DNA Diagnostics Based on Mass Spectrometry

Abstract

Fast and highly accurate mass spectrometry-based processes for detecting a particular nucleic acid sequence in a biological sample are provided. Depending on the sequence to be detected, the processes can be used, for example, to diagnose a genetic disease or chromosomal abnormality; a predisposition to a disease or condition, infection by a pathogenic organism, or for determining identity or heredity.

1 - 103 . (canceled) 
     
     
         104 . A process for characterizing the structure of one or more target nucleic acids from a biological sample, comprising:
 a) determining the base composition of at least one nucleic acid molecule having a known sequence;   b) amplifying one or more target nucleic acid molecules from a biological sample;   c) ionizing and volatilizing the product of step b);   d) analyzing a product of step c) by mass spectrometry to determine an observed molecular mass of said product of step c);   e) obtaining a number of possible base compositions of said product of step c) from the observed molecular mass determined in step d);   f) constraining the number of possible base compositions obtained in step e); and   g) comparing at least one base composition obtained in step e) or at least one base composition constrained in step f) with the base composition determined in step a);   whereby the structure of the one or more target nucleic acids is characterized based on the comparison of (g).   
     
     
         105 . The process of  claim 104  wherein step d) further comprises analyzing a second product of step c) by mass spectrometry to determine an observed molecular mass of the second product of step c), wherein said second product of step c) is the amplification product complementary to the product of step c), and wherein step e) further comprises obtaining a number of possible base compositions of the second product of step c) from the observed molecular mass of the second product of step c), and wherein step f) further comprises constraining the number of possible base compositions of the second product of step c). 
     
     
         106 . The process of  claim 105  wherein step f) comprises constraining the number of possible base compositions of said product of step c) using a composition constraint, said composition constraint comprising the base composition obtained in step e) for the second product of step c), or the base composition constrained in step f) for the second product of step c). 
     
     
         107 . The process of  claim 104  wherein step f) comprises making a mass measurement with an accuracy allowing detection of a mass shift of 0.03% to 0.005%. 
     
     
         108 . The process of  claim 104  wherein electrospray mass spectrometry is used in step d). 
     
     
         109 . The process of  claim 104  wherein step f) comprises the use of a composition constraint. 
     
     
         110 . The process of  claim 109  wherein said composition constraint comprises the length of the product of step c). 
     
     
         111 . The process of  claim 109  wherein said composition constraint comprises at least one known nucleotide in the sequence of the product of step c). 
     
     
         112 . The process of  claim 111  wherein said known nucleotide is a nucleotide in the sequence of an amplification primer used to generate the product of step c). 
     
     
         113 . The process of  claim 109  wherein said composition constraint comprises sequence information of a nucleic acid complementary to the product of step c). 
     
     
         114 . The process of  claim 113  wherein said sequence information comprises at least one known nucleotide in the sequence of the nucleic acid complementary to the product of step c). 
     
     
         115 . The process of  claim 113  wherein said composition constraint comprises the base composition of a second product of step c), wherein said second product of step c) is the amplification product complementary to the product of step c). 
     
     
         116 . The process of  claim 104 , wherein amplification step b) is performed by PCR using primers complementary to sequences of the target nucleic acid molecule. 
     
     
         117 . The process of  claim 116 , wherein step f) comprises the use of a composition constraint, said composition constraint comprising sequence information from a nucleic acid molecule known to be amplifiable using the primers complementary to sequences of the target nucleic acid molecule. 
     
     
         118 . The process of  claim 104 , wherein said determining in step a) comprises calculating the base composition of the at least one nucleic acid molecule having a known sequence. 
     
     
         119 . The process of  claim 104 , wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the molecular mass of one or more target nucleic acids in a biological sample. 
     
     
         120 . The process of  claim 104 , wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the base composition of one or more target nucleic acids in a biological sample. 
     
     
         121 . The process of  claim 104 , wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the length of one or more target nucleic acids in a biological sample. 
     
     
         122 . A process for characterizing the structure of one or more target nucleic acids from a biological sample, comprising:
 a) determining the base composition and molecular mass of at least one nucleic acid molecule having a known sequence;   b) amplifying one or more target nucleic acid molecules from a biological sample;   c) ionizing and volatilizing the product of step b);   d) analyzing a product of step c) by mass spectrometry to determine an observed molecular mass for said product of step c);   e) obtaining a number of possible base compositions of said product of step c) from the observed molecular mass determined in step d);   f) constraining the number of possible base compositions obtained in step e); and   g) comparing at least one base composition obtained in step e) with the base composition determined in step a), or comparing at least one base composition obtained in step f) with the base composition determined in step a), or comparing the observed molecular mass determined in step d) with the molecular mass determined in step a);   whereby the structure of the one or more target nucleic acids is characterized based on the comparison of (g).   
     
     
         123 . The process of  claim 122  wherein step d) further comprises analyzing a second product of step c) by mass spectrometry to determine an observed molecular mass of the second product of step c), wherein said second product of step c) is the amplification product complementary to the product of step c), and wherein step e) further comprises obtaining a number of possible base compositions of the second product of step c) from the observed molecular mass of the second product of step c), and wherein step f) further comprises constraining the number of possible base compositions of the second product of step c). 
     
     
         124 . The process of  claim 123  wherein step f) comprises constraining the number of possible base compositions of said product of step c) using a composition constraint, said composition constraint comprising the base composition obtained in step e) for the second product of step c), or the base composition constrained in step f) for the second product of step c). 
     
     
         125 . The process of  claim 122  wherein step f) comprises making a mass measurement with an accuracy allowing detection of a mass shift of 0.03% to 0.005%. 
     
     
         126 . The process of  claim 122  wherein electrospray mass spectrometry is used in step d). 
     
     
         127 . The process of  claim 122  wherein step f) comprises the use of a composition constraint. 
     
     
         128 . The process of  claim 127  wherein said composition constraint comprises the length of the product of step c). 
     
     
         129 . The process of  claim 127  wherein said composition constraint comprises at least one known nucleotide in the sequence of the product of step c). 
     
     
         130 . The process of  claim 129  wherein said known nucleotide is a nucleotide in the sequence of an amplification primer used to generate the product of step c). 
     
     
         131 . The process of  claim 127  wherein said composition constraint comprises sequence information of a nucleic acid complementary to the product of step c). 
     
     
         132 . The process of  claim 131  wherein said sequence information comprises at least one known nucleotide in the sequence of the nucleic acid complementary to the product of step c). 
     
     
         133 . The process of  claim 131  wherein said composition constraint comprises the base composition of a second product of step c), wherein said second product of step c) is the amplification product complementary to the product of step c). 
     
     
         134 . The process of  claim 122 , wherein said amplification step b) is performed using PCR with primers complementary to sequences of the target nucleic acid molecule. 
     
     
         135 . The process of  claim 134 , wherein step f) comprises the use of a composition constraint, said composition constraint comprising sequence information from a nucleic acid molecule known to be amplifiable using the primers complementary to sequences of the target nucleic acid molecule. 
     
     
         136 . The process of  claim 122 , wherein said determining in step a) comprises calculating the base composition and molecular mass of the at least one nucleic acid molecule having a known sequence. 
     
     
         137 . The process of  claim 122 , wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the molecular mass of one or more target nucleic acids in a biological sample. 
     
     
         138 . The process of  claim 122 , wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the base composition of one or more target nucleic acids in a biological sample. 
     
     
         139 . The process of  claim 122 , wherein said characterizing the structure of one or more target nucleic acids in a biological sample comprises determining the length of one or more target nucleic acids in a biological sample.