Nucleic acid size detection method
First Claim
1. A method for determining the size of a nucleic acid segment in a nucleic acid sample, comprising,fragmenting the nucleic acid in the sample into fragments, wherein said segment is associated with a marker sequence in said fragment,separating said fragments into fractions according to size under conditions in which the fragment(s) containing the segment will be located in said fractions according to the size of the segment, andidentifying those fraction(s) containing the segment by detecting the marker sequence, wherein the size of the segment is determined by the fraction in which it is identified.
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Abstract
The present invention provides methods of determining the size of a particular nucleic acid segment of interest in a sample of nucleic acids through fragmentation of DNA, size fractionation, an optional second fragmentation, and identification using a marker sequence. In particular aspects, an expansion or reduction of tandem repeat sequences can be detected. In further aspects, carriers and individuals afflicted with fragile X syndrome or other diseases associated with tandem repeats can be distinguished from normal individuals.
32 Citations
66 Claims
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1. A method for determining the size of a nucleic acid segment in a nucleic acid sample, comprising,
fragmenting the nucleic acid in the sample into fragments, wherein said segment is associated with a marker sequence in said fragment, separating said fragments into fractions according to size under conditions in which the fragment(s) containing the segment will be located in said fractions according to the size of the segment, and identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the size of the segment is determined by the fraction in which it is identified.
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17. A method according to claim 17, wherein said fractions are chosen to separate fragments with a normal number of tandem repeats from fragments containing an abnormal number of tandem repeats.
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22. A method of detecting a mutation in a tandem repeat segment of a gene in a genomic nucleic acid sample, wherein said mutation is characterized by a change in the number of repeats compared to the number of repeats in the wild type allele, said method comprising
fragmenting the nucleic acid in the sample into fragments, wherein said tandem repeat segment is associated with a marker sequence in said fragment; -
separating said fragments into fractions according to size under conditions in which the fragment(s) containing the tandem repeat segment will be located in said fractions according to the number of repeats in the tandem repeat segment; identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the number of repeats in the tandem repeat segment is determined by the fraction in which it is identified, and comparing the number of repeats in the tandem repeat segment from the nucleic acid sample to the number in the corresponding wild type allele, wherein a number of repeats differing from the number in the wild type allele is indicative of a mutation. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
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44. A method of distinguishing between normal individuals and that are normal, those that are carriers of fragile X syndrome, and those that are afflicted with fragile X syndrome, said method comprising,
fragmenting the nucleic acid in the sample from the individual into fragments, wherein said tandem repeat segment of the FMR1 gene is associated with a marker sequence in said fragment, separating said fragments into fractions according to size under conditions in which the fragment(s) containing the tandem repeat segment having a normal number of repeats will be located in a first fraction; - fragments containing a tandem repeat segment having a premutation will be located in a second fraction; and
fragments having a tandem repeat region having a full mutation will be located in a third fraction,identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the number of repeats in the tandem repeat segment is determined by the fraction in which it is identified, wherein a positive result in only the first fraction indicates the individual is normal; a positive result in the second fraction indicates the individual is a carrier; and a positive result in the third fraction indicates the individual is afflicted with fragile X syndrome. - View Dependent Claims (45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
- fragments containing a tandem repeat segment having a premutation will be located in a second fraction; and
- 63. A kit for detecting the size of a particular nucleic acid segment in a sample comprising a primer pair for amplifying a marker nucleotide sequence upstream or downstream of the particular nucleic acid segment, and one or more restriction endonucleases for cleaving the nucleic acid sample to generate a fragment of the nucleic acid sample which contains the particular nucleic acid segment and the upstream or downstream marker sequence, wherein said particular nucleic acid segment is a tandem repeat sequence.
Specification