Characterizing DNA

• US 6,613,511 B1
• Filed: 12/29/1999
• Issued: 09/02/2003
• Est. Priority Date: 04/21/1997
• Status: Expired due to Fees

• Current Assignee: Xzillion GmbH & Company
• Original Assignee: Xzillion GmbH & Company
• Inventors: Schmidt, Gunter, Thompson, Andrew Hugin
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): SWITZER, JULIET CAROLINE
• Application Number:
  US09/402,542
• Time in Patent Office: 1,343 Days
• Field of Search: 435/6, 435/91.2, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates

Abstract

The present invention is drawn to a method for characterizing cDNA, wherein said method produces a sequence signature having the following structure:

Adaptor Sequence-Restriction Site-Known Length-N_w-Second Known Length-N_x-Poly-A tail (Known Length).

Claims

1. A method for characterising cDNA, wherein said method produces a sequence signature having the following structure:

Adaptor Sequence-Restriction Site-Known Length-N_w-Second Known Length-N_x-Poly-A tail (Known Length),

wherein N is base information and the subscripts w and x indicate the number of bases that are determined, which method comprises;

(a) exposing a sample comprising a population of one or more cDNAs or fragments thereof to a cleavage agent which recognizes a predetermined sequence and cuts at a reference site at a known displacement from the predetermined sequence proximal to the poly-A terminus an end of each cDNA or fragment thereof so as to generate a population of terminal fragments comprising the poly-A terminus;

(b) ligating to each reference site an adaptor oligonucleotide which comprises a recognition site for a sampling cleavage agent;

(c) exposing the population of terminal fragments comprising the poly-A terminus to a sampling cleavage agent which binds to the recognition site and cuts at a sampling site of known displacement from the recognition site so as to generate in each terminal fragment comprising the poly-A terminus a sticky end sequence of a predetermined length of up to 6 bases, and of unknown sequence;

(d) directly after completing step (c), separating the population of terminal fragments comprising the poly-A terminus into sub-populations according to sequence length, thereby determining the second known length of said sequence signature; and

(e) determining each sticky end sequence.

2. A method according to claim 1, wherein each sticky end sequence has 3-5 bases.

3. The method according to claim 1, wherein the cleavage agent of step (a) is a type II restriction endonuclease.

4. A method according to claim 3, wherein the type II restriction endonuclease is selected such that the displacement from the reference site to the predetermined sequence is zero, and the adaptor oligonucleotide is selected such that upon ligation to the reference site the predetermined sequence is not regenerated.

5. A method according to claim 1, wherein each terminal fragment has a poly A tail.

6. A method according to claim 1, wherein the adaptors are ligated using enzymatic ligation.

7. A method according to claim 6, in which T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq ligase, Pfu ligase, or Tth ligase is used.

8. A method according to claim 1, wherein the sampling cleavage agent comprises a type IIs restriction endonuclease.

9. A method according to claim 8, wherein the type IIs restriction endonuclease is the fok1, BstFsI, SfaNI, HgaI, or BbvI enzyme.

10. A method according to claim 1, wherein the population of terminal fragments is separated by capillary electrophoresis, HPLC or gel electrophoresis.

11. A method according to claim 1, wherein each unknown sticky end sequence is determined by a method comprising:

i) probing with an array of labeled hybridization probes, the array containing all possible base sequences of the predetermined length;

ii) ligating those probes which hybridized to the sticky end sequences; and

iii) determining which probes are ligated by identification of the labels.

12. A method according to claim 11, wherein the array comprises a plurality of sub-arrays, and steps i), ii) and iii) are repeated for each sub-array.

13. A method according to claim 12, wherein the predetermined length of the sticky end sequences is 4 bases and the array comprises two sub-arrays, each sub-array containing 128 different probes, the probes in one sub array being complementary to the probes in the other sub-array.

14. A method according to any of claim 11, wherein the labels are cleavably attached to the hybridisation probes.

15. A method according to any of claim 11, wherein each label is specific to the base sequence of the probe to which it is attached.

16. A method according to any of claim 11, wherein the labels are mass labels.

17. A method according to any of claim 11, wherein each hybridisation probe is attached to a known double-stranded primer sequence specific to the base sequence of the probe, such that after hybridisation of the terminal fragments to the probes, each terminal fragment is attached to a primer sequence specific to that fragment, and wherein the terminal fragments are selectively amplified by performing PCR in the presence of one or more of said primer sequences.

18. A method according to any of claim 11, wherein prior to hybridisation, a terminal primer is introduced at the poly A tail of the cDNA, and wherein after hybridisation the terminal fragments are amplified by performing PCR in the presence of said terminal primer.

19. A method of claim 11, wherein said method further comprises the step of quantification of said labels.

20. A method according to claim 1, wherein the cDNA is synthesised using 5-methyl cytosine.

21. A method according to claim 1, wherein the cDNA is immobilised on a solid support.

22. A method of identifying cDNA in a sample, comprising characterising the cDNA by a method as defined in claim 1 and identifying said cDNA.

23. A method of assaying for one or more specific cDNAs in a sample, comprising characterizing the cDNA by a method as defined in claim 1 and assaying said cDNA for one or more specific cDNAs.

24. The method of claim 1, wherein said sequence signature further comprises 3′

to the poly-A tail a primer sequence.

25. A method for characterizing cDNA, which method comprises:

(a) exposing a sample comprising a population of one or more cDNAs or 3′

terminal cDNA fragments of greater than 18 base pairs in length, including the poly-A terminus, to a cleavage agent which recognizes a predetermined sequence and cuts at a reference site at a known displacement from the predetermined sequence proximal to an end of each cDNA or 3′

terminal cDNA fragments of greater than 18 base pairs in length so as to generate a population of terminal fragments;

(b) ligating to each reference site an adaptor oligonucleotide which comprises a recognition site for a sampling cleavage agent;

(c) exposing the population of terminal fragments to a sampling cleavage agent which binds to the recognition site and cuts at a sampling site of known displacement from the recognition site so as to generate in each terminal fragment a sticky end sequence of a predetermined length of up to 6 bases, and of unknown sequence;

(d) separating the population of terminal fragments into sub-populations according to sequence length; and

(e) determining each sticky end sequence.