Detection of mismatches by cleavage of nucleic acid heteroduplexes
First Claim
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1. A method of detecting a mismatch between a first nucleic acid strand and a second nucleic acid strand comprising:
- forming a plurality of duplexes, each duplex including a molecule of said first nucleic acid strand, or a portion thereof, and a molecule of said second nucleic acid strand, or a portion thereof;
contacting a first bacteriophage resolvase and a second bacteriophage resolvase with said plurality of duplexes, wherein said first and said second resolvases are different;
determining if said plurality of duplexes is cleaved by either or both of said first and said second resolvases, wherein cleavage indicates the presence of a mismatch.
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Abstract
Novel methods and kits for detecting mismatches in nucleic acids using one or more resolvase enzymes. These methods can be used in medical procedures or research, veterinary procedures or research, agricultural applications, forensics, and paternity testing. For example, in forensics and paternity testing, methods of the invention can replace or complement RFLP mapping.
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Citations
30 Claims
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1. A method of detecting a mismatch between a first nucleic acid strand and a second nucleic acid strand comprising:
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forming a plurality of duplexes, each duplex including a molecule of said first nucleic acid strand, or a portion thereof, and a molecule of said second nucleic acid strand, or a portion thereof; contacting a first bacteriophage resolvase and a second bacteriophage resolvase with said plurality of duplexes, wherein said first and said second resolvases are different; determining if said plurality of duplexes is cleaved by either or both of said first and said second resolvases, wherein cleavage indicates the presence of a mismatch. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 26, 27, 28, 29, 30)
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11. A method of detecting a mismatch between a first nucleic acid strand and a second nucleic acid strand comprising:
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amplifying a portion of said first strand; providing a plurality of said second strands; forming a first duplex between an amplification product of said first strand and one of said second strands; forming a second duplex between an amplification product of said first strand and one of said second strands; contacting said first duplex with a first bacteriophage resolvase; contacting said second duplex with a second bacteriophage resolvase, wherein said first and said second resolvases are different; determining if at least one strand of said duplex is cleaved, cleavage being indicative of a mismatch.
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12. A method of determining the location of a mismatch between a first nucleic acid strand and a second nucleic acid strand comprising:
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forming a plurality of duplexes, each duplex including a molecule of said first nucleic acid strand, or a portion thereof, and a molecule of said second nucleic acid strand, or a portion thereof; contacting a first bacteriophage resolvase and a second bacteriophage resolvase with said plurality of duplexes, wherein said first and said second resolvases are different; determining the distance between the cleavage site and a second site on the strand (or the complement of the strand).
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13. A method of determining the location of a mismatch between a first nucleic acid strand and a second nucleic acid strand comprising:
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forming a first duplex between a molecule of said first strand, or a portion thereof, and a molecule of said second strand, or a portion thereof; contacting said first duplex with a first bacteriophage resolvase; forming a second duplex between a molecule of said first strand and a molecule of said second strand; contacting said second duplex with a second bacteriophage resolvase wherein said first and said second resolvases are different; determining the distance between the cleavage site and a second site on the strand (or the complement of the strand) by determining the size of a cleavage product, thereby determining the location of said mismatch. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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24. An in vitro reaction mixture comprising:
- a first purified bacteriophage resolvase;
a second purified bacteriophage resolvase, wherein said first and said second resolvases are different; and
a purified nucleic acid molecule.
- a first purified bacteriophage resolvase;
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25. A kit comprising:
- a first purified bacteriophage resolvase;
a second purified bacteriophage resolvase, wherein said first and said second resolvases are different; and
a purified nucleic acid molecule.
- a first purified bacteriophage resolvase;
Specification