Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-05-01
2004-11-09
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S025320, C536S025400, C536S026600
Reexamination Certificate
active
06815167
ABSTRACT:
FIELD OF THE INVENTION
The present disclosure relates generally to methods for generating single-stranded DNA molecules of defined sequence and length from template containing a target nucleotide sequence. Specifically, the present disclosure provides a method for generating short single-stranded DNA molecules of defined sequence and length by linear or non-linear amplification of a template using specially designed primers or probes, conversion of double-stranded amplification products into single-stranded amplification products if necessary, and trimming single-stranded amplification products to yield the desired DNA molecule of defined sequence and length.
BACKGROUND OF THE INVENTION
Amplification of Target Sequences
A number of methods have been developed for amplification of target nucleotide sequences in nucleic acid templates. These include the polymerase chain reaction (PCR), rolling circle amplification (RCA), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), and strand displacement amplification (SDA).
Current methods of PCR amplification involve the use of two primers which hybridize to the regions flanking target nucleotide sequence, such that DNA replication initiated at the primers will replicate the target nucleotide sequence. By separating the replicated strands from the template strand with a denaturation step, another round of replication using the same primers can lead to many-fold amplification of the target nucleotide sequence.
Rolling circle amplification (RCA) is an isothermal amplification method in which a circularizable single-stranded probe is hybridized to a template such as RNA or denatured DNA at regions flanking the target nucleotide sequence, the strand is circularized using primer extension and/or ligation, sequences in the circle are then selectively amplified, and optionally, non-circular products are removed by digestion.
Linear and Nonlinear Amplification of Target Sequences
Amplification of target sequences may be carried out in linear or non-linear mode, for example as described in EP 0971039 to Rabanni et al. Linear amplification of target sequences may be used when a starting mixture contains a large number of copies of a target sequence. Generally, linear amplification utilizes a single initial primer, probe, or other nucleic acid construct to carry out the amplification process.
Non-linear amplification of target sites is often used when the number of copies of a target sequence present in the starting mixture is small. Non-linear amplification results in exponential growth in the number of gene copies present. PCR and RCA, especially RCA in the branching mode, can be used effectively in the non-linear amplification mode. (Lizardi et al., 1998,
Nature Genetics
19:225-232)
Generation of Single Stranded DNA
Many amplification methods generate double-stranded amplification products, while many applications require single-stranded DNA molecules containing the target sequence. Double-stranded DNA can be converted to single-stranded DNA by separating the strands or by removing one strand of the duplex. Strands of a duplex can be separated by thermal or chemical methods of disrupting interstrand bonds. Removing one strand allows recovery of the desired strand and elimination of its complement. One strategy for selectively removing one strand of a DNA duplex is to use exonuclease digestion, preferably 5′→3′ exonuclease digestion, where one strand is protected from attack by the exonuclease.
For example, U.S. Pat. No. 5,518,900 to Nikiforov et al. describes modifying one of two PCR primers used for amplification by incorporating phosphorothioate nucleotide derivatives in the 5′ end of the modified primer, rendering it resistant to exonuclease digestion. After amplifying target sequences using PCR, the double-stranded amplification product is subjected to exonuclease digestion. The unprotected strand is preferentially digested by a 5′→3′ exonuclease, leaving a single-stranded product consisting of the other strand.
In an alternate approach, Shchepinov et al. uses branched PCR primers that are resistant to 5′-exonuclease digestion, with the result that exonuclease digestion of the double-stranded amplification products gave single strands protected from digestion by the exonuclease-resistant branched primers. (Shchepinov et al., 1997,
Nuc Acids Res
25:4447-4454) Disadvantages of this method are that branched primers are difficult to synthesize and the resulting PCR products are branched.
Another approach to generating single-stranded DNA uses phosphorylation of the 5′ end of one strand of a double-stranded amplification product to produce a preferred lambda exonuclease substrate. (Higuchi et al., 1989,
Nuc Acids Res
25: 5685) This method allows selective degradation of the phosphorylated strand and recovery of the nonphosphorylated strand.
Generation of Short Single-stranded DNA Molecules
Short single-stranded DNA molecules of defined sequence and length are needed for applications such as arrays, where the desirable size range is about 45 nucleotides or less. Although methods for generating single-stranded DNA molecules are known in the art, these methods do not necessarily generate small molecules of 45 nucleotides or less. For example, the methods discussed above for generating single-stranded DNA do not provide short single-stranded DNA molecules of defined sequence and length. U.S. Pat. No. 5,518,900 to Nikiforov et al. teaches methods for generating single-stranded DNA molecules from double-stranded PCR amplification products, but the resulting PCR products are typically longer than 45 nucleotides. The method of Shchepinov et al. produces branched PCR products that are typically longer than 45 nucleotides. (Shchepinov et al., 1997,
Nuc Acids Res
25:4447-4454) Likewise, the method of Higuchi et al. yields single-stranded DNA products that are not in the desired size range. (Higuchi et al. 1989,
Nuc Acids Res
17: 5865)
Shaw and Mok disclose cleaving single-stranded DNA into fragments by interaction with a specially designed oligodeoxyribonucleotide adaptor and the class-IIN restriction endonuclease, XcmI. (Shaw and Mok, 1993,
Gene
133:85-89) After hybridizing to the target DNA and addition of XcmI, template DNA is specifically cleaved to near completion; however, hairpin structures on the template close to the hybridization site reduce the efficacy of cleavage.
SUMMARY OF THE INVENTION
The invention described herein is directed to methods for generating a single-stranded DNA molecule of defined sequence and length, where the method includes amplification, conversion, and trimming steps. In accordance with one aspect of the invention, amplification of a template having at least one target nucleotide sequence is directed by one or more primers having at least one exogenous nucleotide sequence not present in the target nucleotide sequence, where the amplification step generates amplification products with at least one target nucleotide sequence and at least one exogenous nucleotide sequence introduced by the primer. In accordance with another aspect of the invention, a conversion step may be performed. When the amplification step generates double-stranded amplification products, the method includes a conversion step wherein each double-stranded amplification product is converted to a single-stranded amplification product. When the amplification step generates single-stranded amplification products, the conversion step is not required. In accordance with another aspect of the invention, the single-stranded amplification product is trimmed to generate a single-stranded DNA molecule of defined sequence and length.
In accordance with one aspect of the invention, polymerase chain reaction (PCR) is used for the amplification step to produce double-stranded amplification products. In one embodiment, multiplex PCR may be used. The amplification step can be carried out in linear or non-linear mode. The template for am
Crothers Donald M.
Koenigsberger Carol
GeneOhm Sciences
Horlick Kenneth R.
Knobbe Martens Olson & Bear LLP
Tung Joyce
LandOfFree
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