Nucleotide sequence composition and method for detection of Neisseria gonorrhoeae and method for screening for a nucleotide sequence that is specific for a genetically distinct group
AssigneeEnzo Biochem, Inc.
InventorsAndrew Lo, Huey-Lang Yang
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Abstract
This invention relates to specific discrete nucleotide sequences. More precisely, this invention relates to a composition comprising discrete nucleotide sequences that are specific for Neisseria gonorrhoeae, as defined hereinbelow, i.e. the compositions of the subject invention can be utilized to detect Neisseria gonorrhoeae chromosomal DNA and thus, Neisseria gonorrhoeae.
Claims6
A composition of matter that is specific for Neisseria gonorrhoeae comprising at least one nucleotide sequence for which the ratio of the amount of said sequence which hybridizes to chromosomal DNA of Neisseria gonorrhoeae to the amount of said sequence which hybridizes to chromosomal DNA of Neisseria meningitidis is greater than about five, said ratio being obtained by a method comprising the folllowing steps;
- (a) providing a radioactively labeled form of said nucleotide sequence;
- (b) providing a serial dilution series of purified chromosomal DNA from each of the N. gonorrhoeae strains;
- (1) ATCC 53420, (2) ATCC 53421, (3) ATCC 53422, (4) STCC 53423, (5) ATCC 53424, (6) ATCC 53425, and forming test dots from each of said dilution series on a matrix;
- (c) providing a serial dilution series of purified nucleotide sequences from each of the N. meningitidis strains:
- (1) ATCC 53414, (2) ATCC 53415, (3) ATCC 53416, l (4) ATCC 53417, (5) ATCC 53418, (6) ATCC 53419, and forming test dots from each of said dilution series on a matrix;
- (d) hybridizing equal portions of the labeled nucleotide sequences to the matrix provided in step (b) and (c), respectively;
- wherein the hybridization is conducted in a solution having a slat concentration of 2X SSC at (i) 65.degree. C. in cases in which the sequence has greater than 50 base pairs or (ii) at Tm (.degree.C.) minus 30.degree. C. in cases in which the sequence has less than 50 base pairs, wherein Tm is the denaturation temperature of the sequence;
- (e) quantifying the labeled nucleotide sequence hybridized in step (d) to each test dot;
- (f) subtracting from the data of step (a) an averaged amount of radioactivity attributable to background to obtain a corrected amount of hybridized radioactivity at each test dot;
- (g) normalizing the data of step (f) by multiplying the amount of corrected radioactivity at each test dot by a factor which adjusts the amount of radioactivity to equal amounts of chromosomal DNA at each test dot;
- (h) selecting two normalized values that are most nearly the same and that correspond to adjacent members of the dilution series for each of the above strains of N. gonorrhoeae and obtaining the average of the selected values;
- (i) selecting two normalized values that are most nearly the same and that correspond to adjacent members of the dilution series for each of the above strains of N. meningitidis and obtaining the average of the selected values;
- (j) dividing the lowest average obtained in step (h) by the highest average obtained in step (i) to obtain said ratio.
Dependencies are inferred from the claim text.