Amplification of DNA to produce single-stranded product of defined sequence and length

• US 20050026208A1
• Filed: 09/01/2004
• Published: 02/03/2005
• Est. Priority Date: 04/25/2002
• Status: Abandoned Application

• Current Assignee: Carol Koenigsberger, Donald M. Crothers
• Original Assignee: Carol Koenigsberger, Donald M. Crothers
• Inventors: Koenigsberger, Carol, Crothers, Donald M.
• Application Number:
  US10/932,518
• Publication Number:
  US 20050026208A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

The present disclosure relates to methods for generating single-stranded DNA molecules of defined sequence and length. Specifically, a region of template containing target sequence is amplified by PCR or RCA, exogenous sequence is introduced by primers or probes used in amplification, double-stranded amplification products are converted to single-stranded amplification products, and single-stranded amplification products are trimmed to produce short single-stranded DNA molecules of defined sequence and length.

Claims

1. A method for identifying at least one organism or individual, comprising:

obtaining template comprising at least one target nucleotide sequence from said organism or individual, wherein said template comprises genomic DNA, cDNA, or RNA;

amplifying said template comprising at least one target nucleotide sequence, said amplification being directed by at least one primer comprising at least one exogenous nucleotide sequence not present in the target nucleotide sequence, wherein said amplification generates a plurality of double-stranded amplification products comprising said at least one target nucleotide sequence and said at least one exogenous nucleotide sequence introduced by said at least one primer;

conversion of said double-stranded amplification products to single-stranded amplification products;

trimming said single-stranded amplification product to generate a single-stranded DNA molecule of defined sequence and length;

determining the mass or nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length; and

using at least one said mass or nucleotide sequence of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

2. The method of claim 1, wherein mass spectroscopy is used to determine said mass or nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length.

3. The method of claim 1, comprising determining said mass of each said single-stranded DNA molecule of defined sequence and length and using at least one said mass of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

4. The method of claim 1, comprising determining said nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length and using at least one said nucleotide sequence of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

5. The method of claim 1, wherein a multiplicity of organisms or individuals is identified.

6. A method for identifying at least one organism or individual, comprising:

obtaining template comprising at least one target nucleotide sequence from said at least one organism or individual, wherein said template comprises genomic DNA, cDNA, or RNA;

amplifying said template comprising at least one target nucleotide sequence, said amplification being directed by at least one primer comprising at least one exogenous nucleotide sequence not present in the target nucleotide sequence, wherein said amplification generates a plurality of single-stranded amplification products comprising said at least one target nucleotide sequence and said at least one exogenous nucleotide sequence introduced by said at least one primer;

trimming each said single-stranded amplification product to generate a single-stranded DNA molecule of defined sequence and length;

determining the mass or nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length; and

using at least one said mass or nucleotide sequence of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

7. The method of claim 6, wherein mass spectroscopy is used to determine said mass or nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length.

8. The method of claim 6, comprising determining said mass of each said single-stranded DNA molecule of defined sequence and length and using at least one said mass of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

9. The method of claim 6, comprising determining said nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length and using at least one said nucleotide sequence of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

10. The method of claim 6, wherein a multiplicity of organisms or individuals is identified.