METHOD TO PRODUCE SINGLE STRANDED DNA OF DEFINED LENGTH AND SEQUENCE AND DNA PROBES PRODUCED THEREBY
First Claim
1. A method for preparing a single stranded DNA molecule of defined sequence and length, having a predefined 5′
- end, a predefined 3′
end and a portion of a first strand of a double stranded precursor molecule, comprising the steps of;
(a) amplifying the double stranded precursor molecule with primers each containing a sequence homologous to a portion of the double stranded molecule and a sequence not homologous to the double stranded precursor molecule, said primers also containing a sequence comprising one of said predefined 5′
end or said predefined 3′
end, said primers further containing restriction enzyme cleavage sites to obtain an amplified double stranded molecule having cleavage sites at the 3′ and
5′
ends;
(b) cleaving the amplified double stranded molecule at the 5′
end with a restriction enzyme;
(c) removing a 5′
terminal phosphate on the first strand;
(d) cleaving the amplified double stranded molecule at the 3′
end; and
(e) digesting the second strand to prepare the single stranded DNA molecule of defined sequence and length.
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Abstract
A method for producing a single stranded DNA (ssDNA) molecule of a defined length and sequence is disclosed. This method enables the preparation of, inter alia, probes of greater length than can be chemically synthesized. The method starts with a double stranded molecule, such as genomic, double stranded DNA (dsDNA) from any organism. A fragment of the starting molecule (dsDNA) is amplified by specific primers engineered to introduce cleavage sites on either side of the desired sequence. Cleavage steps on the amplified, engineered fragment are combined with a phosphate removal step, thereby creating a construct that can be digested with an exonuclease without damage to the desired ssDNA. Probes, which hybridize with large gaps between the ends of the probes, are also disclosed.
23 Citations
14 Claims
-
1. A method for preparing a single stranded DNA molecule of defined sequence and length, having a predefined 5′
- end, a predefined 3′
end and a portion of a first strand of a double stranded precursor molecule, comprising the steps of;(a) amplifying the double stranded precursor molecule with primers each containing a sequence homologous to a portion of the double stranded molecule and a sequence not homologous to the double stranded precursor molecule, said primers also containing a sequence comprising one of said predefined 5′
end or said predefined 3′
end, said primers further containing restriction enzyme cleavage sites to obtain an amplified double stranded molecule having cleavage sites at the 3′ and
5′
ends;(b) cleaving the amplified double stranded molecule at the 5′
end with a restriction enzyme;(c) removing a 5′
terminal phosphate on the first strand;(d) cleaving the amplified double stranded molecule at the 3′
end; and(e) digesting the second strand to prepare the single stranded DNA molecule of defined sequence and length. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
- end, a predefined 3′
-
10. A method for obtaining a molecule for sequencing comprising the step of hybridizing said molecule to a single stranded DNA molecule of defined sequence and length, having a predefined 5′
- end, a predefined 3′
end and a portion of a first strand of a double stranded precursor molecule, said molecule further being prepared by a method comprising the steps of;(a) amplifying the double stranded precursor molecule with primers each containing a sequence homologous to a portion of the double stranded molecule and a sequence not homologous to the double stranded precursor molecule, said primers also containing a sequence comprising one of said predefined 5′
end or said predefined 3′
end, said primers further containing restriction enzyme cleavage sites to obtain an amplified double stranded molecule having cleavage sites at the 3′ and
5′
ends;(b) cleaving the amplified double stranded molecule at the 5′
end with a restriction enzyme;(c) removing a 5′
terminal phosphate on the first strand;(d) cleaving the amplified double stranded molecule at the 3′
end; and(e) digesting the second strand to prepare the single stranded DNA molecule of defined sequence and length. - View Dependent Claims (11, 12)
- end, a predefined 3′
- 13. A primer for performing PCR amplification comprising a homology region for hybridization to a target under annealing conditions and a non-homologous region that contains a restriction endonuclease recognition sequence.
Specification