Method for amplification of DNA

• US 6,207,379 B1
• Filed: 09/11/1998
• Issued: 03/27/2001
• Est. Priority Date: 09/11/1998
• Status: Expired due to Term

• Current Assignee: One Lambda, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: One Lambda, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Blair, Lindley, Lee, Jar-How
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US09/151,465
• Time in Patent Office: 928 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• 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
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention provides methods for detecting target nucleic acid sequences with diagnostic primers including priming regions and probe regions which are complementary to target and reference regions respectively on a sample nucleic acid strand wherein the probe region is located 5′ to the priming region which is complementary to a reference nucleic acid sequence which is 3′ to the target nucleic acid sequence on the sample nucleic acid strand wherein when said reference nucleic acid sequence is contiguous with said target nucleic acid sequence on the sample nucleic acid strand then the priming region and probe region on the diagnostic probe are separated by a spacer region of nucleic acid.

Claims

1. A method for detecting the presence of a target nucleic acid sequence on a sample nucleic acid strand from an individual comprising the steps of:

treating the sample, together or sequentially with appropriate nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, a diagnostic primer and an amplification primer under hybridizing conditions;

wherein the nucleotide sequence of said diagnostic primer comprises (1) a priming region at its 3′

-end which is substantially complementary to the target nucleic acid sequence, and (2) a probe region located 5′

to said priming region which is substantially complementary to a reference nucleic acid sequence which is 3′

to the target nucleic acid sequence on the sample nucleic acid strand wherein when said reference nucleic acid sequence is contiguous with said target nucleic acid sequence on the sample nucleic acid strand then the priming region and probe region on the diagnostic primer are separated by a spacer region of nucleic acid, and further wherein when the priming region and the probe region on the diagnostic primer are contiguous then there exists an intervening sequence between the reference nucleic acid sequence and the target nucleic acid sequence on the sample nucleic acid strand;

whereby for selected hybridization and extension conditions the lengths of the priming region and the probe region are such that an extension product of the diagnostic primer is synthesized when both the priming region is substantially complementary to the target nucleic acid sequence and the probe region is substantially complementary to the reference nucleic acid sequence,

but wherein for said selected hybridization and extension conditions no extension product of the diagnostic primer is synthesized when either the priming region or the probe region are not substantially complementary to the target and reference nucleic acid sequences respectively;

any extension product of the diagnostic primer formed being capable of serving as a template for synthesis of an extension product of said amplification primer after separation from its complement;

amplifying any extension product to produce an amplification product; and

detecting the presence or absence of the target polynucleic acid sequence from the presence or absence of amplification product obtained as above.

2. The method of claim 1 wherein said priming region and said probe region on the diagnostic primer are separated by a spacer region of nucleic acid sequence which is not complementary to the sequence of the sample nucleic acid strand between the target and reference sequences.

3. The method of claim 2 wherein said spacer is from 1 to 30 bases long.

4. The method of claim 2 wherein said spacer is from 8 to 30 bases long.

5. The method of claim 1 wherein said target nucleic acid sequence is from 1 to 350 bases from said reference nucleic acid sequence on a sample nucleic acid sequence.

6. The method of claim 1 wherein the priming region and probe regions are exactly complementary to the target and reference nucleic acid sequences respectively.

7. The method of claim 1 wherein the target nucleic acid sequence is characteristic of a human leukocyte antigen (HLA).

8. The method of claim 1 wherein the nucleotide sequence of said amplification primer comprises (1) a priming region at its 3′

-end which is substantially complementary to a second target nucleic acid sequence on the complement of said sample nucleic acid, and (2) a probe region located 5′

to said priming region which is substantially complementary to a second reference nucleic acid sequence which is 3′

to the second target nucleic acid sequence on the complement of said sample nucleic acid strand wherein when said second reference nucleic acid sequence is contiguous with said second target nucleic acid sequence on the sample nucleic acid strand then the priming region and probe region on the amplification primer are separated by a spacer region of nucleic acid.