Method to produce single stranded DNA of defined length and sequence and DNA probes produced thereby
First Claim
1. A polynucleic acid probe, having a defined sequence, for hybridization to a target polynucleic acid sequence, comprising the following sub-sequences:
- (a) a first target sequence, at one end of the probe, complementary to a first target region of the target polynucleic acid, for specifically binding thereto;
(b) a second target sequence, at an opposite end of the probe, complementary to a second target region of the target polynucleic acid for specifically binding thereto,where said first and second target regions are separated on the target polynucleic acid, when hybridized thereto, by a gap of at least 250 nt of target sequence, said gap adapted to be filled by at least 250 nt of added gap filling nucleotides;
(c) at least one amplification primer site, on the probe, adjacent the target sequence, and connected to a spacer backbone sequence, for specifically binding a PCR primer, said primer oriented in a direction for amplification of target sequences and gap filling nucleotides only when nucleotides are joined to the target sequences as complementary to the target polynucleic acid and further oriented to not amplify the backbone sequence; and
(d) said spacer backbone sequence, of at least 125 nt, chosen to be non-complementary to the target polynucleic acid, and being a random nucleic acid sequence having a length permitting a length of said gap of at least 250 nt, said spacer backbone sequence having a length of at least 50% of the length of said gap.
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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.
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Citations
16 Claims
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1. A polynucleic acid probe, having a defined sequence, for hybridization to a target polynucleic acid sequence, comprising the following sub-sequences:
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(a) a first target sequence, at one end of the probe, complementary to a first target region of the target polynucleic acid, for specifically binding thereto; (b) a second target sequence, at an opposite end of the probe, complementary to a second target region of the target polynucleic acid for specifically binding thereto, where said first and second target regions are separated on the target polynucleic acid, when hybridized thereto, by a gap of at least 250 nt of target sequence, said gap adapted to be filled by at least 250 nt of added gap filling nucleotides; (c) at least one amplification primer site, on the probe, adjacent the target sequence, and connected to a spacer backbone sequence, for specifically binding a PCR primer, said primer oriented in a direction for amplification of target sequences and gap filling nucleotides only when nucleotides are joined to the target sequences as complementary to the target polynucleic acid and further oriented to not amplify the backbone sequence; and (d) said spacer backbone sequence, of at least 125 nt, chosen to be non-complementary to the target polynucleic acid, and being a random nucleic acid sequence having a length permitting a length of said gap of at least 250 nt, said spacer backbone sequence having a length of at least 50% of the length of said gap. - View Dependent Claims (2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15)
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8. A pre-probe for making a probe for hybridization to a target polynucleic acid sequence, comprising a polynucleic acid molecule comprising the following sequences:
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(a) a first target sequence, at a 5′
end of the probe, complementary to a first region of the target polynucleic acid, for specifically binding thereto,(b) a second target sequence, at a 3′
end of the probe, complementary to a second region of target polynucleic acid for specifically binding thereto, said first and second regions of target polynucleic acid separated on the target polynucleic acid by a gap, said gap having a length of at least 250 nt,(c) at least one amplification primer site, adjacent a target sequence, and connected to a spacer backbone sequence, for specifically binding PCR primers, said primers oriented in a direction for amplification of target sequences only when nucleic acids are joined to the target sequences as complementary to the target polynucleic acid and further oriented to not amplify the spacer backbone sequence; (d) said spacer backbone sequence being of at least 250 nt. wherein said backbone sequence is a random nucleic acid sequence chosen to be non-complementary to any target polynucleic acid, said spacer backbone sequence having a length of at least 50% of the length of said gap; and (e) restriction endonuclease recognition sites 5′
of the desired 5′
target sequence and 3′
of the desired 3′
target sequence, further defining cut sites at the desired final 5′
nucleotide and desired final 3′
nucleotide, said restriction endonuclease recognition sites being comprised in a sequence extending 5′
of said desired final 5′
nucleotide and in a sequence extending 3′
of said desired final 3′
nucleotide.
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16. A method for specifically detecting a target polynucleic acid sequence, comprising:
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(a) hybridizing the target polynucleic acid sequence with a probe comprising the following sequences; (i) a first target sequence, at one end of the probe, complementary to a first region of the target polynucleic acid, for specifically binding thereto, (ii) a second target sequence. at an opposite end of the probe, complementary to a second region of target polynucleic acid for specifically binding thereto, said first and second regions of target polynucleic acid separated on the target polynucleic acid by a gap, said gap having a length of at least 250 nt, (iii) at least one amplification primer site, adjacent the target sequence, and connected to a backbone sequence, for specifically binding PCR primers, said primers oriented in a direction for amplification of target sequences only when nucleic acids are joined to the target sequences as complementary to the target polynucleic acid and further oriented to not amplify the backbone sequence; and (iv) a backbone sequence which is at least 125 nt long to permit hybridization of the first target sequence and the second target sequence with said gap, said spacer backbone sequence being at least 50% of the length of said gap and wherein said spacer backbone sequence is a random nucleic acid sequence from a nonhuman organism and does not hybridize to target sequences; (b) filling in said gap of at least 250 nt with gap filling nucleotides to form a circular probe; and (c) amplifying said circular probe without cleaving the probe using said amplification primer site, whereby amplification only occurs if said first target sequence and said second target sequence hybridize to the target polynucleotide, and said gap is filled.
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Specification