Method and assay for detection of the expression of allele-specific mutations by allele-specific in situ reverse transcriptase polymerase chain reaction

5,804,383

08/727,003

October 08, 1996

A method and an allele-specific in situ reverse transcriptase polymerase chain reaction (RT-PCR) amplification assay for detection and differentiation between expression of unmutated wild-type DNA sequences and between endogenous mutated DNA sequences in vitro or in vivo. The method is useful for verification, diagnostic assessment and monitoring of therapeutic small fragment homologous replacement gene therapy, for diagnostic assessment and monitoring of cDNA-based gene therapies, for analysis of gene expression of specific alleles during fetal development and for diagnostic assessment of the expression of alleles involved in cancer mutations.

Gruenert, Dieter C. (Mill Valley, CA); Dohrman, Austin F. (San Francisco, CA)

The Regents of the University of California (Oakland, CA)

What is claimed is

1. An assay for qualitative and quantitative detection of expression of a mutated or nonmutated human cystic fibrosis gene in a tissue or cells of a human subject to be tested, the assay comprising the steps

(a) obtaining

(1) a sample of the tissue or cells of a human subject to be tested for cystic fibrosis or from a human subject previously subjected to gene therapy for cystic fibrosis; and

(2) a sample of nonmutated control tissue cells or wild-type DNA corresponding to the same type of nonmutated tissue to serve as a control;

(b) fixing, digesting and reverse transcribing the samples obtained in step (a) to obtain cDNA;

(c) amplifying the cDNA obtained in step (b) using polymerase chain reaction in the presence of allele-specific primers or allele-nonspecific primers selected from the group of primers identified as SEQ ID NOS: 1-15 and SEO ID NOS: 32-46, under conditions and in a solution comprising all necessary nucleotides to obtain the cDNA in sufficient quantity for assay, wherein at least one nucleotide in the solution or in the primers is labeled with a non-interfering radioactive, immunocytochemical, fluorescent or other labeled marker detectable by spectroscopic, autoradiographic, immunocytochemical or enzymatic detection means;

(d) detecting the presence and quantity of cystic fibrosis gene expression in the samples obtained in step (a) by detecting the presence and quantity of the labeled marker of step (c) in the amplified cDNA; and

(e) comparing qualitatively and quantitatively the results obtained in step (d)

wherein the absence of the labeled marker in the cDNA amplified with mutated allele-specific primers, or wherein the presence of the labeled marker in the cDNA amplified with wild-type allele-specific primers, indicates the detection of a nonmutated cystic fibrosis gene; and

wherein the presence of the labeled marker in the cDNA amplified with mutated allele-specific primers, or wherein the absence of the labeled marker in the cDNA amplified with wild-type allele-specific primers, indicates the detection of a mutated cystic fibrosis gene.

2. The assay of claim 1 wherein the amount or the level of the labeled marker is quantified by counting the number of cells containing the labeled marker.

3. A method for detection of allele-specific mutations of the human cystic fibrosis gene in a human subject by an assay for detection of and differentiation between expression of a mutated cystic fibrosis gene and expression of a nonmutated cystic fibrosis gene in a human subject, said method comprising steps

(a) obtaining a sample of tissue or cells from a human subject to be tested for mutations in the cystic fibrosis gene;

(b) obtaining a control sample of cells of the same tissue or cell type as the tissue or cells of step (a) or a sample of nonmutated wild-type DNA from a healthy human subject;

(c) fixing the cells obtained in steps (a) and step (b);

(d) digesting the cells fixed in step (c) to expose the single stranded mRNA and to eliminate DNA contained in the cells;

(e) subjecting the mRNA exposed in step (d) to reverse transcription to obtain first-strand complementary DNA(cDNA);

(f) subjecting the cDNA obtained in step (e) to polymerase chain reaction amplification to obtain the cDNA in sufficient quantity for a detection and differentiation assay, said amplification performed in the presence of allele-specific or allele-nonspecific primers selected form the group of primers identified as SEQ ID NOS: 1-15 and SEQ ID NOS: 32-46, using a solution comprising at least one non-interfering labeled nucleotide marker detectable by spectroscopic, autoradiographic, immunocytochemical or enzymatic detection means;

(g) detecting the presence and quantity of cystic fibrosis gene expression in the samples obtained in step (a) and step (b) by detecting the presence and quantity of the labeled marker of step (f) in the amplified cDNA; and

(h) comparing qualitatively and quantitatively the results obtained in step (g) wherein the absence of the labeled marker in the cDNA amplified with mutated allele-specific primers, or wherein the presence of the labeled marker in the cDNA amplified with wild-type allele-specific primers, indicates the detection of a nonmutated cystic fibrosis gene; and

4. The method of claim 3 wherein the control sample is the wild-type DNA.

5. The method of claim 3 wherein the sample of step (a) is a bioptic tissue sample from the human subject to be tested.

6. The assay of claim 3 wherein the primers are allele-specific.

7. The assay of claim 3 wherein the primers are allele-nonspecific.

8. A method for assessment of success of a gene therapy for correction of a mutated cystic fibrosis gene in a human subject subjected to the gene therapy by an assay for detection of and differentiation between expression of a mutated cystic fibrosis gene and expression of a nonmutated cystic fibrosis gene in a human subject, said method comprising steps

(a) obtaining a sample of cells from the human subject subjected to the gene therapy for correction of a mutated cystic fibrosis gene;

(b) obtaining a control sample of cells of the same tissue type as the cells obtained in step (a) or a sample of nonmutated wild-type cells from a healthy human subject;

(c) fixing the cells obtained in step (a) and step (b);

(f) subjecting the cDNA obtained in step (e) to polymerase chain reaction amplification to obtain the cDNA in sufficient quantity for a detection and differentiation assay, said amplification performed in the presence of wild-type or mutated allele-specific or allele-nonspecific primers selected from the group of primers identified as SEQ ID NOS: 1-15 and SEQ ID NOS: 32-46, using a solution comprising at least one non-interfering labeled nucleotide marker detectable by spectroscopic, autoradiographic, immunocytochemical or enzymatic detection means;

(g) detecting the presence and quantity of cystic fibrosis gene expression in the sample of cells obtained in step (a) and step (b) by detecting the presence and quantity of the labeled marker of step (f) in the amplified cDNA; and

(h) evaluating qualitatively and quantitatively the results obtained in step (g) wherein the absence of the labeled marker in the cDNA amplified with mutated allele-specific primers, or wherein the presence of the labeled marker in the cDNA amplified with wild-type allele-specific primers, obtained from the human subject subjected to the gene therapy, confirms the correction of the cystic fibrosis gene and success of the gene therapy; and

wherein the presence of the labeled marker in the cDNA amplified with mutated allele-specific primers, or wherein the absence of the labeled marker in the cDNA amplified with wild-type allele-specific primers, obtained from the human subject subjected to the gene therapy, represents unsuccessful gene therapy.

9. The method of claim 8 wherein the sample obtained in step (a) is a bioptic tissue sample obtained from the human subject subjected to gene therapy.

435/6; 435/405; 435/912

C12Q 168; C12P 1934

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