US5683880A
Expired / Lapsed
US
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Linkage analysis of genes with diseases using difference spectrum analysis
Assignee: Myriad Genetics Inc
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Filing: 1995-07-07
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Granted: 1997-11-04
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Expires: 2015-07-07
Abstract
Abstract The present invention is directed to a method for rapidly analyzing whether known genetic markers which are found in various lengths in the population, e.g., those containing (CA) n repeats, are associated with a disease of interest. The method involves using polymerase chain reactions to amplify the DNA in the marker regions and comparing the amplified DNA lengths seen in the normal population with those seen in an affected population of persons. The method involves a pooling of DNA samples from normal patients to average out the normal marker genotype found in the population and also involves a pooling of DNA from affected individuals to give a summing effect to give a stronger signal. The amplified DNA fragments are labeled with two distinguishable markers such as two different colored fluorescent markers, one used to label the amplified DNA from the normal population and the other to label the amplified DNA from the affected population. The amplified products from the normals and the affecteds are pooled, run on a sequencing gel, and a difference spectrum is calculated. Markers which are not associated with the disease will result in a zero or near zero difference spectrum whereas a marker which is associated with the disease will result in a difference spectrum with marked peaks.
Inventors
- 👤 Alexander Kamb
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 05:15
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12Q1/6844; C12Q1/6858
Claims Analysis
Total Claims: 9
Claim 1
Independent Claim
413 words
A method to determine whether a tandem repeat DNA marker is associated with a disease of interest, said method comprising the steps of: (a) preparing two sets of primer pairs for each of several tandem repeat markers, wherein each primer of a primer pair is complementary to sequence flanking a tandem repeat marker such that each pair of primers will amplify a tandem repeat marker in a polymerase chain reaction, wherein at least one primer of each pair from a first set of primer pairs is labeled with a first label and at least one primer of each pair from a second set of primer pairs is labeled with a second label wherein the said first label is distinguishable from said second label; (b) purifying DNA from a population of normal persons to obtain normal DNA; (c) purifying DNA from a population of persons affected with said disease to obtain affected DNA; (d) performing polymerase chain reactions on said normal DNA using said first set of primer pairs to produce a first set of amplified DNA fragments and on said affected DNA using said second set of primer pairs to produce a second set of amplified DNA fragments; (e) pooling said first set of amplified DNA fragments with said second set of amplified DNA fragments and separating the amplified fragments by electrophoresis on a gel; (f) measuring a first signal resulting from said first label in said first set of amplified DNA fragments wherein said first signal indicates relative amounts of each size of amplified normal DNA fragments and measuring a second signal resulting from said second label in said second set of amplified DNA fragments wherein said second signal indicates relative amounts of each size of amplified affected DNA fragments; (g) determining a difference spectrum between said first and second signals; and (h) examining said difference spectrum, wherein a difference spectrum showing a difference of close to zero throughout indicates no association of the tandem repeat markers with the disease and wherein a peak in the difference spectrum indicates an association of one or more tandem repeat markers with said disease; wherein if an association is found, then steps (d) to (h) are repeated using each individual primer pair for each of said several tandem repeat markers from said first and second sets of primer pairs; and wherein tandem repeat markers amplified by said individual primer pairs which yield a peak in the difference spectrum are associated with said disease of interest.
Claim 2
Independent Claim
413 words
A method to determine whether a tandem repeat DNA marker is associated with a disease of interest, said method comprising the steps of: (a) preparing two sets of primer pairs for each of several tandem repeat markers, wherein each primer of a primer pair is complementary to sequence flanking a tandem repeat marker such that each pair of primers will amplify a tandem repeat marker in a polymerase chain reaction, wherein at least one primer of each pair from a first set of primer pairs is labeled with a first label and at least one primer of each pair from a second set of primer pairs is labeled with a second label wherein the said first label is distinguishable from said second label; (b) purifying DNA from a population of normal persons to obtain normal DNA; (c) purifying DNA from a population of persons affected with said disease to obtain affected DNA; (d) performing polymerase chain reactions on said normal DNA using said first set of primer pairs to produce a first set of amplified DNA fragments and on said affected DNA using said second set of primer pairs to produce a second set of amplified DNA fragments; (e) pooling said first set of amplified DNA fragments with said second set of amplified DNA fragments and separating the amplified fragments by electrophoresis on a gel; (f) measuring a first signal resulting from said first label in said first set of amplified DNA fragments wherein said first signal indicates relative amounts of each size of amplified normal DNA fragments and measuring a second signal resulting from said second label in said second set of amplified DNA fragments wherein said second signal indicates relative amounts of each size of amplified affected DNA fragments; (g) determining a difference spectrum between said first and second signals; and (h) examining said difference spectrum, wherein a difference spectrum showing a difference of close to zero throughout indicates no association of the tandem repeat markers with the disease and wherein a peak in the difference spectrum indicates an association of one or more tandem repeat markers with said disease; wherein if an association is found, then steps (d) to (h) are repeated using each individual primer pair for each of said several tandem repeat markers from said first and second sets of primer pairs; and wherein tandem repeat markers amplified by said individual primer pairs which yield a peak in the difference spectrum are associated with said disease of interest.
Claim 3
Dependent Claim
12 words
The method of claim 1 wherein step (f) is performed during electrophoresis.
Claim 4
Dependent Claim
12 words
The method of claim 1 wherein step (f) is performed following electrophoresis.
Claim 5
Dependent Claim
15 words
The method of claim 1 wherein only one primer of each primer pair is labeled.
Claim 6
Dependent Claim
14 words
The method of claim 1 wherein both primers of each primer pair are labeled.
Claim 7
Dependent Claim
42 words
The method of claim 1 wherein the difference spectrum is determined by normalizing said first signal with said second signal to produce a scaled first signal and a scaled second signal and subtracting said scaled first signal from said scaled second signal.
Claim 8
Dependent Claim
17 words
The method of claim 1 wherein the normal DNA is pooled prior to performing polymerase chain reactions.
Claim 9
Dependent Claim
12 words
The method of claim 1 wherein said affected persons are blood relatives.
References Cited (Backward Citations)
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