Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-09-12
2001-03-20
Jones, W. Gary (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C536S024300, C536S024320, C536S024330, C536S025320
Reexamination Certificate
active
06204024
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a method and kit for genotyping the CCR5 chemokine receptor by amplifying the CCR5 RNA transcript using transcription based amplification. The present invention is also directed to oligonucleotides for amplifying the CCR5 RNA transcript and to mutant and wild type specific probes used in the detection of the amplification product.
BACKGROUND OF THE INVENTION
The CCR5 chemokine receptor is known to act as a coreceptor in conjunction with CD4 for the entry of macrophage tropic nonsyncytia-inducing (NSI) strains of HIV-1 during infection. The NSI, or macrophage tropic, strains are apparently the most prevalent strains in HIV positive individuals. Recent studies have indicated that the CCR5 genotype is important in predicting host susceptibility to HIV-1 infection as well as determining disease progression.
A 32 base deletion mutation in the CCR5 gene has been found to occur in some individuals. Individuals who are homozygous for this deletion mutation do not express the CCR5 receptor on the surface of their CD4+ cells and appear not to be injectable by NSI isolates of HIV-1. Individuals who are heterozygous for this deletion mutation, i.e., have a wild type (wt) CCR5 allele and a mutant (mut) CCR5 allele, appear to be injectable, but disease progression is less rapid and less severe than that of individuals who are homozygous for the wild type allele.
In view of the apparent relationship of the CCR5 genotype to HIV-1 infection and progression, an assay method that can determine whether an individual is homozygous mutant, heterozygous, or homozygous wild type at the CCR5 gene can serve as a diagnostic marker for (1) infectability by NSI forms of HIV-1 and (2) disease progression prognoss.
SUMMARY OF THE INVENTION
According to the present invention a method for genotyping the CCR5 chemokine receptor has been discovered that utilizes transcription based amplification, such as the isothermal transcription based amplification commonly known as NASBA (nucleic acid sequence based amplification). In the assay method of the invention, CCR5 transcript RNA is amplified according to a transcription based amplification procedure that uses a pair of oligonucleotides to target and amplify both the wild type and the mutant type transcript RNA's. The amplificate is captured or immobilized and the alleles are detected by means of independent hybridizations using wild type and mutant specific probes.
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Lee Eun Mi
Romano Joseph W.
Akzo Nobel N.V.
Gormley Mary E.
Jones W. Gary
Sullivan Michael G.
Tung Joyce
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