Method of multiplex ligase chain reaction

• US 5,869,252 A
• Filed: 12/18/1996
• Issued: 02/09/1999
• Est. Priority Date: 03/31/1992
• Status: Expired due to Fees

• Current Assignee: Abbott Laboratories
• Original Assignee: Abbott Laboratories
• Inventors: Gordon, Julian, Rhoads, James, Hoijer, Joanell, Bouma, Stanley R., Jou, Cynthia
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Whisenant, Ethan
• Application Number:
  US08/769,176
• Time in Patent Office: 783 Days
• Field of Search: 435/91.2, 435/6, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• 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 invention relates to multiplex ligase chain reaction (LCR). Two or more purative target sequences are selected. For each one, a set of four probes is used simultaneously to amplify the putative sequence if it is present in the sample. Preferably, all the amplicons are labeled with a common label/hapten and, for each different target, with a unique label/hapten. The invention also relates to an immunochromatographic strip device and method employing a diagonal array of capture spots.

Claims

1. A method of performing multiplex ligase chain reaction comprising the steps of:

(a providing a single reaction solution containing nucleic acid of a sample as single-stranded nucleic acid, said sample putatively having one or more of a plurality of target nucleic acid sequences;

b) for each putative target sequence, providing in the reaction solution at least four nucleic acid probes (a probe set), wherein;

i) the first and second of said probes are primary probes, and the third and fourth of said probes are secondary nucleic acid probes;

ii) the first probe is a single strand that hybridizes to a first segment of a primary strand of the target nucleic acid;

iii) the second probe is a single strand that hybridizes to a second segment of said primary strand of the target nucleic acid;

iv) the 5'"'"' end of the first segment of said primary strand of the target is positioned relative to the 3'"'"' end of the second segment of said primary strand of the target to enable joining of the first probe to the second probe when said probes are hybridized to said primary strand of said target nucleic acid, thus forming a reorganized primary molecule having a first portion and a second portion;

v) the third probe hybridizes to a first portion of the reorganized primary molecule; and

vi) the fourth probe hybridizes to a second portion of the reorganized primary molecule, the first portion of the reorganized primary molecule being positioned relative to the second portion of the reorganized primary molecule to enable joining of the third probe to the fourth probe when said third and fourth probes are hybridized to said reorganized primary molecule, thus forming a reorganized secondary molecule; and

wherein for each putative target sequence, said probe set is balanced such that each probe set is provided at a concentration that enables said joining in the presence of each of the other probe sets and wherein each probe set produces a detectable product after the same number of amplification cycles; and

c. repeating the following cycle;

i) hybridizing said probes with nucleic acid in said sample;

ii) performing said joining to form said reorganized molecules; and

iii) denaturing nucleic acid in said sample;

whereby with successive cycles the quantity of reorganized primary and secondary molecules is increased for each putative target sequence present in the reaction solution.

2. The method according to claim 1, wherein said joining is performed by a ligase enzyme, or a ligase enzyme and a polymerase enzyme.

3. The method according to claim 1, wherein the cycle of step c is repeated from 20 to about 60 times.

4. The method according to claim 1, wherein at least one probe from each probe set carries a detectable label, each of said detectable labels being differentiable from the others.

5. The method according to claim 4, wherein said detectable labels comprise haptens which are differentiable based on the specificity of different specific binding members.

6. The method according to claim 1,wherein each of the probe sets are labeled with two distinct labels such that the reorganized molecules, when hybridized together, are labeled with two labels, at least one of which is a unique label that is different for each of the probe sets.

7. The method according to claim 6, wherein the other label is a common label, the same for each probe set.

8. The method according to claim 7, wherein the common label is used for detection and is selected from the group consisting of radioisotopes, fluorophores, chemilumiphores and haptens.

9. The method according to claim 6, wherein the unique labels comprise unique specific binding members such that each unique specific binding member is differentiable from every other specific binding member.

10. The method according to claim 9, wherein the unique labels comprise specific haptens.

11. The method according to claim 9, wherein the unique labels comprise specific hybridization probes.

12. The method according to claim 9, wherein the unique specific binding member is used to separate the reorganized molecules of one probe set from the reorganized molecules of at least one other probe set.

13. The method according to claim 12, wherein the separation is achieved by immobilizing a specific binding partner for each specific binding member at a different location on a solid phase.

14. The method according to claim 12, wherein the separation is achieved by immobilizing a specific binding partner for each specific binding member on a solid phase, said solid phase being characterized by a property which permits differentiation of one specific binding member from another specific binding member.

15. A method of detecting the presence, absence or quantity of each of a plurality of target nucleic acid sequences by a multiplex ligase chain reaction comprising the steps of:

a) providing a reaction solution containing nucleic acid of a sample as single-stranded nucleic acid, said sample putatively having one or more of a plurality of target nucleic acid sequences;

b) for each putative target sequence, providing in the reaction solution a set of at least two and optionally four nucleic acid probes (a probe set), wherein;

i) the first and second of said probes are primary probes, and the optional third and fourth of said probes are secondary nucleic acid probes;

ii) the first probe is a single strand that hybridizes to a first segment of a primary strand of the target nucleic acid;

iii) the second probe is a single strand that hybridizes to a second segment of said primary strand of the target nucleic acid;

iv) the 5'"'"' end of the first segment of said primary strand of the target is positioned relative to the 3'"'"' end of the second segment of said primary strand of the target to enable joining of the first probe to the second probe when said probes are hybridized to said primary strand of said target nucleic acid, thus forming a reorganized primary molecule having a first portion and a second portion;

v) the third probe hybridizes to a first portion of the reorganized primary molecule; and

vi) the fourth probe hybridizes to a second portion of the reorganized primary molecule, the first portion of the reorganized primary molecule being positioned relative to the second portion of the reorganized primary molecule to enable joining the third probe to the fourth probe when said third and fourth probes are hybridized to said reorganized primary molecule, thus forming a reorganized secondary molecule;

wherein at least one probe of each probe set contains a detectable label, the detectable label associated with each probe set being differentiable from the detectable labels associated with the other probe sets, whereby the presence, absence or quantity of each putative target sequence can be determined; and

wherein for each putative target sequence said probe set is balanced such that each probe set is provided at a concentration that enables said joining in the presence of each of the other probe sets and wherein each probe set produces a detectable product after the same number of amplification cycles; and

c. performing the following cycle;

i) hybridizing said probes with nucleic acid in said sample;

ii) performing said joining to form said reorganized molecules; and

iii) denaturing nucleic acid in said sample;

whereby with each cycle the quantity of reorganized primary and, optionally, secondary molecules is increased for each putative target sequence present in the reaction solution; and

d. measuring the detectable label associated with each of said probe sets as a measure of the presence or quantity of target nucleic acid in the reaction solution.

16. The method according to claim 15, wherein said joining is performed by a ligase enzyme, or a ligase enzyme and a polymerase enzyme.

17. The method according to claim 15, wherein said third and fourth probes are provided and the cycle of step c is repeated from 10 to about 100 times.

18. The method according to claim 15, wherein the cycle of step c is repeated from 20 to about 60 times.

19. The method according to claim 15,wherein the probes are labeled with two labels such that the reorganized molecules are labeled with two labels, at least one of which is a unique label that is different for each of the probe sets.

20. The method according to claim 17, wherein the probes are labeled with two labels such that the reorganized molecules are labeled with two labels, at least one of which is a unique label that is different for each of the probe sets and the other of which is a common label.

21. The method according to claim 20, wherein the common label is used for detection and is selected from the group consisting of radioisotopes, fluorophores, chemilumiphores and haptens.

22. The method according to claim 20, wherein the unique labels comprise haptens and wherein each unique hapten differs from every other unique hapten.

23. The method according to claim 20, wherein the unique labels comprise specific hybridization probes.

24. The method according to claim 22, wherein the unique hapten is used to separate the different reorganized molecules.

25. The method according to claim 24, wherein the separation is achieved by immobilizing a specific binding partner for each unique hapten at a different location on a solid phase.

26. The method according to claim 24, wherein the separation is achieved by immobilizing a specific binding partner for each unique hapten on a solid phase, said solid phase being characterized by a property which permits differentiation of one hapten from another hapten.