Nucleic acid detection and amplification by chemical linkage of

Details Nucleic acid detection and amplification by chemical linkage of Nucleic acid detection and amplification by chemical linkage of

1995-05-01

1998-12-01

Zitomer, Stephanie W.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 4, 435 5, 435 911, 435 912, 435 913, 536 231, 536 243, 536 2431, 536 2432, 536 2433, C12Q 168, C12P 1934, C07H 2102, C07H 2104

Patent

active

058436502

ABSTRACT:
The present invention and kits are directed to a method of amplifying and detecting single or double-stranded target nucleic acid molecules in a test sample. Amplification is accomplished through the use of a minimum of two oligonucleotide probe complement pairs, wherein members oligonucleotide probes from both pair of oligonucleotide probe complement pairs form a minimum of two oligonucleotide probe pairs, at least one of which is complementary to a given portion of a target nucleic acid sequence which act as template. One of the oligonucleotide probes of each oligonucleotide probe pair have an additional protecting sequence which is not complementary to the target sequence. These additional protecting sequences are preferably complementary to each other. Chemical functionality groups attached to the oligonucleotide probes covalently combine the probes to form a joined oligonucleotide product. The joined oligonucleotide product is formed without the use of enzymes. The reactivity of the chemical functionality groups on each probe is target dependent. The chemical functionality group on each probe is prevented from reacting with other chemical functionality groups on other probes unless the probes are properly hybridized to the target molecule. The chemical functionality groups are covalently attached to the oligonucleotide probes in such a way that they are sheltered or protected from the chemical functionality groups of other probes while the probes are in solution. Only when the oligonucleotide probes of an oligonucleotide probe pair are hybridized to the target sequence are the chemical functionality groups on the probes brought into close enough proximity to form a covalent bond and join the probes to form a joined oligonucleotide product. Once formed, the joined oligonucleotide product is denatured from the target nucleic acid molecule thereby doubling the amount of target sequences originally present in the sample. The process is repeated a desired number of times to produce detectable amounts of joined oligonucleotide products.

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patent: 5437977 (1995-08-01), Segev
patent: 5451503 (1995-09-01), Hogan et al.
Wu et al, "The ligation amplification reaction (LAR)-Amplification of specific DNA sequences using sequential rounds of template directed ligation", Genomics 4:560-569, May, 1989.
Stratagene Catalog (1994) p. 113, Jan. 1994.
Matthews et al, "Analytical strategies for the use of DNA probes", Anal. Biochem. 169:1-25, Feb. 1988.

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