Categorising nucleic acids

• US 6,297,017 B1
• Filed: 04/10/2000
• Issued: 10/02/2001
• Est. Priority Date: 07/11/1997
• Status: Expired due to Fees

• Current Assignee: Xzillion GmbH & Company
• Original Assignee: Brax Group Limited
• Inventors: Thompson, Andrew Hugin, Schmidt, Günter
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US09/462,636
• Time in Patent Office: 540 Days
• Field of Search: 435/6, 536/25.3
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention concerns a method for characterizing one or more nucleic acids. This method involves immobilizing double-stranded nucleic acids on a solid phase support and cleaving the immobilized nucleic acids with an endonuclease, such that each cleaved nucleic acid has a double-stranded portion. The cleaved nucleic acids are then denatured to form single-stranded cleaved nucleic acids. One or more oligonucleotide sequences are then hybridized to the resulting single-stranded cleaved nucleic acid. The oligonucleotide sequences used each comprise a pre-determined recognition sequence situated such that it recognizes a sequence which was part of the double-stranded portion of the nucleic acid and a label specific to the recognition sequence. The hybridized oligonucleotide sequences are then extended along the single-stranded portion of the immobilized nucleic acid to form an extended strand which is then denatured from the immobilized strand. The immobilized nucleic acid is then characterized by identifying the size of the extended strand and the identity of the recognition sequence.

Claims

1. A method for categorising one or more nucleic acids, which method comprises immobilising double-stranded nucleic acids on a solid phase support. Cleaving the immobilised nucleic acids with an endonuclease such that each cleaved nucleic acid has a double-stranded portion, denaturing the cleaved nucleic acids to form single-stranded cleaved nucleic acid, hybridising one or more oligonucleotide sequences to the resulting single-stranded cleaved nucleic acid, each oligonucleotide sequence comprising a pre-determined recognition sequence situated such that it recognises a sequence which was part of the portion of the nucleic acid which was double-stranded after cleavage with the endonuclease and a label specific to the recognition sequence, extending correctly hybridised oligonucleotide sequences along the single-stranded portion of the immobilised nucleic acid to form an extended strand, denaturing the extended strand from the immobilised strand and characterising the immobilised nucleic acid by identifying the size of the extended stand and the identity of the recognition sequence.

2. A method according to claim 1, wherein the nucleic acid is cDNA.

3. A method according to claim 1, wherein the endonuclease is selected such that each of the cleaved nucleic acids has a sticky end of known common length extending from the free terminal formed by the cleavage.

4. A method according to claim 3, wherein the endonuclease is selected such that each sticky end of each of the cleaved nucleic acids has the same known base sequence.

5. A method according to claim 4, wherein prior to contacting the cleaved nucleic acids with the oligonucleotide sequences, the nucleic acids are contacted with an adaptor to ligate the adaptor to the free terminal of each of the cleaved nucleic acids, wherein the adaptor comprises a double-stranded primer portion having a known base sequence, and a single-stranded portion complementary to the known sticky-end of the cleaved nucleic acids.

6. A method according to claim 5, wherein each oligonucleotide sequence comprises a first sequence, a second sequence attached to the first sequence and a third sequence attached to the second sequence, in which the first sequence is complementary to the sequence of the primer portion of the adaptor, the second sequence is complementary to the known sticky end of the cleaved nucleic acids, and the third sequence comprises the pre-determined recognition sequence.

7. A method according to claim 3, wherein the endonuclease is selected such that the sticky ends of the cleaved nucleic acids have a plurality of different base sequences.

8. A method according to claim 7, wherein prior to contacting the cleaved nucleic acids with the oligonucleotide sequences, the nucleic acids are contacted with an array of adaptors to ligate an adaptor to the free terminal of the cleaved nucleic acids, wherein each adaptor comprises a double-stranded primer portion having a known base sequence, and a single-stranded portion of the same length as the sticky ends of the cleaved nucleic acids, all of the possible base sequences of the single-stranded portion of the adaptor being represented in the array of adaptors.

9. A method according to claim 8, wherein each oligonucleotide sequence comprises a first sequence, a second sequence attached to the first sequence and a third sequence attached to the second sequence, in which the first sequence is complementary to the sequence of the primer portion of the adaptors, the second sequence is of the same length as the sticky ends of the cleaved nucleic acids, and the third sequence comprises the predetermined recognition sequence, and wherein in any one group of oligonucleotides having the same recognition sequence all of the possible base sequences of the second sequence are represented.

10. A method according to claim 1, wherein an array of oligonucleotide sequences is contacted with the cleaved nucleic acids, each of the oligonucleotides in the array having a recognition sequence of a common length, all possible recognition sequences of that length being represented in the array.

11. A method according to claim 1, wherein the recognition sequence comprises 4 bases.

12. A method according to claim 1, wherein the steps of hybridising the oligonucleotide sequences to the cleaved nucleic acids, extending correctly hybridised oligonucleotide sequences and denaturing the extended strand from the captured strand are repeated to linearly amplify the quantity of extended strands.

13. A method according to claim 1, wherein characterising the immobilised nucleic acid by identifying the extended strand and the identity of the recognition sequence is carried out by capillary electrophoresis mass spectrometry.

14. A method according to claim 1, wherein the oligonucleotide sequences have equalised melting temperatures.

15. A method according to claim 14, wherein the melting temperatures are equalised by incorporating one or more analogues of natural nucleotides into the oligonucleotide sequences, the analogues comprising base modifications, sugar modifications and/or backbone modifications.

16. A method according to claim 1, wherein the endonuclease is selected such that it cuts the nucleic acid at a site within the recognition site of the endonuclease.

17. A method according to claim 1, wherein the labels on the oligonucleotide sequences are mass labels.

18. A kit for categorising a nucleic acid, which kit comprises one or more adaptors and one or more sets of oligonucleotide sequences, wherein the adaptors comprise nucleic acid having a double-stranded primer portion of a known sequence and a single-stranded portion of a pre-determined length, either each single-stranded portion of each nucleic acid in the adaptors having the same pre-determined sequence or all possible sequences of the single-stranded portion being represented in the adaptors, and wherein each oligonucleotide sequence comprises a first sequence, a second sequence attached to the first sequence and a third sequence attached to the second sequence, in which the first sequence is complementary to the sequence of the primer portion of the adaptor, the second sequence is the same sequence as the single-stranded portion of the adaptors or all possible second sequences of the same length as the single-stranded portion of the adaptors are represented within the set of oligonucleotides, and the third sequence comprises a pre-determined recognition sequence.

19. A kit according to claim 18, wherein the recognition sequence comprises 4 bases.

20. A kit according to claim 18, additionally comprising an endonuclease.