Detection of nucleic acids in cells by thermophilic strand displacement amplification
AssigneeBecton, Dickinson and Company
InventorsKenton L. Lohman, Natalie V. Ostrerova, Mark V. Cleve, Robert A. Reid
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Abstract
Thermophilic Strand Displacement Amplification (tSDA) for amplification of nucleic acid target sequences in situ in cells in suspension, on slides or in tissues is described. Excellent specimen morphology is preserved, and either DNA targets, RNA targets, or both may be selectively amplified. In situ amplification by tSDA is compatible with immunochemical techniques, so that both amplification of target sequences and immunological staining can be performed on the same specimen.
Claims35
A method for amplifying a double stranded HIV gag target sequence comprising the steps of:
- a) hybridizing a first amplification primer comprising the target binding sequence of SEQ ID NO:1 3' to the target sequence on a first strand of the target sequence and hybridizing a second amplification primer comprising the target binding sequence of SEQ ID NO:2 3' to the target sequence on a second strand of the target sequence;
- b) extending the first and second amplification primers with polymerase to produce first and second amplification primer extension products;
- c) displacing the first and second amplification primer extension products from the target sequence, and;
- d) repeating the hybridizing, extending and displacing steps such that the target sequence is amplified.
A method for amplifying a double stranded HIV gag target sequence comprising the steps of:
- a) hybridizing a first amplification primer consisting of SEQ ID NO:1 3' to the target sequence on a first strand of the target sequence and hybridizing a second amplification primer consisting of SEQ ID NO:2 3' to the target sequence on a second strand of the target sequence, and hybridizing external primers upstream of the first and second amplification primers;
- b) extending the first and second amplification primers and the external primers in the presence of a 5'.fwdarw.3' exonuclease deficient polymerase with strand displacing activity, the polymerase being active between about 50.degree. C. and 75.degree. C., an .alpha.-thio deoxynucleoside triphosphate, and BsoBI, thereby producing first and second amplification primer extension products comprising BsoBI recognition sites which are displaced from the first and second strands of the target sequence by extension of the external primers;
- c) making the first and second amplification primer extension products and the BsoBI recognition sites double stranded by synthesizing complementary strands, thereby nicking the double stranded BsoBI recognition sites with the BsoBI;
- d) extending from the nicks using the polymerase, thereby displacing a copy of the target sequence from the double stranded first and second amplification primer extension products, and;
- e) repeating the nicking, extending and displacing steps such that the target sequence is amplified.
A method for amplifying a double stranded HLA-DQ.alpha. exon 3 target sequence comprising the steps of:
- a) hybridizing a first amplification primer comprising the target binding sequence of SEQ ID NO:6 3' to the target sequence on a first strand of the target sequence and hybridizing a second amplification primer comprising the target binding sequence of SEQ ID NO:7 3' to the target sequence on a second strand of the target sequence;
- b) extending the first and second amplification primers with polymerase to produce first and second amplification primer extension products;
- c) displacing the first and second amplification primer extension products from the target sequence, and;
- d) repeating the hybridizing, extending and displacing steps such that the target sequence is amplified.
A method for amplifying a double stranded HLA-DQ.alpha. exon 3 target sequence comprising the steps of:
- a) hybridizing a first amplification primer consisting of SEQ ID NO:6 3' to the target sequence on a first strand of the target sequence and hybridizing a second amplification primer consisting of SEQ ID NO:7 3' to the target sequence on a second strand of the target sequence, and hybridizing external primers upstream of the first and second amplification primers;
- b) extending the first and second amplification primers and the external primers in the presence of a 5'.fwdarw.3' exonuclease deficient polymerase with strand displacing activity, the polymerase being active between about 50.degree. C. and 75.degree. C., an .alpha.-thio deoxynucleoside triphosphate, and BsoBI, thereby producing first and second amplification primer extension products comprising BsoBI recognition sites which are displaced from the first and second strands of the target sequence by extension of the external primers;
- c) making the first and second amplification primer extension products and the BsoBI recognition sites double stranded by synthesizing complementary strands, thereby nicking the double stranded BsoBI recognition sites with BsoBI;
- d) extending from the nicks using the polymerase, thereby displacing a copy of the target sequence from the double stranded first and second amplification primer extension products, and;
- e) repeating the nicking, extending and displacing steps such that the target sequence is amplified.
An isolated oligonucleotide comprising the target binding sequence of SEQ ID NO:
- 1, the target binding sequence of SEQ ID NO:2, the target binding sequence of SEQ ID NO:6 or the target binding sequence of SEQ ID NO:7.
An isolated oligonucleotide consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:9.
An isolated oligonucleotide consisting of SEQ ID NO:
- 5, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.
Dependencies are inferred from the claim text.