Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-08
2001-02-13
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S235100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06187538
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of nucleic acid diagnostics and the identification of single base or two base variation in target nucleotide sequences. More specifically, this invention relates to a method of using nucleic acid amplification and specific nucleic acid probes to determine the relative amounts of two variants of a nucleotide sequence in a target population.
BACKGROUND OF THE INVENTION
Various single base or two base point mutations in the human immunodeficiency virus (HIV) genome have been described which are associated with decreased susceptibility of HIV to drug therapy. Larder et al.,
Science
(1989) 246:1155-1158; Kellam et al.,
Proc. Natl. Acad. Sci
. (
USA
) (1992) 89:1934-1938. The identification of drug resistant strains has been associated with disease progression. One known example of this type of mutation is the two base mutation of amino acid 215 of the HIV-1 reverse transcriptase gene associated with zidovudine (AZT) resistance. Larder et al., supra.
Diagnosis of disease progression, as determined by onset of resistance mutations such as those described above, would be aided by the ability to determine the degree of conversion to the resistant mutant. Current methods, such as “selective PCR” are designed to detect presence or absence of the mutant sequence or wild type/mutant mixture, but are unable to accurately quantify amounts. Boucher et al.,
Lancet
(1990) 336:585-590; Larder et al.,
AIDS
(1991) 5:137-144.
A method for determining the presence or absence of allelic variants in a samples is described in Saiki et al.,
Nature
(1986) 324:163-166; and Farr et al.,
Proc. Natl. Acad. Sci
. (
USA
) (1987) 85:1629-1633. This method uses the polymerase chain reaction (PCR) technique to amplify a specific region of DNA. The amplified DNA is then used as a target for various radioactively-labelled oligonucleotide probes to identify point mutations and allelic sequence variation. This method has also been used to analyze the allelic genotype of a single sperm at the DNA level. Li et al.,
Nature
(1988) 335:414-417.
An improved method is disclosed herein which provides a method for actually comparing the relative amounts of two variants (e.g., a wild type and a mutant) of a target nucleotide sequence of a target genome in a sample.
SUMMARY OF THE INVENTION
A method of measuring the relative populations of first and second variants of a target nucleotide sequence of a target genome in a sample is provided, the method comprising the steps of: (a) amplifying a region of the target genome containing the target nucleotide sequence and a control nucleotide sequence to obtain amplified target polynucleotides; (b) separating the amplified target polynucleotides into at least a first and second portion; (c) contacting the first portion of the amplified target polynucleotide with a first labelled polynucleotide probe complementary to the first variant of the target nucleotide sequence to obtain a first hybridized labelled polynucleotide probe, and contacting the second portion of the amplified target polynucleotide with a second labelled polynucleotide probe complementary to the second variant of the target nucleotide sequence to obtain a second hybridized labelled polynucleotide probe; (d) quantifying the amount of the first and second hybridized labelled polynucleotide probes; (e) contacting the first and second portions of the amplified target polynucleotide with a third labelled polynucleotide probe complementary to the control nucleotide sequence to obtain a third labelled polynucleotide probe; (f) quantifying the amount of the third hybridized labelled polynucleotide probe; (g) measuring the relative populations of the first and second variants by determining the relative amounts of the first and second hybridized labelled polynucleotide probes compared to the third hybridized labelled polynucleotide probe.
REFERENCES:
patent: 5176995 (1993-01-01), Sninksy et al.
patent: 5827648 (1998-10-01), Eastman et al.
Gingeras et al J. Inf. Dis. vol. 164 pp. 1066-1074, 1991.
Eastman P. Scott
Kolberg Janice A.
Urdea Michael S.
Bayer Corporation
Horlick Kenneth R.
Reed Dianne E.
Reed & Associates
Siew Jeffrey
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