Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-09-11
2001-03-27
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091100
Reexamination Certificate
active
06207379
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to methods for the detection of the presence or absence of nucleic acid sequences which are characteristic of pathogens and the like as well as of gene variations and mutations including those relating to the human leukocyte antigen (HLA) which is of interest in the field of human transplantation.
The HLA locus is highly polymorphic in nature. As disclosed in the Nomenclature for Factors of the HLA System 1996 (Tissue Antigens 1997: 49:297-321), there are 83 HLA-A alleles, 186 HLA-B alleles, 42 HLA-C alleles, 184 HLA-DRB1 alleles, 11 DRB3 alleles, 8 DRB4 alleles, 12 DRB5 alleles, 18 DQA1 alleles and 31 DQB1 alleles, with new alleles being discovered continuously. All HLA-A, -B, and -C alleles have similar sequences. The same holds for DRB1, DRB3, DRB4 and DRB5 sequences. Because of these similarities, very often when a primer pair is used in the practice of polymerase chain reaction sequence-specific priming (PCR-SSP), two or more alleles will be amplified. Therefore, for each allele to have a unique PCR-SSP pattern many pairs of primers must be used. Accordingly, in clinical use of PCR-SSP for HLA typing there exists a desire to use a limited number of PCR reactions to achieve as much resolution as possible whereby the number of alleles amplified by a pair of primers would be reduced (i.e., the specificity of the primers increased). Simultaneously, all of the primer pairs must have optimal annealing temperatures within a very restricted range.
PCR requires a pair of primers flanking the region on the DNA template for that region to be amplified. The ability of a primer to anneal to the desired sequence depends on the length of the primer and the annealing temperature set in the PCR thermocycling program. The longer the primer, the higher the annealing temperature it needs to achieve specific amplification of a DNA sequence. If the annealing temperature for a PCR is above the optimal range for the primer to anneal to its target, little or no amplification will occur. If the annealing temperature for a PCR is below the optimal range for the primer to anneal to its target, non-specific amplification will occur. PCR-SSP uses a balance between primer length and annealing temperature to achieve the specificity of the primer-directed sequence amplification. This technique can be used to characterize the sequence on the target DNA template—if amplification occurs, the template DNA contains the same sequences as the primers used; if no amplification occurs, the sequences on the template DNA are different from the primer sequences. Of interest to the present application are the disclosures of Olerup et al., Tissue Antigens 41: 119-134 (1993) and Bunce et al., Tissue Antigens 43: 7-17 (1994) which teach methods of PCR-SSP for HLA typing.
Newton et al., U.S. Pat. No. 5,595,890 disclose PCR diagnostic methods for typing including molecular typing of HLA using PCR-SSP. According to this method, an unknown allele is assigned based on the pattern of positive or negative reactions from multiple PCR. The methods disclosed by Newton are limited in their effectiveness for HLA typing, however, due to the high degree of polymorphism in HLA as described above. As a consequence two primers, each with specific sequences, frequently amplify many HLA alleles, thus increasing the required number of PCR in order to assign an unknown allele.
Accordingly, there exists a desire in the art for improved methods of PCR-SSP based molecular typing whereby the specificity of the typing can be increased so as to reduce the number of PCR reactions required for each typing.
SUMMARY OF THE INVENTION
The present invention relates to improved methods for detecting and/or amplifying target nucleic acid sequences and in particular for the detection and amplification of human leukocyte antigens (HLA) through methods such as polymerase chain reaction whereby the specificity of diagnostic primers is increased such that at least one primer is capable of recognizing two or more regions on the template and is preferably capable of doing so without increasing the annealing temperature of the primer to the template DNA. The increased specificity of the primer set reduces the number of alleles amplified by that primer pair and provides improved resolution over conventional PCR-SSP at lower cost.
Specifically, the invention provides 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 or absence of amplification product obtained as above. According to a preferred aspect of the present invention, the priming region and probe regions are exactly complementary to the target and reference nucleic acid sequences respectively.
While the priming region and the probe region can be contiguous on the diagnostic primers of the invention they need not be. Thus, according to one preferred embodiment of the invention the priming region and the probe region may be 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 sequence. Specifically, the spacer region is selected so as to provide a discontinuity in the complementarity of the nucleotide sequences of the priming and probe regions of the diagnostic primer such that hybridization and extension of the primer region will not occur unless the probe region is substantially complementary to the reference nucleic acid sequence.
The spacer region can be from 1 to 30 nucleotides or more in length with lengths of from 8 to 30 nucleotides being preferred although those of skill in the art will recognize that in general the longer the spacer region the longer the priming region must be in order to successfully hybridize to the target nucleic acid sequence under the hybridization conditions selected for practice of the invention. Nevertheless, the length of the spacer region, as well as the length of intervening sequence between the target nucleic acid sequence and the reference nucleic acid sequence on the sample nucleic acid strand which can be from 0 to 350 bases or more should not be so long that the priming region is able to hybridize independently to the target nucleic
Blair Lindley
Lee Jar-How
Jones W. Gary
Marshall O'Toole Gerstein Murray & Borun
One Lambda
Souaya Jehanne
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