Quantative method for early detection of mutant alleles and diagnostic kits for carrying out the method

• US 5,512,441 A
• Filed: 11/15/1994
• Issued: 04/30/1996
• Est. Priority Date: 11/15/1994
• Status: Expired due to Fees

• Current Assignee: American Health Foundation
• Original Assignee: American Health Foundation
• Inventors: Ronai, Zeey A.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/339,786
• Time in Patent Office: 532 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/18, 435/91.53, 536/24.33, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

There is disclosed a quantitative sensitive method to enable the detection of point mutations at a known site to a diagnostic kit which uses a multi step (for example, four steps) or a single step reaction. The method uses selective polymerase chain reaction (PCR) amplification of mutant test gene sequences involving first stage amplification of both mutant and wild-type sequences, first stage restriction enzyme digestion of only wild-type sequences, second stage amplification of undigested amplified fragments enriched in mutant sequences and second stage digestion of previously undigested wild-type sequences. Long and short tail primers are used in the first and second stages of amplification respectively to enable selective amplification (in the second stage) of only previously amplified material and none of the original test genomic DNA. The short tail primers are labelled with biotin and fluorescence at their respective 5'"'"' and 3'"'"' ends to enable easy detection and quantitation of mutations in the test gene via automated fluorescence readers. The use of multi steps as well as a single step reaction is disclosed. The process is exemplified with respect to its use in detecting mutations in the human K-ras gene, yet it is applicable for any given mutation in a defined site.

Claims

1. A process for detection in a nucleic acid test sample taken from the genome of an organism of a mutant nucleotide sequence in a specific region of the genome, wherein said region can contain said mutant nucleotide sequence or a wild-type nucleotide sequence, wherein the test sample is suspected of containing a first genomic strand of nucleic acid having said region with the mutant nucleotide sequence together with a second genomic strand of nucleic acid having said region with the wild-type nucleotide sequence, wherein the first genomic strand, if present in the test sample, is present or is caused to be present in the form of a first genomic duplex consisting of the first genomic strand and a first complementary strand, and the second genomic strand is present or is caused to be present in the test sample in the form of a second genomic duplex consisting of the second genomic strand and a second complementary strand, the process comprising:

(i) a first amplification step comprising amplifying material in the first and second genomic duplexes present in the test sample in a first polymerase chain reaction in which upstream and downstream long tail primers, DNA polymerase, four ,different nucleotide triphosphates and a buffer are used in a repetitive series of reaction steps involving template denaturation, primer annealing and extension of annealed primers to form first and second synthesized nucleic acid duplexes, said first synthesized nucleic acid duplexes having said region with the mutant nucleotide sequence, and said second synthesized nucleic acid duplexes having said region with the wild-type nucleotide sequence, said upstream and downstream long tail primers being selected such that synthesized strands formed in the first polymerase chain reaction using the upstream and downstream long tail primers can anneal with Upstream and downstream short tail primers which do not anneal with any nucleic acid strands in the first or second genomic duplexes, said long tail upstream primers also being selected such that the second synthesized duplexes have a restriction site which is not present in said first synthesized duplexes due to the presence in said first synthesized duplexes of said region with the mutant nucleotide sequence, said restriction site being cleavable with a first restriction enzyme,

ii) a first digestion step comprising treating at least a portion of the test sample containing the first and second synthesized duplexes with said first restriction enzyme whereby selectively to cleave said second synthesized duplexes while leaving said first synthesized duplexes uncleaved,

iii) a second amplification step comprising amplifying material in the uncleaved first synthesized duplexes in a second polymerase chain reaction in which the upstream and downstream short tail primers are used selectively further to amplify nucleic acid strands not cleaved in step (ii) whereby to form further synthesized duplexes, said upstream and downstream short tail primers being selected such that they anneal with nucleic acid strands synthesized in said first amplification step but do not anneal with any strands of the first or second genomic duplexes whereby the upstream and downstream short tail primers can be used in the second amplification step selectively to amplify material in duplexes synthesized in the first amplification step but cannot amplify material in the first or second genomic duplexes, each of said upstream short tail primers being labelled with a first substance that binds tightly with a second substance such that upstream ends of the further synthesized duplexes bind to a supporting surface coated with the second substance, each of said downstream short tail primers being labelled with a detectable marker;

iv) a binding step comprising causing contact between the test sample and the supporting surface coated with said second substance whereby further synthesized duplexes labelled with the first substance bind thereto;

v) a second digestion step wherein the test sample is again treated with the first restriction enzyme selectively to cleave synthesized duplexes containing nucleic acid strands having said region with the wild type sequence; and

vi) a detection step comprising washing to remove unbound duplexes and assaying for the detectable marker to detect the presence of the mutant nucleotide sequence on uncleaved duplexes bound to the supporting surface.

2. A process as claimed in claim 1, wherein the first substance comprises biotin and the second substance comprises avidin or strepavidin, wherein each of said downstream short tail primers is labelled with a fluorescent label such that downstream ends of the further synthesized duplexes have the fluorescent label, and wherein the detection step comprises assaying for the presence of the fluorescent label.

3. A process as claimed in claim 2, wherein the test sample is placed on a first microtiter for the first amplification step following which the test sample, or a portion thereof, is transferred to a second microtiter plate which is coated with the avidin or strepavidin, the test sample being left in said second microtiter plate for at least the digestion step, and the second amplification step.

4. A process as claimed in claim 1, wherein steps i-iv are performed in a polymerase chain reaction microcycler, wherein the detection step comprises denaturing the uncleaved duplexes bound to the supporting surface and performing hybridization with a labelled probe to detect said mutant nucleotide sequence.

5. A process as claimed in claim 1, wherein the first and second amplification steps and the first and second digestion steps are performed in a single reaction mix, wherein the region comprising the mutant nucleic acid sequence comprises codon 12 of a human K-ras gene, and wherein the restriction enzyme is BstN1, the polymerase is Taq polymerase and the buffer is a diluted solution comprising 60-80% of BstN1 buffer.

6. A process as claimed in claim 1 wherein the region comprising the mutant nucleic acid sequence, is a codon of K-ras, H-ras or N-ras gene selected from the group consisting of codon 12, codon 13 and codon 61.

7. A method as claimed in claim 1 wherein the upstream and downstream short tail primers consist of DNA sequences from polyoma virus.

8. A process as claimed in claim 7 wherein the upstream and downstream short tail primers are between about 15 and 25 bases in length, whereby the further synthesized duplexes are longer than the genomic duplexes by between about 30 and 50 bases pairs.

9. In a process for detecting mutations in nucleic acid duplexes, wherein synthesized duplexes synthesized from genomic duplexes in a first amplification stage are present in a test sample together with the genomic duplexes, wherein the synthesized duplexes are synthesized in the first amplification stage from the genomic duplexes in a first polymerase chain reaction in which upstream and downstream primers are used to provide the synthesized duplexes with nucleotide sequences which differ from nucleotide sequences in the genomic duplexes, wherein the synthesized duplexes are treated with a restriction enzyme selectively to cleave duplexes containing a specific nucleotide sequence and wherein material in the synthesized duplexes present in the test sample with the genomic duplexes is further amplified in a second amplification stage using said upstream and downstream primers, the improvement wherein the upstream and downstream primers in the first amplification stage are upstream and downstream long tail primers and the upstream and downstream primers in the second amplification stage are upstream and downstream short tail primers, the upstream and downstream long tail and short tail primers being selected such that synthesized nucleic acid strands formed in the first polymerase chain reaction using the upstream and downstream long tail primers anneal with one of either the upstream or downstream short tail primers, and such that the short tail primers do not anneal with any nucleic acid strands in the genomic duplexes whereby the upstream and downstream short tail primers can be used in the second amplification stage selectively to amplify material in duplexes synthesized in the first amplification stage but cannot amplify material in the first or second genomic duplexes.

10. A process as claimed in claim 9 wherein the upstream and downstream short tail primers consist of DNA sequences from polyoma virus.

11. A process as claimed in claim 10 wherein the upstream and downstream short tail primers are between about 15 and 25 bases in length, whereby the further synthesized duplexes are longer than the genomic duplexes by between about 30 and 50 bases pairs.

12. In a process for detecting mutations in nucleic acid duplexes comprising amplification of material in the duplexes in a polymerase chain reaction in a first reaction mix to form synthesized nucleic acid duplexes and subsequent treatment of the synthesized duplexes with a restriction enzyme in a second reaction mix selectively to cleave duplexes containing a specific nucleotide sequence, the first reaction mix comprising a nucleic acid polymerase with a first buffer suitable for enabling the polymerase to facilitate amplification of material in the nucleic acid duplexes in a polymerase chain reaction, the second reaction mix comprising the restriction enzyme with a second buffer suitable for enabling the restriction enzyme selectively to cleave duplexes containing the specific nucleotide sequence, said polymerase chain reaction comprising repetitive cycles of denaturation, annealing and extension, the improvement comprising:

conducting the amplification and treatment steps in a single reaction mix by a) including in the single reaction mix a thermostable nucleic acid polymerase, a thermostable restriction enzyme and an intermediate buffer suitable for enabling the polymerase to facilitate amplification of the material in the nucleic acid duplexes while also enabling the restriction enzyme to cleave duplexes containing the specific nucleotide sequence, said intermediate buffer consisting essentially of BstN1 buffer diluted to between 60-80% concentration, said thermostable restriction enzyme being selected such that it can selectively cleave duplexes containing the specific nucleotide sequence when used with said intermediate buffer; and

b) regulating the times and temperatures at which the cycles of the polymerase chain reaction are performed so as to amplify the nucleic acid duplexes without inactivating the thermostable restriction enzyme.

13. A process as claimed in claim 12 wherein the material amplified in the polymerase chain reaction comprises codon 12 of the human K-ras gene, the restriction enzyme is BstNI, the polymerase is Taq polymerase and the intermediate buffer is a diluted solution comprising 60-80% of BstNI buffer.

14. A process as claimed in claim 12 wherein the temperature at which the denaturation cycle of the polymerase chain reaction is performed is between about 86°

C.-92°

C.