Method, sequences, compositions and kit for detection of mutations in the htert gene promoter

Abstract

The present invention relates to a method for detecting c.-124 C>T and c.-146 C>T mutations in the Htert gene promoter. The method uses reaction compositions that comprise amplification primers and genotyping probes. Another aspect of this invention relates to the primers and probes used to implement the aforementioned method, with sequences, identified as SEQ ID no.1 to SEQ ID no.6, that displayhigh specificity for these mutations, and to the compositions containing these primers and probes. The present invention further relates to a kit comprising the above-mentioned compositions for detecting c.-124 C>T and c.-146 C>T mutations in the Htert gene promoter by carrying out the method according to the present invention. The method, gene sequences, compositions and kit of the present invention can be advantageously used for detecting trace amounts of c.-124 C>T and c.-146 C>T mutations in biological samples, due to their high sensitivity and specificity for such mutations. The present invention can thus be applied in early detection, identification, detection of recurrence or prediction and monitoring of diseases associated with this type of mutations, such as bladder carcinomas, thyroid carcinomas, squamous cell carcinomas, basal cell carcinomas, melanomas, gliomas and hepatocellular carcinomas, inter alia, and ultimately provide the basis for defining a suitable treatment. Thus, the present invention pertains to the technical fields of medicine, pharmaceutics, molecular biology, biochemistry, human genetics and the like.

Description

technical field

[0001] The present invention relates to a method for ultrasensitive detection of c.-124C>T and c.-146C>T mutations in the Htert gene promoter, comprising a reaction combination of primers for amplification and probes for genotyping 6, which are designed to show high specificity for these mutations, and kits comprising such compositions for carrying out the methods.

[0002] The present invention can be advantageously used to detect such c.-124C>T and c.-146C>T mutations present in trace amounts in biological samples and in vitro, due to the high sensitivity of the method and the resulting gene Sequence specificity and thus allow early detection, recurrence identification or prediction and monitoring of diseases associated with those mutations, e.g. bladder cancer, thyroid cancer, squamous cell carcinoma, basal cell carcinoma, skin cancer, central nervous system cancer and hepatocellular carcinoma, among others, and finally provide the basis for app...

Examples

Embodiment 1

[0151] Example 1 - Preparation of primer and probe sequences

[0152] Two primer sequences: SEQ ID NO.1 and SEQ ID NO.2, and 4 additional probe sequences: SEQ ID NO.3 to SEQ ID NO.6 were prepared as follows:

[0153] The above sequences were prepared by solid phase nucleotide synthesis using phosphoramidite nucleosides. The synthesis was carried out on a column packed with a solid support functionalized with the first base at the 3' end of each oligonucleotide. The preparation of each oligonucleotide is carried out according to several synthetic cycles, and each synthetic cycle is composed of four chemical reactions:

[0154] Release (detritylation); coupling; protection and oxidation. In each cycle, nucleotide residues corresponding to the desired sequence are added incrementally at the 5' end of the growing chain.

[0155] Probe concentrations were adjusted to values ​​of 400-1600 nM by diluting the lyophilized preparations in double distilled and double deionized water. ...

Embodiment 2

[0156] Embodiment 2-Preparation of PCR reaction composition

[0157] Several PCR reaction compositions were prepared containing different concentrations of the nucleotide mixture according to SEQ ID NO. 1 to SEQ ID NO. 6 (eg 400nm, 800nm, 1600nM), as described below. For comparison purposes, solutions with alternative probe concentrations for the "wild type" allele (eg 250 nM, 500 nM) were also prepared.

[0158] I - Composition for detecting mutations at base -124 upstream of the ATG initiation codon of the hTERT gene

[0159] Composition Ia according to the invention:

[0160] ·Reagent solution for real-time PCR" Universal PCR Master Mix", concentration 1X;

[0161] Oligonucleotide, having sequence: SEQ ID NO.1, concentration 900nM;

[0162] Oligonucleotide, having sequence: SEQ ID NO.2, concentration 900nM;

[0163] Oligonucleotide probe with sequence: SEQ ID NO.3, concentration 250nM, which contains modifications, such as the introduction of YAKIMA at the 5' and 3' e...

Embodiment 3

[0209] Example 3 - Preparation of compositions for detecting mutations

[0210] The composition of the reaction mixture for the detection of mutations c.-124C>T and c.-146C>T in the promoter of the gene hTERT is obtained by combining DNA (about 100ng of DNA, which can be 1ng-500ng A smaller amount used) was added to the above PCR composition to prepare, and the concentration of the probes having the sequences SEQ ID NO.3 and SEQ ID NO.6 was adjusted to a value of 400-1600 nM, as described in the previous examples. The composition for detection is thus prepared with a DNA sample containing the c.124C>T mutation or with a "serial dilution" of the c.146C>T mutation in a "wild type" DNA sample.

[0211] Also, a detection composition for comparison purposes was prepared by adding DNA from a biological test sample to the comparison solution of the preceding examples.

technical field

[0001] The present invention relates to a method for ultrasensitive detection of c.-124C>T and c.-146C>T mutations in the Htert gene promoter, comprising a reaction combination of primers for amplification and probes for genotyping 6, which are designed to show high specificity for these mutations, and kits comprising such compositions for carrying out the methods.

[0002] The present invention can be advantageously used to detect such c.-124C>T and c.-146C>T mutations present in trace amounts in biological samples and in vitro, due to the high sensitivity of the method and the resulting gene Sequence specificity and thus allow early detection, recurrence identification or prediction and monitoring of diseases associated with those mutations, e.g. bladder cancer, thyroid cancer, squamous cell carcinoma, basal cell carcinoma, skin cancer, central nervous system cancer and hepatocellular carcinoma, among others, and finally provide the basis for app...

Examples

Embodiment 1

[0151] Example 1 - Preparation of primer and probe sequences

[0152] Two primer sequences: SEQ ID NO.1 and SEQ ID NO.2, and 4 additional probe sequences: SEQ ID NO.3 to SEQ ID NO.6 were prepared as follows:

[0153] The above sequences were prepared by solid phase nucleotide synthesis using phosphoramidite nucleosides. The synthesis was carried out on a column packed with a solid support functionalized with the first base at the 3' end of each oligonucleotide. The preparation of each oligonucleotide is carried out according to several synthetic cycles, and each synthetic cycle is composed of four chemical reactions:

[0154] Release (detritylation); coupling; protection and oxidation. In each cycle, nucleotide residues corresponding to the desired sequence are added incrementally at the 5' end of the growing chain.

[0155] Probe concentrations were adjusted to values ​​of 400-1600 nM by diluting the lyophilized preparations in double distilled and double deionized water. ...

Embodiment 2

[0156] Embodiment 2-Preparation of PCR reaction composition

[0157] Several PCR reaction compositions were prepared containing different concentrations of the nucleotide mixture according to SEQ ID NO. 1 to SEQ ID NO. 6 (eg 400nm, 800nm, 1600nM), as described below. For comparison purposes, solutions with alternative probe concentrations for the "wild type" allele (eg 250 nM, 500 nM) were also prepared.

[0158] I - Composition for detecting mutations at base -124 upstream of the ATG initiation codon of the hTERT gene

[0159] Composition Ia according to the invention:

[0160] ·Reagent solution for real-time PCR" Universal PCR Master Mix", concentration 1X;

[0161] Oligonucleotide, having sequence: SEQ ID NO.1, concentration 900nM;

[0162] Oligonucleotide, having sequence: SEQ ID NO.2, concentration 900nM;

[0163] Oligonucleotide probe with sequence: SEQ ID NO.3, concentration 250nM, which contains modifications, such as the introduction of YAKIMA at the 5' and 3' e...

Embodiment 3

[0209] Example 3 - Preparation of compositions for detecting mutations

[0210] The composition of the reaction mixture for the detection of mutations c.-124C>T and c.-146C>T in the promoter of the gene hTERT is obtained by combining DNA (about 100ng of DNA, which can be 1ng-500ng A smaller amount used) was added to the above PCR composition to prepare, and the concentration of the probes having the sequences SEQ ID NO.3 and SEQ ID NO.6 was adjusted to a value of 400-1600 nM, as described in the previous examples. The composition for detection is thus prepared with a DNA sample containing the c.124C>T mutation or with a "serial dilution" of the c.146C>T mutation in a "wild type" DNA sample.

[0211] Also, a detection composition for comparison purposes was prepared by adding DNA from a biological test sample to the comparison solution of the preceding examples.

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