Polymorphic DNA fragments and uses thereof
First Claim
1. A method of making a reference library comprising a mixture of heterogeneous nucleic acid fragments, comprising:
- digesting pooled nucleic acid from a plurality of individuals, comprising first restriction sites s, with a first restriction endonuclease S, to produce a mixture of restriction fragments;
forming a first population of single stranded nucleic DNA fragments from a first subpopulation of said restriction fragments, wherein said first subpopulation of restriction fragments comprises a second restriction site t which is different from said first restriction site;
forming a second population of single stranded DNA fragments from a second subpopulation of said restriction fragments, wherein said second subpopulation of said restriction fragments do not contain said second restriction site, and wherein said first single stranded DNA fragments are complementary to said second single stranded DNA fragments when said single stranded DNA fragments are derived from the same restriction fragment;
hybridizing the first and second populations of single stranded DNA fragments to form a population of duplexes; and
isolating said duplexes to form a reference population of restriction fragments.
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Abstract
The invention provides methods and materials for generating a reference library of restriction fragments from pooled nucleic acids that contain a sequence polymorphism. Preferably, such a library is formed by digesting genomic DNA from a pool of individuals with a first and a second restriction endonuclease to form a population of restriction fragments; isolating restriction fragments of the population digested by both the first and second restriction endonucleases and forming a first single stranded fragment population therefrom; separately isolating restriction fragments from the population digested by the first restriction endonuclease but not the second restriction endonuclease and forming a second single stranded fragment population therefrom; hybridizing the first and second single stranded fragment populations to form a population of duplexes; and isolating the population of duplexes to form a reference library of restriction fragments that contain sequence polymorphism. An important aspect of the invention is the use of the reference population of restriction fragments to compare the frequencies of polymorphic sequences between different population pools. Such comparisons may be accomplished by competively hybridizing DNA from the respective pools which has been enriched for the presence of a restriction site polymorphism with DNA from the reference population. Preferably, such competitive hybridization reactions are carried out the reference library attached to one or more solid phase supports. Most preferably, members of the reference library are attached to individual microparticles so that each microparticle has a unique fragment attached. After competitive hybridization, the microparticles may be analyzed and sorted for identifying those microparticles carrying sequences for which the pools being compared exhibit different polymorphic frequencies.
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Citations
5 Claims
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1. A method of making a reference library comprising a mixture of heterogeneous nucleic acid fragments, comprising:
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digesting pooled nucleic acid from a plurality of individuals, comprising first restriction sites s, with a first restriction endonuclease S, to produce a mixture of restriction fragments;
forming a first population of single stranded nucleic DNA fragments from a first subpopulation of said restriction fragments, wherein said first subpopulation of restriction fragments comprises a second restriction site t which is different from said first restriction site;
forming a second population of single stranded DNA fragments from a second subpopulation of said restriction fragments, wherein said second subpopulation of said restriction fragments do not contain said second restriction site, and wherein said first single stranded DNA fragments are complementary to said second single stranded DNA fragments when said single stranded DNA fragments are derived from the same restriction fragment;
hybridizing the first and second populations of single stranded DNA fragments to form a population of duplexes; and
isolating said duplexes to form a reference population of restriction fragments. - View Dependent Claims (2, 3, 4, 5)
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Specification