Process for analyzing length polymorphisms in DNA regions
DC CAFCFirst Claim
1. A method for determining length polymorphisms in a simple or cryptically simple sequence in one or more DNA regions of one or more subjects, which comprises:
- a) providing at least one DNA sample, comprising a template DNA having a nucleotide sequence that includes a simple or cryptically simple sequence comprising trinucleotide repeats, from at least one subject;
b) annealing at least one primer pair to the template DNA of each of said DNA samples, wherein said primer pair is composed of a first primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 5′
side of said simple or cryptically simple DNA sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 3′
side of said simple or cryptically simple DNA sequence;
wherein said first and second primers each anneal to a single site in said template DNA and the sequence of the template DNA between the sites where said primers anneal is 50 to 500 nucleotides in length;
c) performing at least one primer-directed polymerase chain reaction upon said template DNA having said primers annealed thereto, so as to form at least one polymerase chain reaction product;
d) separating the products of each polymerase chain reaction according to their lengths; and
e) analyzing the lengths of the separated products to determine the length polymorphisms of the simple or cryptically simple sequences.
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Abstract
A process for analyzing length polymorphism in DNA regions wherein the following steps are carried out:
(a) annealing at least one primer pair to the DNA to be analyzed, wherein one of the molecules of the primer pair is substantially complementary to one of the complementary strands of the 5′ or 3′ flank of a simple or cryptically simple DNA sequence, and wherein the annealing occurs in such an orientation that the synthesis products obtained by a primer-controlled polymerisation reaction with one of said primers can serve as template for annealing the other primer after denaturation;
(b) primer-controlled polymerase chain reaction; and
(c) separating and analyzing the polymerase chain reaction products.
5 Citations
42 Claims
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1. A method for determining length polymorphisms in a simple or cryptically simple sequence in one or more DNA regions of one or more subjects, which comprises:
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a) providing at least one DNA sample, comprising a template DNA having a nucleotide sequence that includes a simple or cryptically simple sequence comprising trinucleotide repeats, from at least one subject;
b) annealing at least one primer pair to the template DNA of each of said DNA samples, wherein said primer pair is composed of a first primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 5′
side of said simple or cryptically simple DNA sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 3′
side of said simple or cryptically simple DNA sequence;
wherein said first and second primers each anneal to a single site in said template DNA and the sequence of the template DNA between the sites where said primers anneal is 50 to 500 nucleotides in length;
c) performing at least one primer-directed polymerase chain reaction upon said template DNA having said primers annealed thereto, so as to form at least one polymerase chain reaction product;
d) separating the products of each polymerase chain reaction according to their lengths; and
e) analyzing the lengths of the separated products to determine the length polymorphisms of the simple or cryptically simple sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
a) at least one vessel containing an equimolar mixture of primers constituting between 1 and 50 of said primer pairs;
b) a vessel containing a polymerizing enzyme suitable for performing a primer-directed polymerase chain reaction;
c) a vessel containing the deoxynucleotide triphosphates adenosine, guanine, cytosine and thymidine;
d) a vessel containing a buffer solution suitable for performing a polymerase chain reaction, or a concentrate of said buffer solution;
e) a vessel containing a template DNA that has a nucleotide sequence including a simple or cryptically simple sequence for assaying positive performance of the method, wherein each simple or cryptically simple DNA sequence comprises at least one trinucleotide motif.
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13. The kit of claim 12, wherein at least one primer of each primer pair is labelled with a fluorescent or a radioactive label.
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14. The method of claim 1, wherein the kinship of at least two subjects is determined by comparing length polymorphisms determined in step (e).
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15. A method for analyzing length polymorphisms in at least one locus in an DNA sample obtained from at least one subject, wherein said DNA sample comprises a DNA template having at least one locus comprising a simple or cryptically simple DNA sequence, said method comprising:
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a) annealing said DNA template with at least one pair of primers, wherein said primer pair is composed of a first primer complementary to a nucleotide sequence flanking said simple or cryptically simple DNA sequence on the 5′
side of said simple or cryptically simple DNA sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 3′
side of said simple or cryptically simple DNA sequence;
wherein said first and second primers each anneal to a single site in said DNA template and wherein the annealing sites are separated by 50 to 500 nucleotides of template DNA;
b) performing at least one primer-directed polymerase chain reaction upon said template DNA having said primers annealed thereto, so as to form at least one polymerase chain reaction product;
c) separating the products of each polymerase chain reaction according to their lengths; and
d) analyzing the lengths of the separated products to determine the length polymorphisms of said simple or cryptically simple sequences;
- View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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35. A kit for performing analysis of polymorphism in simple or cryptically simple sequences, comprising:
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a) at least one vessel containing a mixture of primers constituting 1 to 50 primer parts;
wherein each of said primer pairs is composed of a first primer complementary to a nucleotide sequence flanking said simple or cryptically simple DNA sequence on the 5′
side of said simple or cryptically simple DNA sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 3′
side of said simple or cryptically simple DNA sequence;
wherein said first and second primers each anneal to a single site in said DNA template and wherein the annealing sites are separated by 50 to 500 nucleotides of template DNA;
b) a vessel containing a template DNA that has a nucleotide sequence including a simple or cryptically simple sequence for assaying positive performance of the method. - View Dependent Claims (36, 37, 38, 39)
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40. A method for determining length polymorphisms in a simple or cryptically simple sequence in one or more DNA regions of one or more subjects, which comprises:
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a) providing at least one DNA sample, comprising a template DNA consisting essentially of a nucleotide sequence that includes i) a simple or cryptically simple sequence having a trinucleotide repeat motif and ii) nucleotide sequences flanking the simple or cryptically simple sequence, from at least one subject;
b) annealing at least one primer pair to the template DNA of each of said DNA samples, wherein said primer pair is composed of a first primer complementary to the nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 5′
side of said simple or cryptically simple DNA sequence and a second primer complementary to the nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 3′
side of said simple or cryptically simple DNA sequence;
wherein said first and second primers each anneal to a single site in said template DNA and the sequence of the template DNA between the sites where said primers anneal is 50 to 500 nucleotides in length;
c) performing at least one primer-directed polymerase chain reaction upon said template DNA having said primers annealed thereto, so as to form at least one polymerase chain reaction product;
d) separating the products of each polymerase chain reaction according to their lengths;
e) analyzing the lengths of the separated products to determine the length polymorphisms of the simple or cryptically simple sequences.
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41. A method for analyzing polymorphism in at least one locus in an DNA sample comprising a DNA template, said method comprising:
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a) annealing said DNA template with at least one pair of primers, wherein said primer pair is composed of a first primer complementary to a nucleotide sequence flanking said simple or cryptically simple DNA sequence on the 5′
side of said simple or cryptically simple DNA sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 3′
side of said simple or cryptically simple DNA sequence;
wherein said first and second primers each anneal to a single site in said DNA template and wherein the annealing sites are separated by 50 to 500 nucleotides of template DNA;
b) performing at least one primer-directed polymerase chain reaction upon said template DNA having said primers annealed thereto, so as to form at least one polymerase chain reaction product;
c) separating the products of each polymerase chain reaction product according to their lengths; and
d) analyzing the lengths of the separated products to determine the length polymorphisms of said simple or cryptically simple sequences,
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42. A kit for analyzing polymorphism in at least one locus in an DNA sample, comprising:
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a) at least one vessel containing a mixture of primers constituting between 1 and 50 of said primer pairs;
b) a vessel containing a polymerizing enzyme suitable for performing a primer-directed polymerase chain reaction;
c) a vessel containing the deoxynucleotide triphosphates adenosine, guanine, cytosine and thymidine;
d) a vessel containing a buffer solution for performing a polymerase chain reaction;
e) a vessel containing a template DNA comprising i) a simple or cryptically simple nucleotide sequence having a repeat motif length of 3 to 10 nucleotides and ii) nucleotide sequences flanking said simple or cryptically simple nucleotide sequence that are effective for annealing at least one pair of said primers, for assaying positive performance of the method.
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Specification