Method for determining alleles

• US 20030082549A1
• Filed: 08/30/2001
• Published: 05/01/2003
• Est. Priority Date: 08/30/2000
• Status: Abandoned Application

• Current Assignee: Invitrogen Dynal AS (Invitrogen Dynal Holding AS)
• Original Assignee: Dynal Biotech ASA
• Inventors: Liu, Xiangjun
• Application Number:
  US09/943,416
• Publication Number:
  US 20030082549A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention provides methods and kits for separating and identifying alleles, and thereby the haplotype, in genomic DNA samples. The method generally involves hybridizing primers specific to polymorphic sites within the alleles to the DNA sample, elongating the primers by one or more nucleic acids, separating the elongated primers and identifying the alleles utilizing the elongated primer. The method also allows for a ligation of two primers, their separation and subsequent use in identifying the targeted allele. The method further provides that another primer can be used as a blocking site for elongation of the first primer such that a stretch of DNA that includes a polymorphic site is replicated and identified. The unextended or extended primers can be labeled so that the primer can be easily separated and/or identified.

Claims

1. A method for separating nucleic acid molecules which have specific alleles, comprising:

(a) hybridizing a nucleic acid comprising a heterosequence site with at least one nucleic acid primer specific to the heterosequence site to form a hybridized nucleic acid sequence, wherein the at least one specific nucleic acid primer is capable of undergoing elongation only when hybridized to the heterosequence site;

(b) subjecting the hybridized nucleic acid sequence to conditions which permit elongation of the at least one nucleic acid primer; and

(c) separating the hybridized nucleic acid sequences which have undergone elongation from the nucleic acid sequences from unhybridized nucleic acid sequences and the nucleic acid primers which have not undergone elongation.

2. The method of claim 1, wherein the 3′

end of the at least one nucleic acid primer corresponds in position to a polymorphic base within the heterosequence site and the nucleic acid primer is capable of undergoing elongation only when the 3′

end of the at least one nucleic acid primer is complementary to and hybridized to the polymorphic base within the heterosequence site.

3. The method of claim 2, wherein one of the primer or one of the elongated primer is labeled with a detection molecule and (d) further comprises separating the hybridized nucleic acid sequences which have undergone elongation utilizing the detection molecule.

4. The method of claim 1, further comprising:

(d) amplifying the nucleic acid molecule comprising the heterosequence site prior to hybridization with the at least one nucleic acid primer; and

(e) identifying the heterosequence site.

5. The method of claim 1 wherein the heterosequence site comprises a single nucleotide polymorphism.

6. A kit for separating nucleic acid molecules which have specific alleles comprising instructions for carrying out the method of claim 1.

7. A method for separating a nucleic acid molecule which has a specific allele, comprising:

(a) hybridizing a nucleic acid comprising one or more heterosequence sites with at least one nucleic acid primer specific to the heterosequence site and a ligation primer to form a hybridized nucleic acid sequence, wherein the 3′

end of the at least one nucleic acid primer corresponds in position to a polymorphic base within the heterosequence site and the 5′

end of the ligation primer is adjacent to the 3′

end of the at least one nucleic acid primer;

(b) subjecting the at least one nucleic acid primer and the ligation primer to conditions which permit ligation of the at least one nucleic acid primer and the ligation primer; and

(c) separating the hybridized nucleic acid molecule in which the primers have undergone ligation.

8. The method of claim 7 wherein one of the at least one nucleic acid primer and the ligation primer is labeled with a detection molecule and (c) further comprises separating the hybridized nucleic acid sequences which have undergone elongation utilizing the detection molecule.

9. The method of claim 8 wherein the at least one nucleic acid primer comprises a plurality of primers having different sequences and each sequence is associated with a particular detection molecule such that no two sequences are associated with the same detection molecule.

10. A kit for separating a nucleic acid molecule which has a specific allele comprising instructions for carrying out the method of claim 7.

11. A method for separating a nucleic acid molecule which has a specific allele, comprising:

(a) hybridizing a nucleic acid comprising one or more heterosequence sites with at least one nucleic acid primer specific to the heterosequence site to form hybridized nucleic acid complexes; and

(b) separating the hybridized nucleic acid complexes which have complete complementary hybridization from the hybridized nucleic acid complexes which do not have complete complementary hybridization.

12. The method of claim 11 further comprising determining the sequence of the nucleic acid comprising the one or more heterosequence sites.

13. The method of claim 11 wherein step (b) comprises heating the hybridized nucleic acid complexes to a temperature at which the nucleic acid complexes which do not have complete complementary hybridization dissociate and which the nucleic acid complexes which have complete complementary hybridization do not dissociate.

14. The method of claim 11 wherein the at least one nucleic acid primer is labeled with a detection molecule.

15. A kit for separating a nucleic acid molecule which has a specific allele comprising instructions for carrying out the method of claim 11.

16. A method for separating a nucleic acid molecule which has a specific allele, comprising:

(a) hybridizing a nucleic acid comprising at least a 5′

heterosequence site and a 3′

heterosequence site with a hetero primer specific to the 3′

heterosequence site and a homo primer to form a hybridized nucleic acid sequence, wherein the 3′

end of the hetero primer corresponds in position to a polymorphic base within the 3′

heterosequence site, the homo primer is capable of hybridizing to the nucleic acid at a position located 5′

of the 5′

heterosequence site and the hetero primer is capable of undergoing elongation only when the 3′

end of the hetero primer is complementary to and hybridized to the polymorphic base within the 3′

heterosequence site;

(b) elongating the hybridized hetero primer such that the nucleic acid sequence between the hetero primer and the homo primer is produced and includes the 5′

heterosequence site; and

(c) determining the identity of the 5′

heterosequence site.

17. The method of claim 16 further comprising:

(d) utilizing the identity of the 5′

heterosequence site to produce another hetero primer and another homo primer, wherein the 3′

end of the another hetero primer corresponds in position to a polymorphic base within the 5′

heterosequence site, the 5′

heterosequence is located 3′

to another 5′

heterosequence, the homo primer is capable of hybridizing to the nucleic acid at a position located 5′

of the another 5′

heterosequence site and the hetero primer is capable of undergoing elongation only when the 3′

end of the hetero primer is complementary to and hybridized to the polymorphic base within the 5′

heterosequence site;

(e) hybridizing nucleic acid sequence with the another hetero primer and the another homo primer; and

(f) repeating steps (a) through (e) one or more times.

19. A kit for separating a nucleic acid molecule which has a specific allele comprising instructions for carrying out the method of claim 16.

18. The method of 16 further comprising determining the haplotype of the nucleic acid molecule.

20. A method for identifying an allele in a nucleic acid molecule, comprising:

(a) hybridizing a nucleic acid comprising a plurality of heterosequence sites with at least one primer to a produce a hybridized nucleic acid, wherein the at least one primer is attached to a bead;

(b) elongating the hybridized primer to produce an extended primer;

(c) dissociating the nucleic acid from the extended primer;

(d) hybridizing the extended primer with a second primer attached to the bead;

(e) elongating the second primer to produce a second extended primer; and

(f) identifying any heterosequence sites of the nucleic acid utilizing the extended primer, the second extended primer or both.

21. A method for identifying an allele in a nucleic acid molecule comprising instructions for carrying out the method of claim 20.