# US 11,306,352 B2 · Nucleic acid amplification method and primers for use therein > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,306,352 B2 - Document ID: US11306352B2 - Status: Granted - Kind: B2 - Application number: 16/478,786 - Filed: 2019-06-07 - Published: 2021-03-25 - Granted: 2022-04-19 - Earliest claimed: 2018-06-11 - Assignees: Monoquant Pty Ltd. - Applicants: Monoquant Pty Ltd. - Inventors: Alexander Alan Morley - Web page: https://patentgrep.com/patent/US11306352B2 ## CPC - C12Q 1/6858: Allele-specific amplification - C12Q 1/6853: using modified primers or templates - C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - C12Q 2527/107: Temperature of melting, i.e. Tm - C12Q 2600/156: Polymorphic or mutational markers - C12Q 2600/158: Expression markers - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention relates generally to an improved method of amplifying a nucleic acid region of interest and to primers for use therein. More particularly, the present invention is directed to an improved method of amplifying a nucleic acid region which has resulted from the recombination of two or more immunoglobulin or T cell receptor gene segments and primers for use therein. The method of the present invention is based on the determination that performing the amplification step at an annealing temperature determined relative to the critical annealing temperature unique to a given reaction and/or using optimised primers enables higher levels of sensitivity than have previously been achievable in the context of prior art methods of amplifying rearranged immunological or T cell receptor genes. The method of the present invention is particularly useful where the subject recombination target comprises only one N region. The provision of a highly sensitive yet simple means of detecting specific immunological and T cell receptor nucleic acid recombination events is useful in a range of applications including, but not limited to, the diagnosis and/or monitoring of clonal lymphoid cell populations or disease conditions which are characterised by specific V/D/J recombination events (such as detecting minimal residual disease in leukaemias) or the analysis or identification of immunological or T cell receptor gene regions of interest. ## Claims ### Claim 1 A method of amplifying an Ig or TCR nucleic acid region which is characterised by the rearrangement of two or more V, D or J gene segments said method comprising contacting a nucleic acid sample of interest with forward and reverse primers directed to said rearranged Ig or TCR nucleic acid region and amplifying said nucleic acid sample using an annealing temperature within the range between Tc and (Tc-3° C.) of the subject amplification reaction, and optionally: (i) at least one primer which comprises at least one A and/or T nucleotide at the 3′ terminal nucleotide position; and/or (ii) conducting a melting curve analysis. ### Claim 2 A method of detecting and/or monitoring a clonal population of cells in a mammal, which clonal cells are characterised by an Ig or TCR nucleic acid region which is characterised by the rearrangement of two or more V, D or J gene segments, said method comprising: (i) contacting DNA from a biological sample derived from a mammal with forward and reverse primers directed to said rearranged Ig or TCR nucleic acid region under conditions sufficient to facilitate interaction of said primers with said nucleic acid region; (ii) amplifying said nucleic acid region using an annealing temperature within the range between Tc and (Tc-3° C.) of the subject amplification reaction, and optionally (a) at least one primer which comprises at least one A and/or T nucleotide at the 3′ terminal nucleotide position of said primer; and/or (b) a means to conduct a melting curve analysis; and (iii) detecting the product amplified in accordance with step (ii). ### Claim 3 (depends on 1) The method according to claim 1 wherein said method comprises using both an annealing temperature within the range between Tc and (Tc-3° C.) of the subject amplification reaction and one or more primers which comprise an A or T nucleotide at the 3′ terminal nucleotide position of said primer. ### Claim 4 (depends on 1) The method according to claim 1 wherein said nucleic acid region is DNA. ### Claim 5 (depends on 1) The method according to claim 1 wherein there is an A and/or T nucleotide at each of the two, three, four, five or six most 3′ terminal nucleotide positions of said primer. ### Claim 6 (depends on 1) The method according to claim 1 wherein either or both of the forward or reverse primers are designed to include said A and/or T nucleotides. ### Claim 7 (depends on 1) The method according to claim 1 wherein only said reverse primer is designed to include said A and/or T nucleotides. ### Claim 8 (depends on 1) The method according to claim 1 wherein said VDJ rearrangement is a partial rearrangement and there is an N region situated at the junction between one or more of the rearranged V and D gene segments, the rearranged D and J gene segments or the rearranged V and J gene segments. ### Claim 9 (depends on 1) The method according to claim 1 wherein said VDJ rearrangement is a complete rearrangement and there is an N region situated at the junction between one or more of the rearranged V and D gene segments, the rearranged D and J gene segments or the rearranged V and J gene segments. ### Claim 10 (depends on 1) The method according to claim 1 wherein said VDJ rearrangement is a partial rearrangement and there is an N region situated within one or more of the V gene segment, D gene segment or J gene segment. ### Claim 11 (depends on 1) The method according to claim 1 wherein said VDJ rearrangement is a complete rearrangement and there is an N region situated within one or more of the V gene segment, D gene segment or J gene segment. ### Claim 12 (depends on 1) The method according to claim 1 wherein at least one of said primers comprises a hybridisation subregion directed to one N gene segment of the rearranged V, D and J gene segments. ### Claim 13 (depends on 1) The method according to claim 1 wherein at least one of said primers comprises hybridisation subregions directed to at least two N gene segments of the rearranged V, D and J gene segments. ### Claim 14 (depends on 1) The method according to claim 1 wherein the primer directed to the J gene segment comprises at least one A and/or T nucleotide at its 3′ end. ### Claim 15 (depends on 1) The method according to claim 1 wherein a primer subregion is modified to substitute one or more of the nucleotides of said subregion with a nucleotide from an N mixture. ### Claim 16 (depends on 15) The method according to claim 15 wherein every fourth nucleotide of said subregion is substituted. ### Claim 17 (depends on 16) The method according to claim 16 wherein a sequence of 3-7 adjacent nucleotides of said subregion are substituted. ### Claim 18 (depends on 1) The method according to claim 1 wherein a melting curve analysis is performed. ### Claim 19 (depends on 18) The method according to claim 18 wherein said melting curve analysis is performed using a dye. ### Claim 20 (depends on 19) The method according to claim 19 wherein said dye is a fluorophore. ### Claim 21 (depends on 1) The method according to claim 1 wherein said method includes a Ct determination step. ### Claim 22 (depends on 21) The method according to claim 21 wherein said Ct determination is performed using a probe directed to said rearranged Ig or TCR region and which probe is coupled to a detection means. ### Claim 23 (depends on 22) The method according to claim 22 wherein said detection means is a fluorescent molecule. ### Claim 24 (depends on 1) The method according to claim 1 wherein said at least one primer is an ASO primer. ### Claim 25 (depends on 1) The method according to claim 1 wherein said reverse primer is directed to a J segment. ### Claim 26 (depends on 1) The method according to claim 1 wherein said amplification is polymerase chain reaction. ### Claim 27 (depends on 2) The method according to claim 2 wherein said clonal cells are neoplastic cells. ### Claim 28 (depends on 27) The method according to claim 27 wherein said neoplastic cells are lymphoid neoplastic cells. ### Claim 29 (depends on 28) The method according to claim 28 wherein said lymphoid neoplastic cells are malignant. ### Claim 30 (depends on 29) The method according to claim 29 wherein said lymphoid malignant cells are indicative of lymphoid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia or myeloma. ### Claim 31 (depends on 2) The method according to claim 2 wherein said method is used to detect minimum residual disease. ### Claim 32 (depends on 31) The method according to claim 31 wherein said minimum residual disease is detected in the context of lymphoid leukaemia.