Method of detecting nucleic acid sequences

Claim 1

A probe-vector molecule used for detecting a target nucleic acid containing a particular base sequence X 1 X 2 X 3 . . . X m . . . Y 1 Y 2 Y 3 . . . Y n , said probe-vector comprising a linear partially single stranded DNA molecule comprised of two strands, a long strand containing the sequence X' m X' m-1 . . . X' 3 X' 2 X' 1 . . . Z' 1 Z' 2 Z' 3 . . . Z'hd p . . . Y' n Y' n-1 . . . Y' 3 Y' 2 Y' 1 , and a short strand containing the sequence Z 1 Z 2 Z 3 . . . Z p , wherein m, n, p and k are integers, and in which for any k, X' k is the base complementary to X k is the base complementary to Y k , and Z' k is the base complementary to Z K , the ends of said long strand of said probe-vector being substantially complementary to sections of said target nucleic acid, m and n are sufficiently large so that when said probe-vector is added to said target nucleic acid under hybridizing conditions, stable hybridization will occur between the ends of said long strand of said probe-vector and the said substantially complementary section of said target nucleic acid forming thereby a circular hybrid, the probe-vector being circularized if the particular target sequence is present and has hybridized to said probe-vector, and the regions Z 1 Z 2 Z 3 . . . Z p and Z' 1 Z' 2 Z' 3 . . . Z' p of said probe-vector strands contain a replicon and confer a detectable phenotype such that bacteria transformed by said hybrid are detectable and distinguishable from untransformed bacteria by detection of said phenotype.

Claim 4

A method for determining the presence, in a mixture of DNA, of a target nucleic acid containing a particular base sequence X 1 X 2 X 3 . . . X m . . . Y 1 Y 2 Y 3 . . . Y n , said method requiring the use of a probe-vector having a long strand and a short strand or just a long strand, wherein the long strand comprises the sequence X' m X' m-1 . . . X' 3 X' 2 X' 1 . . . Z' 1 Z' 2 Z' 3 . . . Z' p . . . Y' n Y' n-1 . . . Y' 3 Y' 2 Y' 1 , and the short strand comprises the sequence Z 1 Z 2 Z 3 . . . Z p , where m, n, p and k are integers and wherein for any k, X' k is the base complementary to X k , Y' k is the base complementary to Y k , and Z' k is the base complementary to Z k , the ends of said long strand of said probe-vector being substantially complementary to sections of said target nucleic acid, m and n are sufficiently large so that when said probe-vector is added to said target nucleic acid under hybridizing conditions, stable hybridization will occur between the ends of said long strand of said probe-vector and the said substantially complementary section of said target DNA forming thereby a circular hybrid, the probe-vector being circularized if the particular target sequence is present and has hybridized to said prove-vector, and the regions Z 1 Z 2 Z 3 . . . Z p and Z' 1 Z' 2 Z' 3 . . . Z' p of said probe vector strands contain a replicon and confer a detectable phenotype such that bacteria transformed by said hybrid are detectable and distinguishable from untransformed bacteria by detection of said phenotype, said method comprising the following steps: (A) introducing said probe-vector to the sample containing nucleic acids, said nucleic acids being single stranded or being made single stranded before or after the addition of probe-vector, said mixture of sample and probe-vector comprising the test mixture; (B) adjusting the conditions of the test mixture to hybridization conditions, such conditions being favorable for the formation of circular hybrids between said long strand and short strand or only with said long strand together with said target if said target is present in the sample, but such conditions being unfavorable for the formation of hybrids between non-target nucleic acids and probe-vector, the test mixture after hybridization comprising the hybridization mixture; (C) introducing said hybridization mixture to bacterial cells, said bacterial cells being predisposed to transformation by circular DNA molecules but not by linear DNA molecules, said introduction being made under conditions which allow transformation by said circular hybrid but not by linear probe-vector, said bacterial cells lacking the detectable phenotype conferred by said probe-vector, said mixture of bacterial cells and hybridization mixture comprising the transformation mixture; (D) adjusting the conditions of the transformation mixture to allow detection of the phenotypic marker, said phenotypic marker being exhibited only by transformed cells, such a mixture comprising the detection mixture; and (E) detecting the phenotypic marker in the detection mixture.

Claim 5

A method for determining the presence, in a mixture of DNA, of a target nucleic acid containing a particular base sequence X 1 X 2 X 3 . . . X m . . . Y 1 Y 2 Y 3 . . . Y n , said method requiring the use of a probe-vector having a long strand and a short strand or just a long strand, wherein the long strand comprises the sequence X' m X' m-1 . . . X' 3 X' 2 X' 1 . . . Z' 1 Z' 2 Z' 3 . . . Z' p . . . Y' n Y' n-1 . . . Y' 3 Y' 2 Y' 1 , and the short strand comprises the sequence Z 1 Z 2 Z 3 . . . Z p , where m, n, p and k are integers and wherein for any k, X' k is the base complementary to X k , Y' k is the base complementary to Y k , and Z' k is the base complementary to Z k , the ends of said long strand of said probe-vector being substantially complementary to sections of said target nucleic acid, m and n are sufficiently large so that when said probe-vector is added to said target nucleic acid under hybridizing conditions, stable hybridization will occur between the ends of said long strand of said probe-vector and the said substantially complementary section of said target DNA forming thereby a circular hybrid, the probe-vector being circularized if the particular target sequence is present and has hybridized to said prove-vector, and the regions Z 1 Z 2 Z 3 . . . Z p and Z' 1 Z' 2 Z' 3 . . . Z' p of said probe vector strands contain a replicon and confer a detectable phenotype such that bacteria transformed by said hybrid are detectable and distinguishable from untransformed bacteria by detection of said phenotype, said method comprising the following steps: (A) introducing said probe-vector to the sample containing nucleic acids, said nucleic acids being single stranded or being made single stranded before or after the addition of probe-vector, said mixture of sample and probe-vector comprising the test mixture; (B) adjusting the conditions of the test mixture to hybridization conditions, such conditions being favorable for the formation of circular hybrids between said long strand and short strand or only with said long strand together with said target if said target is present in the sample, but such conditions being unfavorable for the formation of hybrids between non-target nucleic acids and probe-vector, the test mixture after hybridization comprising the hybridization mixture; (C) introducing said hybridization mixture to bacterial cells, said bacterial cells being predisposed to transformation by circular DNA molecules but not by linear DNA molecules, said introduction being made under conditions which allow transformation by said circular hybrid but not by linear probe-vector, said bacterial cells lacking the detectable phenotype conferred by said probe-vector, said mixture of bacterial cells and hybridization mixture comprising the transformation mixture; (D) adjusting the conditions of the transformation mixture to allow detection of the phenotypic marker, said phenotypic marker being exhibited only by transformed cells, such a mixture comprising the detection mixture; and (E) detecting the phenotypic marker in the detection mixture.