Restriction endonuclease enhanced polymorphic sequence detection

• US 8,206,926 B2
• Filed: 03/25/2009
• Issued: 06/26/2012
• Est. Priority Date: 03/26/2008
• Status: Expired due to Fees

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Ehrich, Mathias, Van den Boom, Dirk Johannes
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US12/411,329
• Publication Number:
  US 20090317818A1
• Time in Patent Office: 1,189 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

Provided in part herein is an improved method for the detection of specific polymorphic alleles in a mixed DNA population. The method comprises enriching the relative percentage of a given polymorphic allele that is exponentially amplifiable by PCR. Provided also are methods for selectively enriching target nucleic acid, for example, fetal nucleic acid in a maternal sample. In the case of detecting fetal nucleic acid in a maternal sample, a restriction enzyme is introduced that can discriminate between the alleles of a polymorphic site. In some embodiments, the maternal allele is digested and nucleic acid comprising the paternal allele is relatively enriched.

Claims

1. A method for detecting in nucleic acid the presence or absence of one or more target alleles of paternal origin, comprising:

a) cleaving nucleic acid from a sample with a Tsp509I restriction enzyme;

b) exposing the nucleic acid after (a) to amplification conditions that amplify uncleaved nucleic acid but not cleaved nucleic acid, and which amplification conditions are capable of generating amplification products comprising some or all of the single nucleotide polymorphic loci selected from the group consisting of;

2. The method of claim 1, wherein part (b) is conducted using amplification primer pairs selected the group consisting of:

3. The method of claim 1, further comprising contacting the amplification products with one or more extension primers.

4. The method of claim 3, wherein the extension primers are selected from the group consisting of:

5. The method of claim 1, wherein the sample is from a pregnant female or a female suspected of being pregnant.

6. The method of claim 5, wherein the sample is blood.

7. The method of claim 1, wherein 20 or more of the single nucleotide polymorphic loci are assayed.

8. The method of claim 1, wherein 60 or more of the single nucleotide polymorphic loci are assayed.

9. The method of claim 1, wherein 90 or more of the single nucleotide polymorphic loci are assayed.

10. The method of claim 1, wherein the target allele concentration is 10% or less of total nucleic acid concentration prior to cleaving and amplifying the nucleic acid.

11. The method of claim 1, wherein four or more of the single nucleotide polymorphic loci are informative of the presence or absence of the one or more target alleles.

12. The method of claim 1, further comprising determining whether fetal nucleic acid is present in the sample.

13. A method for detecting in nucleic acid the presence or absence of one or more target alleles of paternal origin, comprising:

a) cleaving nucleic acid from a sample with a Tsp509I restriction enzyme;

b) exposing the nucleic acid after (a) to amplification conditions that amplify uncleaved nucleic acid but not cleaved nucleic acid, and which amplification conditions are capable of generating amplification products comprising the following single nucleotide polymorphic loci;

14. The method of claim 13, wherein part (b) is conducted using amplification primer pairs:

15. The method of claim 13, further comprising contacting the amplification products with extension primers.

16. The method of claim 15, wherein the extension primers are:

17. The method of claim 13, wherein the sample is from a pregnant female or a female suspected of being pregnant.

18. The method of claim 17, wherein the sample is blood.

19. The method of claim 13, wherein the target allele concentration is 10% or less of total nucleic acid concentration prior to cleaving and amplifying the nucleic acid.

20. The method of claim 13, wherein four or more of the single nucleotide polymorphic loci are informative of the presence or absence of the one or more target alleles.

21. The method of claim 13, further comprising determining whether fetal nucleic acid is present in the sample.

22. A method for detecting in nucleic acid the presence or absence of one or more target alleles of paternal origin, comprising:

a) cleaving nucleic acid from a sample with a Tsp509I restriction enzyme;

b) exposing the nucleic acid after (a) to amplification conditions that amplify uncleaved nucleic acid but not cleaved nucleic acid, and which amplification conditions are capable of generating amplification products comprising some or all of the single nucleotide polymorphic loci selected from the group consisting of;

23. A method for detecting in nucleic acid the presence or absence of one or more target alleles of paternal origin, comprising:

a) cleaving nucleic acid from a sample with a Tsp509I restriction enzyme;

b) exposing the nucleic acid after (a) to amplification conditions that amplify uncleaved nucleic acid but not cleaved nucleic acid, and which amplification conditions are capable of generating amplification products comprising some or all of the single nucleotide polymorphic loci selected from the group consisting of;

24. A method for detecting in nucleic acid the presence or absence of one or more target alleles of paternal origin, comprising:

a) cleaving nucleic acid from a sample with a Tsp509I restriction enzyme;

b) exposing the nucleic acid after (a) to amplification conditions that amplify uncleaved nucleic acid but not cleaved nucleic acid, and which amplification conditions are capable of generating amplification products comprising some or all of the single nucleotide polymorphic loci selected from the group consisting of;