Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-01-08
2003-08-26
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300, C536S025300
Reexamination Certificate
active
06610492
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to organic chemistry, analytical chemistry, biochemistry, molecular biology, genetics, diagnostics and medicine. In particular, it relates to novel base-modified nucleotides that can be substituted into a polynucleotide at greater than 90% of the points of incorporation of a corresponding natural nucleotide and than can be cleaved at greater than 90% of their points of incorporation.
BACKGROUND OF THE INVENTION
The following is offered as background information only and is not intended nor admitted to be prior art to the present invention.
The ability to detect DNA sequence variances in an organism's genome has become an important tool in the diagnosis of diseases and disorders and in the prediction of response to potential therapeutic regimes. It is becoming increasingly possible, using early variance detection, to diagnose and treat, even prevent, a disorder before it has physically manifested itself. Furthermore, variance detection can be a valuable research tool in that it may lead to the discovery of genetic bases for disorders the cause of which were hitherto unknown or thought to be other than genetic.
It is estimated that sequence variations in human DNA occur with a frequency of about 1 in 100 nucleotides when 50 to 100 individuals are compared. Nickerson, D. A.,
Nature Genetics,
1998, 223-240. This translates to as many as 30 million variances in the human genome. However, very few of these variances have any effect on the physical well-being of humans. Detecting these 30 million variances and then determining which of them are relevant to human health is clearly a formidable task.
Once the DNA sequence of a DNA segment; e.g., a gene, a cDNA or, on a larger scale, a chromosome or an entire genome, has been determined, the existence of sequence variances in that DNA segment among members of the same species can be explored. Complete DNA sequencing is the definitive procedure for accomplishing this task. However, current DNA sequencing technology is costly, time consuming and, in order to assure accuracy, highly redundant. Most sequencing projects require a 5- to 10-fold coverage of each nucleotide to reach an acceptable error rate of 1 in 2,000 to 1 in 10,000 bases. In addition, DNA sequencing is an inefficient way to detect variances. A variance between two copies of a gene, for example when two chromosomes are being compared, may occur as infrequently as one in 1,000 or more bases. Thus, only a small segment of the gene is of interest. If full sequencing is employed, a tremendous number of nucleotides have to be sequenced to arrive at the desired information contained in that segment. For example, to compare ten versions of a 3,000 nucleotide DNA sequence for the purpose of detecting four variances among them, even if only 2-fold redundancy is employed (each strand of the double-stranded 3,000 nucleotide DNA segment from each individual is sequenced once), 60,000 nucleotides would have to be sequenced (10×3,000×2). In addition, sequencing problems are often encountered that can require additional runs with new primers. Thus, as many as 100,000 nucleotides might have to be sequenced to determine four variances.
What is needed is a rapid, inexpensive, yet accurate method to identify variances such as SNPs among related polynucleotides. The present invention provides such a method and materials for its implementation.
SUMMARY OF THE INVENTION
Thus, in one aspect the present invention relates to a modified heterocyclic nitrogen base comprising the chemical formula:
R is 1′-ribose, 1′-(2′-deoxy)ribose, a 1′-ribose-5′-monophosphate, a 1′-(2′-deoxy)ribose-5′-monophosphate, a 1′-ribose-5′-triphosphate, a 1′-(2′-deoxy)ribose-5′-triphosphate or a 1′-ribose or 1′-(2′-deoxy)ribose moiety of an oligonucleotide or of a polynucleotide. R
1
and R
2
are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl and alkaryl, wherein if R
1
or R
2
contains two or more contiguous methylene (—CH
2
—) groups, any two such methylene groups may have interjected between them another group selected from the group consisting of —O—, —C(O)NH—, —C(O)NHC(O)—, —NH—, —C(S)NH—, —CO—, —CS—, —S— and (—CF
2
—)
m
. The subscript m is 1-10. R
3
is hydrogen or —NH
2
. Finally, n is 0, 1 or 2.
An aspect of this invention is a method for cleaving a polynucleotide, comprising replacing a natural nucleotide at greater than 90% of its points of occurrence in a polynucleotide with a base-modified nucleotide to form a base-modified polynucleotide. The base-modified polynucleotide is then contacted with a reagent or combination of reagents that cleaves it at greater than 90% of the points of occurrence of the base-modified nucleotide. In this method, the base-modified nucleotide has the chemical structure:
R is a ribose or 2′-deoxyribose moiety of an oligonucleotide or of a polynucleotide. R
1
and R
2
are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl and alkaryl, wherein if R
1
or R
2
contains two or more contiguous methylene (—CH
2
—) groups, any two such methylene groups may have interjected between them another group selected from the group consisting of —O—, —C(O)NH—, —C(O)NHC(O)—, —NH—, —C(S)NH—, —CO—, —CS—, —S— and (—CF
2
—)
m
, wherein m is 1-10. R
3
is hydrogen or —NH
2
. Finally, n is 0, 1 or 2.
In the above method contacting the modified polynucleotide with a reagent or reagents comprises contacting the polynucleotide with a chemical base in an aspect of this invention.
The chemical base is an amine in an aspect of this invention.
The amine has a boiling point greater than 100° C. at atmospheric pressure in as aspect of this invention.
The amine has a boiling point greater than 200° at atmospheric pressure in an aspect of this invention.
The amine is a secondary amine in an aspect of this invention.
The secondary amine is selected from the group consisting of 3-pyrrolidinol, 2-pyrrolidinemethanol, 3-pyrrolidinemethanol, 4-hydroxypiperidine and 4-piperidineethanol in an aspect of this invention.
In an aspect of this invention the modified polynucleotide is contacted with a chemical oxidant prior to contact with the chemical base.
A further aspect of this invention is a method for cleaving a polynucleotide, comprising replacing a natural nucleotide at greater than 90% of its points of occurrence in a polynucleotide with a base-modified nucleotide to form a base-modified polynucleotide. The base-modified polynucleotide is then contacted with a secondary amine having a boiling point greater than 100° C. at atmospheric pressure whereby the polynucleotide is cleaved at greater than 90% of the sites of incorporation of the modified nucleotide.
The secondary amine has a boiling point greater than 150° at atmospheric pressure in an aspect of this invention.
The secondary amine has a boiling point greater than 200° at atmospheric pressure in as aspect of this invention.
The secondary amine is selected from the group consisting of 3-pyrrolidinol, 2-pyrrolidinemethanol, 3-pyrrolidinemethanol, 4-hydroxypiperidine and 4-piperidineethanol in an aspect of this invention.
In an aspect of this invention in the above method, the modified polynucleotide is contacted with a chemical oxidant prior to contact with the secondary amine.
In any of the above methods, the percentage replacement of a natural nucleotide with a modified nucleotide, the percentage cleavage of a modified polynucleotide or both the percentage replacement and the percentage cleavage is greater than 95% in an aspect of this invention.
In any of the above methods, the percentage replacement of a natural nucleotide with a modified nucleotide, the percentage cleavage of a modified polynucleotide or both the percentage replacement and the percentage cleavage is greater than 99% in an aspect of this invention.
DETAILED DESCRIPTION OF THE INVENTION
Allerson Charles
Kawate Tomohiko
Stanton, Jr. Vincent P.
Verdine Gregory L.
Wolfe Jia Liu
Bingham & McCutchen LLP
Riley Jezia
Rose, Esq. Bernard F.
Variagenics Inc.
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