Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-29
2002-01-22
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091200
Reexamination Certificate
active
06340566
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of molecular biology, and more particularly, detection of single nucleotide polymorphisms, DNA sequence variation DNA mutations, DNA damage and DNA base pair mismatches. In particular, the invention relates to the use of DNA mutation binding proteins to detect single nucleotide polymorphisms, DNA sequence variations, DNA mutations, damaged DNA and DNA with mismatched base pairs.
BACKGROUND OF THE INVENTION
Natural DNA sequence variation exists in identical genomic regions of DNA among individual members of a species. It is of interest to identify similarities and differences in such genomic regions of DNA because such information can help identify sequences involved in susceptibility to disease states as well as provide genetic information for characterization and analysis of genetic material.
When a cell undergoes reproduction, its DNA molecules are replicated and precise copies are passed on to its descendants. The linear base sequence of a DNA molecule is maintained during replication by complementary DNA base pairing. Occasionally, an incorrect base pairing does occur during DNA replication, which, after further replication of the new strand, results in a double-stranded DNA offspring with a sequence containing a heritable single base difference from that of the parent DNA molecule. Such heritable changes are called “genetic polymorphisms,” “genetic mutations,” “single base pair mutations,” “point mutations” or simply, “DNA mismatches”. In addition to random mutations during DNA replication, organisms are constantly bombarded by endogenous and exogenous genotoxic agents which injure or damage DNA. Such DNA damage or injury can result in the formation of DNA mismatches or DNA mutations such as insertions or deletions.
The consequences of natural DNA sequence variation, DNA mutations, DNA mismatches and DNA damage range from negligible to lethal, depending on the location and effect of the sequence change in relation to the genetic information encoded by the DNA. In some instances, natural DNA sequence variation, DNA mutations, DNA mismatches and DNA damage can lead to cancer and other diseases of which early detection is critical for treatment.
There is thus a tremendous need to be able to rapidly identify differences in DNA sequences among individuals. In addition there is a need to identify DNA mutations, DNA mismatches and DNA damage to provide for early detection of cancer and other.
SUMMARY OF THE INVENTION
In order to meet these needs, the present invention concerns the use of proteins that function biologically to recognize DNA mutations to detect and map single nucleotide polymorphisms, DNA mutations, DNA mismatches and DNA damage.
In one embodiment, the present invention is directed to a method for detecting a DNA mutation in a DNA molecule comprising the steps of: (a) obtaining a solid support to which the DNA molecule is coupled; (b) forming a mixture by mixing the solid support (with DNA attached) and a labeled DNA mutation binding protein, the labeled DNA mutation binding protein being capable of detecting DNA mutations and binding to such mutated DNA; (c) forming a reacted sample by incubating the mixture under conditions wherein if the DNA molecule includes mutated DNA, the DNA damage binding protein binds to the mutated DNA; (d) analyzing the reacted sample by detecting the label on the solid support to detect the DNA mutation or absence thereof.
In another embodiment, the present invention is directed to a method for detecting a DNA mutation in a DNA molecule, said method comprising the steps of: (a) obtaining a solid support to which the DNA molecule is coupled wherein the DNA molecule is labeled; (b) forming a mixture by mixing the solid support (with labeled DNA attached) and a chimeric protein wherein the chimeric protein includes a DNA mutation binding protein and a nuclease and wherein the labeled DNA mutation binding protein is capable of detecting DNA mutations and binding to such mutated DNA; (c) forming a reacted sample by incubating the mixture under conditions wherein if the DNA molecule includes mutated DNA, the DNA damage binding protein binds to the mutated DNA and the nuclease cleaves the DNA thereby removing the label from DNA molecule coupled to said solid support and (d) analyzing the reacted sample by detecting the label or absence thereof on the solid support to detect the DNA mutation.
In another embodiment, the present invention is directed to a method of detecting a DNA mutation by a) obtaining a DNA molecule; b) coupling the DNA molecule to a flow cytometry bead to form a DNA-bead complex; c) forming a mixture by mixing the DNA-bead complex with a labeled DNA mutation binding protein; d) forming a reacted sample by incubating the mixture under conditions wherein if the DNA molecule includes mutated DNA the DNA mutation binding protein binds to the mutated DNA and e) analyzing the reacted sample by flow cytometry to determine the amount of label on the beads.
The present invention is also directed to a method for flow cytometric analysis to detect a DNA mutation in a DNA molecule by a) obtaining flow cytometry beads coupled to the DNA molecule; b) forming a mixture by mixing the beads and a labeled DNA mutation binding protein wherein the DNA mutation binding protein is capable of detecting DNA mutations and binding to such mutated DNA; c) forming a reacted sample by incubating said mixture under conditions wherein if the DNA molecule includes mutated DNA the DNA mutation binding protein binds to the mutated DNA; d) analyzing the reacted sample by flow cytometry to determine the amount of label on the bead; and e) detecting the DNA mutation or absence thereof by determining the amount of label on the beads.
In an alternative embodiment, the present invention is directed to a method for detecting a DNA mutation in a DNA molecule comprising the steps of: (a) obtaining a first DNA molecule; (b) coupling the first DNA molecule to a solid support to form a DNA-support complex; (c) obtaining a second DNA molecule; (d) forming a first mixture by mixing the second DNA molecule with the DNA-support complex; (e) incubating the first mixture under conditions such that the second DNA molecule hybridizes to the first DNA molecule thereby forming a hybrid double stranded DNA molecule coupled to the support wherein the hybrid DNA molecule includes one DNA strand from said the DNA molecule and one strand from the second DNA molecule; (f) obtaining a labeled DNA mutation binding protein, wherein the labeled DNA mutation binding protein is capable of detecting DNA mutations and binding to such mutated DNA; (g) forming a second mixture by mixing the labeled DNA mutation binding protein with the hybrid double stranded DNA molecule coupled to said support; (h) forming a reacted sample by incubating the second mixture under conditions wherein if the hybrid double stranded DNA molecule includes mutated DNA, the labeled DNA mutation binding protein binds to the mutated DNA and forms a labeled, hybrid double stranded DNA-support complex; (i) analyzing the reacted sample to detect the label or absence thereof on the hybrid double stranded DNA-support complex to thereby identify the DNA mutation.
The first DNA molecule may be coupled to the bead as single stranded DNA or as double stranded DNA and then converted to single stranded DNA by increasing the temperature or by placing the coupled DNA under conditions sufficiently stringent to convert the double stranded DNA to single stranded DNA. Similarly, the second DNA molecule may be added to the first mixture as single stranded DNA or as double stranded DNA and then converted to single stranded DNA by increasing the temperature or by placing the first mixture under conditions sufficiently stringent to convert the double stranded DNA to single stranded DNA.
In this embodiment, the nucleotide sequence of the first, single stranded DNA molecule may be known and the nucleotide sequence of said second, single stranded DNA molecule may be unknown. Whe
Horlick Kenneth R.
The Regents of the University of California
Thompson Alan H.
Ward Michael R.
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