Method for amplification of DNA

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.

What is claimed is:  
     
         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 probe are separated by a spacer region of nucleic acid,    whereby for selected hybridization and extension conditions an extension product of the diagnostic primer is synthesized when the priming region is substantially complementary to the target nucleic acid sequence and when 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; and    detecting the presence or absence of the target polynucleic acid sequence from the presence of 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.  
     
     
         9 . A diagnostic primer for detecting the presence of a target nucleic acid sequence on a sample nucleic acid strand comprising (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 probe are separated by a spacer region of nucleic acid.  
     
     
         10 . The diagnostic primer of    claim 9    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  
     
     
         11 . The diagnostic primer of    claim 10    wherein said spacer is from 1 to 30 bases long.  
     
     
         12 . The diagnostic primer of    claim 10    wherein said spacer is from 8 to 30 bases long.  
     
     
         13 . The diagnostic primer of    claim 9    wherein the target nucleic acid sequence is characteristic of a human leukocyte antigen (HLA).  
     
     
         14 . The diagnostic primer of    claim 9    wherein the priming region and probe regions are exactly complementary to the target and reference nucleic acid sequences respectively.  
     
     
         15 . A kit comprising a diagnostic primer for detecting the presence of a target nucleic acid sequence on a sample nucleic acid strand comprising (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; 
 each of four different nucleoside triphosphates; and  
 an agent for polymerization of the nucleoside triphosphates.  
 
     
     
         16 . The kit of    claim 15    wherein said priming region and said probe region on the diagnostic probe 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.  
     
     
         17 . The kit of    claim 15    further comprising an amplification primer corresponding to said diagnostic primer, the nucleotide sequence of the amplification primer being such that any extension product of the corresponding diagnostic primer may, after separation from its complement serve as a template for synthesis of an extension product of the amplification primer.  
     
     
         18 . The kit of    claim 15    wherein the priming region and probe regions of said diagnostic primer are exactly complementary to the target and reference nucleic acid sequences respectively.  
     
     
         19 . The kit of    claim 15    wherein the nucleotide sequence of said amplification primer comprises (1) a priming region at its 3′-end which is substantially complementary to a 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 diagnostic primer are separated by a spacer region of nucleic acid.  
     
     
         20 . The kit of    claim 16    wherein said spacer is from 1 to 30 bases long.  
     
     
         21 . The kit of    claim 16    wherein said spacer is from 8 to 30 bases long.