Methods for the survey and genetic analysis of populations
First Claim
1. A method to determine the genetic diversity of a population comprising the steps:
- a) providing a sample of interest containing a population of interest;
b) isolating genomic DNA from the sample;
c) performing PCR on the genomic DNA to amplify a subregion comprising a polymorphic site of interest using an upstream primer comprising a binding site for a solid support and a downstream primer to produce an amplified fragment;
d) cleaving the amplified fragment with a restriction enzyme that cleaves the amplified fragment at a site close to the polymorphic region;
e) binding the cleaved amplified fragment to the solid support to form an immobilized product;
f) splitting the immobilized product into two pools;
g) attaching a double-stranded linker to the immobilized product of each pool, wherein the linker comprises a Type IIS restriction enzyme site;
h) digesting the product with the Type IIS restriction enzyme to form a tag;
i) ligating the tags to one another to form ditags;
j) amplifying the ditags by PCR;
k) cleaving the ditags with the restriction enzyme of step c) and ligating the ditags to form a concatemer;
l) cloning the concatemer into a vector; and
m) determining the sequence the concatemer.
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Abstract
The present invention relates to methods for performing surveys of the genetic diversity of a population. The invention also relates to methods for performing genetic analyses of a population. The invention further relates to methods for the creation of databases comprising the survey information and the databases created by these methods. The invention also relates to methods for analyzing the information to correlate the presence of nucleic acid markers with desired parameters in a sample. These methods have application in the fields of geochemical exploration, agriculture, bioremediation, environmental analysis, clinical microbiology, forensic science and medicine.
172 Citations
44 Claims
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1. A method to determine the genetic diversity of a population comprising the steps:
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a) providing a sample of interest containing a population of interest;
b) isolating genomic DNA from the sample;
c) performing PCR on the genomic DNA to amplify a subregion comprising a polymorphic site of interest using an upstream primer comprising a binding site for a solid support and a downstream primer to produce an amplified fragment;
d) cleaving the amplified fragment with a restriction enzyme that cleaves the amplified fragment at a site close to the polymorphic region;
e) binding the cleaved amplified fragment to the solid support to form an immobilized product;
f) splitting the immobilized product into two pools;
g) attaching a double-stranded linker to the immobilized product of each pool, wherein the linker comprises a Type IIS restriction enzyme site;
h) digesting the product with the Type IIS restriction enzyme to form a tag;
i) ligating the tags to one another to form ditags;
j) amplifying the ditags by PCR;
k) cleaving the ditags with the restriction enzyme of step c) and ligating the ditags to form a concatemer;
l) cloning the concatemer into a vector; and
m) determining the sequence the concatemer. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 40)
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15. A method for analyzing a marker diversity profile (MDP) by comparison to a marker diversity profile matrix database to identify at least one parameter associated with the MDP, comprising the steps of:
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a) constructing a marker diversity profile comprising a plurality of nucleic acid markers, wherein each marker is an identifier for one member or a limited number of members of a population, wherein the abundance of said marker provides an indication as to the abundance of the corresponding member of said population, and wherein said population is associated with at least one parameter;
b) detecting the abundance of each marker;
c) transducing the abundance of each marker into an electrical signal;
d) storing a marker diversity profile comprising the marker electrical signals in a marker diversity profile matrix data structure;
e) determining at least one parameter associated with said marker diversity profile by comparing said marker diversity profile with a marker diversity profile matrix data structure comprising marker diversity profiles that are each known to be associated with at least one parameter.
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28. A method for analyzing a marker diversity profile database to identify one or more robust indicator markers for a desired parameter, comprising the steps of:
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a) constructing a marker diversity profile comprising a plurality of nucleic acid markers, wherein each marker is an identifier for one member or a limited number of members of a population, wherein the presence or abundance of said marker provides an indication as to the presence or abundance of the corresponding member of said population, and wherein said population is associated with said desired parameter;
b) detecting the presence or abundance of each marker;
c) transducing the presence or abundance of each marker into an electrical signal;
d) storing the marker diversity profile comprising marker electrical signals in a marker diversity profile matrix data structure;
e) correlating the presence or abundance of each marker in the marker diversity profile database with the presence or abundance of each said parameters;
f) ranking the markers for each parameter based upon correlation values; and
g) designating one or more markers as robust indicators for the parameter as those that have a correlation value of at least 0.4 to the parameter.
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37. A method for analyzing a MDP database to identify groups of correlated markers, comprising the steps of
a) constructing a marker diversity profile comprising a plurality of nucleic acid markers, wherein each marker is an identifier for one member or a limited number of members of a population, wherein the presence or abundance of said marker provides an indication as to the presence or abundance of a corresponding member of said group, and wherein said group is associated with a desired parameter; -
b) detecting the presence or abundance of each marker;
c) transducing the presence or abundance of each marker into an electrical signal;
d) storing the marker diversity profile comprising marker electrical signals in a marker diversity profile matrix data structure;
e) correlating the presence or abundance of each marker in the marker diversity profile database with the presence or abundance of other markers in pairwise comparison to form a correlation matrix;
f) separating the markers into correlated groups based upon values from correlation matrix.
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38. A method of constructing a matrix diversity profile database, comprising the steps of
a) constructing a marker diversity profile comprising a plurality of nucleic acid markers, wherein each marker is an identifier for one member or a limited number of members of a population, wherein the presence or abundance of said marker provides an indication as to the presence or abundance of the corresponding member of said population, and wherein said population is associated with at least one parameter; -
b) detecting the presence or abundance of each marker;
c) transducing the presence or abundance of each marker into an electrical signal;
d) storing the marker diversity profile comprising marker electrical signals in a marker diversity profile matrix data structure; and
e) indexing the marker diversity profile with the associated parameter to form a matrix diversity profile database.
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39. A method for identifying a marker that correlates with a desired parameter, comprising the steps of
a) constructing a marker diversity profile comprising a plurality of nucleic acid markers, wherein each marker is an identifier for one member or a limited number of members of a population, wherein the presence or abundance of said marker provides an indication as to the presence or abundance of the corresponding member of said population, and wherein said population is associated with said desired parameter; -
b) detecting the presence or abundance of each marker;
c) correlating the presence or abundance of each marker in the marker diversity profile database with the parameter;
d) ranking the markers for the parameter based upon correlation values; and
e) designating one or more markers as a robust indicator for the parameter as those that have a correlation value of at least 0.4 to the parameter.
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41. A method for identifying a marker that correlates with a desired parameter, comprising the steps of
a) constructing a marker diversity profile comprising a plurality of nucleic acid markers, wherein each marker is an identifier for one member or a limited number of members of a population, wherein the presence or abundance of said marker provides an indication as to the presence or abundance of the corresponding member of said population, and wherein said population is associated with said desired parameter; -
b) detecting the presence or abundance of each marker;
c) correlating the presence or abundance of each marker in the marker diversity profile database with the parameter;
d) ranking the markers for the parameter based upon correlation values;
e) designating one or more markers as a potential indicator for the parameter as those that have a correlation value of at least 0.4 to the parameter; and
f) determining whether the potential indicator is present or abundant in a population that is not associated with said desired parameter; and
g) designating one or more markers as a robust indicator for the parameter if the potential indicator is not present or is less abundant in a population that is not associated with said desired parameter than in a population that is associated with said desired parameter. - View Dependent Claims (42)
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43. A method for performing mineral deposit exploration or subsurface petroleum or natural gas reserve exploration, comprising the steps of:
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a) isolating nucleic acids from an environmental sample;
b) amplifying a specific DNA molecule that has been shown to correlate with the presence of mineral deposits and/or petroleum or natural gas reserves;
c) detecting the presence of said amplified DNA molecule; and
c) inferring the presence of said mineral deposits and/or said petroleum or natural gas reserves from the presence of said amplified DNA molecule.
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44. A method for performing mineral deposit exploration or subsurface petroleum or natural gas reserve exploration, comprising the steps of:
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a) isolating nucleic acids from an environmental sample;
b) amplifying a specific RNA molecule that has been shown to correlate with the presence of mineral deposits and/or petroleum or natural gas reserves;
c) detecting the presence of said amplified RNA molecule; and
c) inferring the presence of said mineral deposits and/or said petroleum or natural gas reserves from the presence of said amplified RNA molecule.
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Specification