Electrophoretic detection and separation of mutant DNA using replaceable polymer matrices
First Claim
1. A method of resolving mutant DNA from non-mutant DNA in a DNA sample which, as obtained or modified, contains double stranded mutant DNA and double stranded non-mutant DNA, each with two iso-melting domains, a first iso-melting domain referred to as a low temperature iso-melting domain, which melts at a first temperature and a second iso-melting domain, referred to as a high temperature iso-melting domain, which melts at a second, higher temperature, wherein the mutant DNA contains a mutation of interest in the low temperature iso-melting domain, comprising the steps of:
- a) isolating the DNA sample from a biological source;
b) fragmenting DNA in the DNA sample isolated in a) to obtain fragmented double-stranded DNA of interest;
c) melting and reannealing the fragmented double-stranded DNA of interest obtained in step b) under conditions appropriate to form duplexed DNA, thereby producing a mixture of DNA heteroduplexes and DNA homoduplexes;
d) introducing the mixture produced in step c) into a replaceable polymer matrix contained within a capillary column, thereby producing a replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes;
e) subjecting the replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes to a high electrical field and partially denaturing conditions, wherein the partially denaturing conditions are constant denaturing conditions, whereby the DNA heteroduplexes partially melt and the DNA homoduplexes do not melt and the DNA heteroduplexes migrate at a slower velocity in the replaceable polymer matrix then the DNA homoduplexes, resulting in the separation of DNA heteroduplexes from DNA homoduplexes.
5 Assignments
0 Petitions
Accused Products
Abstract
The disclosure relates to a method for resolving double-stranded DNA species differing by at least one base pair. Each of the species is characterized by an iso-melting domain with a unique melting temperature contiguous with a melting domain of higher thermal stability.
-
Citations
51 Claims
-
1. A method of resolving mutant DNA from non-mutant DNA in a DNA sample which, as obtained or modified, contains double stranded mutant DNA and double stranded non-mutant DNA, each with two iso-melting domains, a first iso-melting domain referred to as a low temperature iso-melting domain, which melts at a first temperature and a second iso-melting domain, referred to as a high temperature iso-melting domain, which melts at a second, higher temperature, wherein the mutant DNA contains a mutation of interest in the low temperature iso-melting domain, comprising the steps of:
-
a) isolating the DNA sample from a biological source; b) fragmenting DNA in the DNA sample isolated in a) to obtain fragmented double-stranded DNA of interest; c) melting and reannealing the fragmented double-stranded DNA of interest obtained in step b) under conditions appropriate to form duplexed DNA, thereby producing a mixture of DNA heteroduplexes and DNA homoduplexes; d) introducing the mixture produced in step c) into a replaceable polymer matrix contained within a capillary column, thereby producing a replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes; e) subjecting the replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes to a high electrical field and partially denaturing conditions, wherein the partially denaturing conditions are constant denaturing conditions, whereby the DNA heteroduplexes partially melt and the DNA homoduplexes do not melt and the DNA heteroduplexes migrate at a slower velocity in the replaceable polymer matrix then the DNA homoduplexes, resulting in the separation of DNA heteroduplexes from DNA homoduplexes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
-
-
9. A method of resolving mutant DNA from non-mutant DNA in a DNA sample, which, as obtained or modified, contains double stranded mutant DNA and double stranded non-mutant DNA, each with one iso-melting domain but the iso-melting domain is not contiguous with an iso-melting domain of higher thermal stability, comprising the steps of:
-
a) isolating the DNA sample from a biological source; b) fragmenting DNA in the DNA sample in step a) to obtain fragmented double-stranded DNA of interest; c) partially isolating the fragmented DNA of interest by size separation to obtain partially isolated DNA fragments; d) attaching to the fragmented DNA of interest partially isolated in c) a DNA sequence which is an iso-melting domain of higher thermal stability than the iso-melting domain contained in the DNA as obtained, thereby producing DNA fragments of interest which comprise DNA sequences which contain an iso-melting domain contiguous with an iso-melting domain of higher thermal stability; e) melting and reannealing the DNA fragments obtained in step d) under conditions appropriate to form duplexed DNA, thereby producing a mixture of DNA heteroduplexes and DNA homoduplexes; f) introducing the mixture obtained in step e) into a replaceable polymer matrix contained within a capillary column, thereby producing a replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes; g) subjecting the replaceable polymer matrix in the capillary column containing the mixture of DNA heteroduplexes and DNA homoduplexes to a high electrical field and partially denaturing conditions, wherein the partially denaturing conditions are constant denaturing conditions, whereby the DNA heteroduplexes partially melt and the DNA homoduplexes do not melt and the DNA heteroduplexes migrate at a slower velocity in the replaceable polymer matrix than the DNA homoduplexes, resulting in the separation of DNA heteroduplexes from DNA homoduplexes. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16)
-
-
17. A method of resolving mutant DNA from non-mutant DNA in a DNA sample which, as obtained or modified, contains double stranded mutant DNA and double stranded non-mutant DNA, each with one iso-melting domain but the iso-melting domain is not contiguous with an iso-melting domain of higher thermal stability, comprising the steps of:
-
a) isolating the DNA sample from a biological source; b) fragmenting DNA in the DNA sample to obtain fragmented double-stranded DNA of interest; c) partially isolating the fragmented DNA by size separation to obtain partially isolated DNA fragments; d) melting and reannealing the DNA fragments obtained in step c) with synthetic DNA comprising the non-mutant DNA sequence containing an iso-melting domain as obtained with a DNA sequence of higher thermal stability attached thereto, under conditions appropriate to form duplexed DNA comprising two DNA strands, one strand being shorter than the other strand; e) extending the shorter strand of the duplexed DNA obtained in step d) with DNA polymerase to produce duplexes comprising two strands of the same length, thereby producing a mixture of DNA heteroduplexes and DNA homoduplexes comprising DNA containing an iso-melting domain contiguous with an iso-melting domain of higher thermal stability; f) introducing the mixture obtained in step e) into a replaceable polymer matrix contained within a capillary column, thereby producing a replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes; g) subjecting the replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes to a high electrical field and partially denaturing conditions, wherein the partially denaturing conditions are constant denaturing conditions, whereby the DNA heteroduplexes partially melt and the DNA homoduplexes do not melt and the DNA heteroduplexes migrate at a slower velocity in the replaceable polymer matrix than the DNA homoduplexes, resulting in the separation of DNA heteroduplexes from DNA homoduplexes. - View Dependent Claims (18, 19, 20, 21, 22, 23)
-
-
24. A method of resolving mutant DNA from non-mutant DNA in a DNA sample which, as obtained or modified, contains double stranded mutant DNA and double stranded non-mutant DNA which both lack a suitable iso-melting domain, comprising the steps of:
-
a) isolating the DNA sample of interest from a biological source; b) fragmenting DNA in the DNA sample to obtain fragmented double-stranded DNA of interest; c) partially isolating the fragmented DNA by size separation to obtain partially isolated DNA fragments; d) attaching to one end of the fragmented DNA of interest partially isolated in step c) a DNA sequence which is an iso-melting domain of higher thermal stability than the fragmented DNA, and to the other end, a DNA sequence which is an iso-melting domain which melts at, or near, the average melting temperature of the fragmented DNA sequence thereby producing DNA fragments which comprise a suitable iso-melting domain containing the DNA sequence of interest contiguous with an iso-melting domain of higher thermal stability; e) melting and reannealing the DNA fragments of interest obtained in step d) under conditions appropriate to form duplexed DNA, thereby producing a mixture of DNA heteroduplexes and DNA homoduplexes; f) introducing the mixture obtained in step e) into a replaceable polymer matrix contained within a capillary column, thereby producing a replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes; g) subjecting the replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes to a high electrical field and partially denaturing conditions, wherein the partially denaturing conditions are constant denaturing conditions, whereby the DNA heteroduplexes partially melt and the DNA homoduplexes do not melt and the DNA heteroduplexes migrate at a slower velocity in the polymer matrix than the DNA homoduplexes, resulting in the separation of DNA heteroduplexes from DNA homoduplexes. - View Dependent Claims (25, 26, 27, 28, 29, 30)
-
-
31. A method of resolving mutant DNA from non-mutant DNA in a DNA sample which, as obtained or modified, contains double stranded mutant DNA and double stranded non-mutant DNA, each with two iso-melting domains, a first iso-melting domain referred to as low temperature iso-melting domain, which melts at a first temperature and a second iso-melting domain, referred to as a high temperature iso-melting domain, which melts at a second, higher temperature and wherein the mutant DNA contains a mutation of interest is in the low temperature iso-melting domain, comprising the steps of:
-
a) isolating the DNA sample from a biological source; b) fragmenting the DNA in DNA sample to obtain fragmented double-stranded DNA of interest; c) melting and reannealing the double-stranded DNA fragments obtained in step b) under conditions appropriate to form duplexed DNA, thereby producing a mixture of DNA heteroduplexes and DNA homoduplexes; d) introducing the mixture obtained in step c) into a replaceable polymer matrix contained in a capillary column, thereby producing a replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes; e) subjecting the replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes to a high electrical field and partially denaturing conditions, wherein the partially denaturing conditions are constant denaturing conditions, whereby the DNA heteroduplexes partially melt and the DNA homoduplexes do not melt and the DNA heteroduplexes migrate at a slower velocity in the replaceable polymer matrix than the DNA homoduplexes, resulting in the separation of DNA heteroduplexes from DNA homoduplexes; f) detecting the relative movement of the DNA heteroduplexes and DNA homoduplexes as they exit from the replaceable polymer matrix and collecting fractions containing the DNA heteroduplexes and DNA homoduplexes as they exit from the replaceable polymer matrix; g) removing the replaceable polymer matrix from the channel and introducing fresh replaceable polymer matrix into the channel; and h) reintroducing the DNA heteroduplexes collected in step f) into the fresh replaceable polymer matrix, thereby producing a fresh replaceable polymer matrix containing DNA heteroduplexes and subjecting the fresh replaceable polymer matrix to a high electrical field and, as in step e), collecting fractions containing the DNA heteroduplexes as they exit from the fresh replaceable polymer matrix. - View Dependent Claims (32, 33, 34, 35)
-
-
36. A method of screening a DNA sample for mutations which, as obtained or modified, contains non-mutant DNA and is suspected of containing mutant DNA, wherein the mutant DNA and the non-mutant DNA contain two iso-melting domains, a first iso-melting domain, referred to as a low-temperature iso-melting domain, which melts at a first temperature, and a second iso-melting domain, referred to as a high temperature iso-melting domain, which melts at a second, higher temperature and the mutant DNA contains a mutation of interest in the low temperature iso-melting domain, comprising the steps of:
-
a) isolating the DNA sample from a biological source; b) fragmenting DNA in the DNA sample to obtain fragmented double-stranded DNA of interest; c) labeling the fragmented DNA of interest obtained in step b) with a detectable reporter group thereby producing labeled DNA of interest; d) melting and reannealing the labeled DNA of interest under conditions appropriate to form duplexed DNA, thereby producing DNA heteroduplexes, if mutant DNA is present, and DNA homoduplexes; e) introducing the duplexes produced in step d) into a replaceable polymer matrix contained within a capillary column; f) subjecting the replaceable polymer matrix containing the DNA duplexes to a high electrical field and partially denaturing conditions, wherein the partially denaturing conditions are constant denaturing conditions, whereby if DNA heteroduplexes are present, the DNA heteroduplexes partially melt and the DNA homoduplexes do not melt and the DNA heteroduplexes migrate at a slower velocity in the replaceable polymer matrix than the DNA homoduplexes, resulting in the separation of DNA heteroduplexes from DNA homoduplexes; and g) detecting the relative movement of the DNA heteroduplexes, if present, and DNA homoduplexes as they exit from the replaceable polymer matrix and collecting fractions containing the DNA heteroduplexes, if present, and DNA homoduplexes as they exit from the replaceable polymer matrix, wherein the presence of DNA heteroduplexes is indicative of the presence of mutations in the DNA sample. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43)
-
-
44. A method for resolving two or more species of double-stranded DNA fragments which differ by at least one base pair, the double-stranded DNA fragments comprising two iso-melting domains, a first iso-melting domain, referred to as a low-temperature iso-melting domain, which melts at a first temperature, and a second iso-melting domain, referred to as a high temperature iso-melting domain, which melts at a second, higher temperature and the mutant DNA contains a mutation of interest in the low temperature iso-melting domain, comprising the steps of:
-
a) providing a liquid sample containing two or more species of double-stranded DNA fragments which differ by at least one base pair labeled with a fluorescent reporter group; b) melting and reannealing the labeled double-stranded DNA fragments under conditions appropriate to form duplexed DNA, thereby producing a mixture of labeled DNA heteroduplexes and labeled DNA homoduplexes; c) introducing the labeled mixture of duplexes produced in step b) into a replaceable polymer matrix contained within a capillary column thereby producing a replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes; d) subjecting the replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes to constant denaturant capillary electrophoresis whereby the DNA heteroduplexes partially melt and the DNA homoduplexes do not melt and the DNA heteroduplexes migrate at a slower velocity in the replaceable polymer matrix than the DNA homoduplexes resulting in the separation of DNA heteroduplexes from DNA homoduplexes; e) detecting the relative movement of the labeled DNA heteroduplexes and labeled DNA homoduplexes as they exit the replaceable polymer matrix by exposing an end portion of the capillary column to light energy of a wavelength appropriate for the stimulation of fluorescence of the fluorescent reporter group and detecting the fluorescence with a detector thereby detecting the relative movement of the separated labeled DNA duplexes as they exit the replaceable polymer matrix; and f) collecting fractions containing the labeled DNA heteroduplexes as they exit the replaceable polymer matrix. - View Dependent Claims (45, 46, 47, 48, 49, 50, 51)
-
Specification