METHOD TO PRODUCE SINGLE STRANDED DNA OF DEFINED LENGTH AND SEQUENCE AND DNA PROBES PRODUCED THEREBY

• US 20110086393A1
• Filed: 12/17/2010
• Published: 04/14/2011
• Est. Priority Date: 05/25/2006
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Davis, Ronald W., Krishnakumar, Sujatha, Mindrinos, Michael
• Granted Patent: US 8,795,968 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

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.

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.

2. The method of claim 1 wherein the cleaving is done with a type IIs restriction enzyme.

3. The method of claim 1 further comprising the steps of digesting with BsaI to generate a 5′

overhang outside of BsaI'"'"'s recognition site.

4. The method of claim 1 wherein multiple probes, having different sequences, are prepared using single cleaving, removing and digesting steps.

5. The method of claim 1 wherein the cleaving with a restriction enzyme at the desired 5′

end done with a restriction enzyme to create either a 5′

overhang or blunt end on the first strand at the desired 5′

end.

6. The method of claim 1 wherein the cleaving with a restriction enzyme at the desired 3′

end is done with a restriction enzyme to create a 3′

blunt end or no 5′

overhang.

7. The method of claim 1 wherein the predefined 5′

end and predefined 3′

end are separated by at least 125 nucleotides of spacer backbone from the precursor molecule.

8. The method of claim 1 wherein the spacer backbone is prepared from a nonhuman organism.

9. The method of claim 8 wherein the nonhuman organism is a microorganism.

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.

11. The method of claim 10 wherein said molecule for sequencing spans an exon.

12. The method of claim 10 further comprising the step of sequencing said molecule.

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.

14. The primer of claim 13 further comprising a non-homologous region forming a primer-binding site to another primer.