Methods and kits for isolating primer extension products using modular oligonucleotides
First Claim
Patent Images
1. A method for isolating primer extension products, produced from a template vector, said products containing sequences corresponding or complementary to i) a primer binding region, ii) an insert and iii) vector-derived sequence(s), wherein said method comprises:
- a) binding a modular oligonucleotide comprising at least two modules to adjacent stretches on said primer extension products, wherein said modular oligonucleotide is complementary to and capable of binding to said vector-derived sequence of said primer extension products, wherein at least one of said modules is a capture module which is immobilized or has means for immobilization; and
b) isolating said primer extension products bound to said modular oligonucleotide.
3 Assignments
0 Petitions
Accused Products
Abstract
The invention provides a method of improving the binding of a series of consecutive nucleotide bases to a complementary target nucleic acid molecule in a sample, wherein said method comprises at least the step or steps of binding a complementary modular oligonucleotide of at least two parts (modules) including said nucleotide bases to adjacent stretches of said target nucleic acid molecule in said sample, especially methods of detection/isolation, particularly in the isolation of primer extension products and methods in which the modular oligonucleotide is a primer, modular oligonucleotides themselves and their use in methods of the invention.
-
Citations
16 Claims
-
1. A method for isolating primer extension products, produced from a template vector, said products containing sequences corresponding or complementary to i) a primer binding region, ii) an insert and iii) vector-derived sequence(s), wherein said method comprises:
-
a) binding a modular oligonucleotide comprising at least two modules to adjacent stretches on said primer extension products, wherein said modular oligonucleotide is complementary to and capable of binding to said vector-derived sequence of said primer extension products, wherein at least one of said modules is a capture module which is immobilized or has means for immobilization; and
b) isolating said primer extension products bound to said modular oligonucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16)
derived from any region of a particular vector and contains a portion which remains intact after longing of the insert, regardless of restriction site of a multiple cloning site of a particular vector into which the insert is cloned, and to which the modular oligonucleotide anneals. -
4. A method as claimed in claim 1 or 2 wherein said modular oligonucleotide consists of two or three modules.
-
5. A method as claimed in claims 1 or 2 wherein each module has ≧
- 9≧
18 nucleotides.
- 9≧
-
6. A method as claimed in claim 1 or 2 wherein said modular oligonucleotide comprises a total of at least 18 nucleotides.
-
7. A method as claimed in claim 1 or 2 wherein the modules when bound to said primer extension products are less than 2 bases apart.
-
8. A method as claimed in claim 7 wherein said modules are directly adjacent.
-
9. A method as claimed in claim 1 or 2 wherein immobilization is via the streptavidin:
- biotin binding system.
-
10. A method as claimed in claim 1 or 2 wherein said primer extension products are contacted directly with all modules of said modular oligonucleotide in a single hybridization step.
-
11. A method as claimed in claim 1 or 2 comprising the steps ofcontacting a sample containing the primer extension products with all modules of the modular oligonucleotide, wherein the capture module is immobilized on a solid support;
-
2) binding said modules by hybridization;
3) separating target primer extension products bound to said solid support; and
4) washing said solid support.
-
-
12. A method as claimed in claim 1 or 2 wherein said binding is performed at between 40°
- C. and 60°
C. for between 15 to 90 minutes.
- C. and 60°
-
13. A method as claimed in claim 1 or 2 wherein said modular oligonucleotide comprises a pair of oligonucleotides, wherein said pair of oligonucleotides is selected from the group consisting of:
-
5′
-NNAAGCTTACT-3′
(SEQ ID NO.
7) and 5′
-GGCCGTCGTTTTACAACG-3′
(SEQ ID NO.
8)5′
-NNNNAATTCGCCCTATAG-3′
(SEQ ID NO.
9) and 5′
-TGAGTCGTATTAC-3′
(SEQ ID NO.
6)5′
-GAATTCACGGAAATCATG-3′
(SEQ ID NO.
10) and 5′
-GTCATAGCTGTTTCCTGT-3′
(SEQ ID NO.
11)5′
-TCCAGCTTTTGTTCC-3′
(SEQ ID NO.
12) and 5′
-CTTTAGTGAGGGTTAATT-3′
(SEQ ID NO.
13)5′
-NNTTGAGTATTCTATAG-3′
(SEQ ID NO.
14) and 5′
-TGTCACCTAAATAGCT-3′
(SEQ ID NO.
15)5′
-TCTAGAGTCGACCTGCAG-3′
(SEQ ID NO.
16) and 5′
-GCATGCAAGCTT-3′
(SEQ ID NO.
17)5′
-ACCCAATTCGCCCTATAG-3′
(SEQ ID NO.
5) and 5′
-TGAGTCGTATTAC-3′
(SEQ ID NO.
6)and analogs or derivatives thereof with modified or derivatized nucleotide bases but which retain their complementarity.
-
-
16. A method of determining the nucleotide sequence of a nucleic acid insert in a vector wherein sequencing products are generated by performing primer extension reactions on said vector, the sequencing products are isolated by a method as claimed in claim 1 or 2, the products thus isolated are separated and the labels carried on said sequencing products are visualized to allow determination of the sequence of said insert or a portion thereof.
-
-
14. A modular oligonucleotide wherein said modular oligonucleotide comprises a pair of oligonucleotides, wherein said pair of oligonudleotides is selected from the group consisting of:
-
5′
-NNAAGCTTACT-3′
(SEQ ID NO.
7) and 5′
-GGCCGTCGTTTTACAACG-3′
(SEQ ID NO.
8)5′
-NNNNAATTCGCCCTATAG-3′
(SEQ ID NO.
9) and 5′
-TGAGTCGTATTAC-3′
(SEQ ID NO.
6)5′
-GAATTCACGGAAATCATG-3′
(SEQ ID NO.
10) and 5′
-GTCATAGCTGTTTCCTGT-3′
(SEQ ID NO.
11)5′
-TCCAGCTTTTGTTCC-3′
(SEQ ID NO.
12) and 5′
-CTTTAGTGAGGGTTAATT-3′
(SEQ ID NO.
13)5′
-NNTTGAGTATTCTATAG-3′
(SEQ ID NO.
14) and 5′
-TGTCACCTAAATAGCT-3′
(SEQ ID NO.
15)5′
-TCTAGAGTCGACCTGCAG-3′
(SEQ ID NO.
16) and 5′
-GCATGCAAGCTT-3′
(SEQ ID NO.
17)5′
-ACCCAATTCGCCCTATAG-3′
(SEQ ID NO.
5) and 5′
-TGAGTCGTATTAC-3′
(SEQ ID NO.
6)and analogs or derivatives thereof with modified or derivatized nucleotide bases but which retain their complementarity. - View Dependent Claims (15)
a modular oligonucleotide as defined in claim 14.
-
Specification
-
- Resources
Litigation Campaign Assessment
Thank you for your request. You will receive a custom alert email when the Litigation Campaign Assessment is available.
×
-
Current AssigneeLife Technologies AS (Perspecta, Inc.)
-
Original AssigneeDynal AS (Thermo Fisher Scientific Incorporated)
-
InventorsUhlen, Mathias, Lundeberg, Joakim
-
Primary Examiner(s)Siew, Jeffrey
-
Application NumberUS09/153,242Time in Patent Office1,526 DaysField of Search435/6, 435/91.1, 435/91.2, 536/23.1, 536/25.32, 536/24.3, 536/24.31, 536/24.32, 536/24.33US Class Current435/6.12CPC Class CodesC12Q 1/6832 Enhancement of hybridisatio...C12Q 1/707 non-A, non-B Hepatitis, exc...C12Q 2525/185 incorporating bases where t...C12Q 2537/162 Helper probe
