary nucleotide sequences and in that the separation of the hybrids formed between the primary probes and the secondary probes is obtained by controlled denaturing by raising the temperature. 
     
     
         73 . The method according to  claims 67 , wherein at least one detector is used to measure the variations of impedance related to the hybridisation of the polynucleotide portions of the primary and secondary probes. 
     
     
         74 . The method according to  claim 67 , wherein at least one detector is used to measure the variations of polarised light caused by the hybridisation of the polynucleotide portions of the primary and secondary probes. 
     
     
         75 . The method according to  claim 67 , wherein at least one detector is used to measure the hybridisation of the polynucleotide portions of the primary and secondary probes by PSR. 
     
     
         76 . The method according to  claim 56 , wherein the molecular targets to be analysed are nucleic sequences representative of a transcriptome. 
     
     
         77 . The method according to  claim 56 , wherein the molecular targets to be analysed constitute a proteome. 
     
     
         78 . The method according to  claim 56 , wherein step a) comprises putting the mixture liable to contain proteins to be analysed into contact with a set of primary probes of different types, consisting of a set of antibodies or fragments of antibodies containing an antigen binding site, each bound to a specific polynucleotide sequence (called sequence or polynucleotide tag), each type of antibody or fragment of antibody comprising the set being suitable to recognise a single type of protein to be analysed, in conditions allowing a specific bond between said proteins and said antibodies or fragments of antibodies. 
     
     
         79 . The method according to  claim 78 , wherein step c) comprises separating the proteins and the antibodies or fragments of antibodies bound by a specific bond, then separating each antibody or fragment of antibody and its specific polynucleotide tag, so as to recover the set of polynucleotide tags representing a fingerprint of the proteins to be analysed. 
     
     
         80 . The method according to  claim 79 , wherein analysis step d) comprises putting the sequence tags separated at step c) into contact with secondary probes of different types, each type of secondary probe being suitable to be bound by a specific bond to one type of polynucleotide tag. 
     
     
         81 . The method according to  claim 80 , wherein step e) comprises quantitative detection of the proteins from the detection, and/or the recovery and/or the analysis of polynucleotide tags bound to the secondary probes. 
     
     
         82 . The method according to  claim 56 , wherein the polynucleotides of the primary probes all have different masses and that at least one mass spectrometer is used to analyse these polynucleotides. 
     
     
         83 . The method according to  claim 78 , wherein the polynucleotides of the primary probes all have different sizes and that the polynucleotides are analysed by electrophoresis, or chromatography, or filtration. 
     
     
         84 . The method according to  claim 56 , wherein said step d. is performed by detecting an effect of the polynucleotide of each eluted primary probe, said effect being due to the nucleotide sequence, to the size or to the mass of the polynucleotide