Parallel methods for genomic analysis

• US 6,480,791 B1
• Filed: 10/26/1999
• Issued: 11/12/2002
• Est. Priority Date: 10/28/1998
• Status: Expired due to Fees

• Current Assignee: Michael P. Strathmann
• Original Assignee: Michael P. Strathmann
• Inventors: Strathmann, Michael P.
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): Moran, Marjorie A.
• Application Number:
  US09/427,834
• Time in Patent Office: 1,113 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78, 436/89, 702/20
• US Class Current: 506/3
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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 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

The present invention provides parallel methods for determining nucleotide sequences and physical maps of polynucleotides associated with sample tags. This information can be used to determine the chromosomal locations of sample-tagged polynucleotides. In one embodiment, the polynucleotides are derived from genomic DNA coupled to insertion elements. As a result, the invention also provides parallel methods for locating the integration sites of insertion elements in the genome.

Claims

1. A parallel method for polynucleotide sequencing, comprising:

a) preparing a library comprising a collection of two or more sample-tagged polynucleotide clones;

b) carrying out a nucleic acid sequencing reaction on the library wherein a first sequencing primer binding site or no sequencing primer binding site is used to generate a plurality of tagged reaction products from the sample-tagged clones in the collection;

c) separating the reaction products according to size;

d) collecting fractions of the separated reaction products;

e) amplifying the products collected in step (d) to generate tagged amplicons;

f) hybridizing the tagged amplicons to an array comprising tag complements; and

g) determining a plurality of polynucleotide sequences of the sample-tagged clones by detecting the hybridizations to deconvolute a plurality of sequence ladders for the sample-tagged clones in the collection.

2. The method of claim 1, wherein the library further comprises a second collection of sample-tagged clones, and a second plurality of tagged sequencing reaction products is generated from each clone in the second collection using a second sequencing primer binding site.

3. The method of claim 1, wherein the collection comprises a pool of the sample-tagged clones.

4. The method of claim 3, wherein the library preparation step (a) comprises pooling a first pool comprising sample-tagged polynucleotides with a second pool comprising sample polynucleotides, carrying out a reaction to join polynucleotides from the first pool with polynucleotides from the second pool, and cloning the reaction products.

5. The method of claim 4, wherein the sample-tagged polynucleotides in the first pool are cloning vectors.

6. The method of claim 1, wherein after the hybridization step (f) the tagged amplicons are amplified in-situ.

7. The method of claim 1, wherein the sequencing reaction does not comprise the use of sequencing primers.

8. The method of claim 7, wherein the sequencing reaction comprises chemical cleavage of the sample-tagged clones.

9. The method of claim 1, wherein the sequencing reaction comprises a plurality of reactions that are carried out on separate aliquots of the library to generate one pool of the tagged reaction products from each aliquot.

10. The method of claim 9, wherein the pools of the tagged reaction products are independently separated in the separation step (c).

11. The method of claim 9, wherein at least two of the pools of the tagged reaction products are pooled before the separation step (c).

12. The method of claim 11, wherein the tagged reaction products comprise tags that identify the pools.

13. The method of claim 1, wherein the tagged reaction products are pooled before the separation step.

14. The method of claim 1, wherein the sample-tagged clones comprise genomic tags.

15. The method of claim 1, wherein the sample-tagged clones comprise adapter tags.

16. The method of claim 15, wherein each sample-tagged clone comprises a distinct sequence element, two common sequence elements and a sample sequence element such that the order of elements in the polynucleotide is the first common element, the distinct element, the second common element and the sample sequence element.

17. The method of claim 16, wherein the tagged amplicons comprise the distinct sequence elements.

18. The method of claim 17, wherein the tag complements hybridize to the distinct sequence elements.

19. The method of claim 18, wherein the tag complements and the distinct sequence elements form perfectly matched duplexes.

20. The method of claim 16, wherein the first common sequence element comprises a sequencing primer binding site.

21. The method of claim 16, wherein the second common sequence element comprises a primer binding site for use in the amplification step (e).

22. The method of claim 1, wherein the separation step (c) comprises gel electrophoresis.

23. The method of claim 1, wherein the amplification step (e) comprises a polymerase chain reaction.

24. The method of claim 1, wherein the amplification step comprises the use of an RNA polymerase.

25. The method of claim 1, wherein the collection comprises greater than 100 sample-tagged polynucleotide clones.

26. The method of claim 25, wherein the collection comprises greater than 1000 sample-tagged polynucleotide clones.

27. The method of claim 26, wherein the collection comprises greater than 100,000 sample-tagged polynucleotide clones.

28. A parallel method for polynucleotide sequencing, comprising:

a) preparing a library comprising sample-tagged polynucleotide clones;

b) carrying out a nucleic acid sequencing reaction on the library to generate a plurality of tagged reaction products;

c) separating the reaction products according to size;

d) collecting fractions of the separated reaction products;

e) hybridizing the products collected in step (d) to an array comprising tag complements;

f) amplifying the hybridized reaction products in situ; and

g) determining a plurality of polynucleotide sequences of the sample-tagged clones by detecting the amplified reaction products to deconvolute a plurality of sequence ladders for the sample-tagged clones.

29. The method of claim 28, wherein the amplification step (f) comprises the use of an RNA polymerase.

30. The method of claim 28, wherein the amplification step (f) comprises a rolling-circle type amplification.