Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1992-01-28
2001-03-13
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023500
Reexamination Certificate
active
06200747
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to methods for amplifying, detecting and cloning nucleic acid sequences, particularly to methods that are based on a polymerase chain reaction (PCR). It relates to a process for amplifying nucleic acid sequences in a test sample. One can also employ the present invention to determine whether a specific nucleic acid sequence is present in a test sample. More specifically, this invention relates to a process for amplifying a selected GC-rich nucleic acid sequence to facilitate detection and/or cloning of the sequence. The process of the present invention uses, in one embodiment, a thermostable polymerase to catalyze the extension of a primer bound to a template. The present invention also relates to the development of diagnostic assays for inherited or sporadic genetic defects. One application of the present invention provides an assay for the genetic defect that causes the fragile X syndrome in carriers, persons afflicted, fetuses and embryos.
BACKGROUND
The fragile X syndrome is the most common inherited form of mental retardation and developmental disability. This condition afflicts approximately 1 in 1250 males and 1 in 2000 females.
As the name implies, fragile X is an X chromosome-linked condition. The fragile X phenotype is characterized by a visible constriction near the end of the X chromosome, at locus q27.3, and there is a tendency for the tip of the X-chromosome to break off under certain conditions in tissue culture. These tissue culture procedures form the basis of the assay most commonly used for fragile X at present.
The pattern of inheritance of this condition is atypical of that associated with X-linked conditions. Typically, there is a 50% probability that the son of a woman who carries an X-linked genetic defect will be afflicted by the defect. Additionally, all males who carry the abnormal gene are afflicted by the X-linked condition in the typical pattern. Furthermore, since females have two X chromosomes, they normally do not suffer the effects of a single damaged X chromosome.
In fragile X, however, some carrier males are phenotypically normal. Moreover, about one third of the females who inherit the fragile X chromosome are afflicted. The incidence of carrier males in different generations of a family varies. Daughters of carrier males are generally non-expressing carriers, but may have afflicted sons. Furthermore, afflicted daughters occur more frequently among the offspring of carrier mothers than among the offspring of carrier fathers (Brown,
The Fragile X: Progress toward Solving the Puzzle
, Am. J. Human Genet. 47 175-80, 1990).
Researchers recently identified the genomic region associated with this condition. (Oberle, et al.,
Instability of a
550-
Base Pair DNA Segment and Abnormal Methylation in Fragile X Syndrome
, Science 252 1097-1102, 1991; Kremer, et al.,
Mapping of DNA Instability at the Fragile X to a Trinucleotide Repeat Sequence p
(
CCG
)
n
, Science 252 1711-14, 1991; and Bell, et al.,
Physical Mapping across the Fragile X Hypermethylation and Clinical Expression of the Fragile X Syndrome
, Cell 64 861-66, 1991). Additionally, researchers have sequenced a partial cDNA clone derived from this region, called FMR-I. (Verkerk, et al.,
Identification of a Gene
(
FMR
-1)
Containing a CGG Repeat Coincident with a Breakpoint Cluster Region Exhibiting Length Variation in Fragile X Syndrome
, Cell 65 905-14, 1991). The Oberle, Kremer, Bell and Verkerk papers are hereby incorporated by reference.
These studies provide an explanation for the atypical pattern of inheritance of fragile X. The mutation that ultimately results in the fragile X phenotype occurs in stages. In the early stages, the gene is not fully defective, rather there is a “pre-mutation” of the gene. Carriers of the premutation have a normal phenotype. A further mutation occurs in carrier females that produces the phenotype in their offspring.
The coding sequence for FMR-I contains a variable number of CGG repeats. Individuals who are not carriers have approximately 30 CGG repeats in their FMR-I. Carriers, however, have between 50 and 200 CGG repeats. This amplification of the FMR-I CGG sequence is the pre-mutation. Afflicted individuals have even more CGG repeats. As many as several thousand CGG repeats have been observed in afflicted individuals. (Oberle, et al., 1991).
However, most affected individuals do not express the FMR-1 mRNA (Pieretti, et al., Absence of Expression of the FMR-1 Gene in Fragile X Syndrome, Cell 66 1-201991). A CpG island, located upstream of the CGG repeat region, is methylated when the number of CGG repeats is above a threshold of about 200 copies (Oberle, et al., 1991; Kremer, et al., 1991, Bell, et al., 1991). This methylation inactivates the gene.
Until now, the only way to diagnose the fragile X syndrome has been to examine microscopically an afflicted individual's chromosomes after cell growth and treatment in tissue culture. In such an examination, the laboratory examined the X chromosome to ascertain whether it was characteristically constricted, or had a broken tip. This method is both costly and not reliable. For example, this method misses almost all male carriers and half of the female carriers of the fragile X syndrome. (THE FRAGILE X SYNDROME, Oxford Univ. Press (Davies, ed. 1989)) Another method for detecting fragile X carriers and genotypes employs a Southern blot methodology but lacks sensitivity and speed. (Rousseau et al. Direct Diagnosis by DNA Analysis of the Fragile X Syndrome of Mental Retardation, N.E. J. Med. 1673-81 (1991))
The present invention provides a fast, inexpensive genetic test for reliably identifying carriers of the fragile X genotype based on molecular structure of the gene defect. The method of the present invention determines whether the number of CGG repeats in the test individual's X-chromosome are characteristic of a normal, carrier or afflicted person.
The test method of the present invention is based on the polymerase chain reaction (PCR). PCR-based assays are ideal for detecting specific DNA sequences that are present in low abundance relative to the total DNA. In brief, a PCR method amplifies the specific DNA sequence, for example, one hundred thousand to a million fold. Once amplified to this level, the specific DNA sequence, if present, is readily detected.
Prior attempts to develop PCR-based methods to directly identify the CGG repeat sequence at the genomic level have been unsuccessful (Kremer, et al., 1991), or only partially successful (Fu et al. Variations of the CGG Repeat at the Fragile X Site Results in Genetic Instability: Resolution of the Sherman Paradox, Cell 67: 1047-58 (1991)). This region appears unstable and difficult to clone or to analyze directly.
The inability of PCR-based methods to detect GC-rich sequences has hindered the development of an assay for other conditions. For example, clonality in Epstein-Barr virus infection, the androgen receptor gene, the beta-adrenergic receptor and the CMV genome are each characterized by a GC-rich nucleic acid sequence. It has not been possible to identify clonality of the Epstein-Barr virus with conventional PCR methods. Moreover, as the androgen receptor has a CAG repeat region, the beta-adrenergic receptor has an 80% GC rich region and the CMV genome has portions that are more than 75% GC, none of these nucleic acids are amplifiable by conventional PCR methods.
We have solved the problem of using PCR-based methods with GC-rich nucleic acid sequences. Using our method, we have amplified and detected the GC-rich region of the FMR-1 gene in normals, carriers and afflicted individuals.
SUMMARY OF THE INVENTION
The present invention amplifies selected GC-rich nucleic acid sequences present in a test sample. In one embodiment of the present invention, 7-deaza dGTP was substituted for dGTP.
The present invention can be used to assay for the GC-rich nucleic acid sequence characteristic of carriers of, and persons afflicted with, the fragile X syndrome.
REFERENCES:
patent: 4683195 (1
Brown W. Ted
Erster Susan H.
Pergolizzi Robert G.
Houtteman Scott W.
Kenyon & Kenyon
North Shore University Hospital Research Corp.
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