Screening for mutations by expressing cDNA segments

• US 5,656,425 A
• Filed: 08/17/1993
• Issued: 08/12/1997
• Est. Priority Date: 08/17/1993
• Status: Expired due to Term

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: Kraus, Jan P.
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/108,216
• Time in Patent Office: 1,456 Days
• Field of Search: 435/6, 435/91.41, 435/91.42, 435/69.1, 435/320.1
• US Class Current: 435/6.13
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

Abstract

A rapid screening method is described for detecting, localizing, and expressing pathogenic mutations in patients suffering from diseases which can include genetically inherited diseases. The screening method allows simultaneous localization and expression of the mutation. By identifying the types and locations of the mutations causing a disease, the method can aid in determining the course of treatment for the disease. The method involves making an expression vector containing a sequence encoding an inactive enzyme by deleting a segment of the coding sequence of the enzyme in the vector, replacing the deleted segment with a corresponding segment from a patient'"'"'s enzyme coding sequence to make a hybrid enzyme, expressing the hybrid enzyme and assaying the hybrid enzyme for activity to determine whether the Patient'"'"'s enzyme cDNA contains a pathogenic mutation.

Claims

1. A method of locating the position of a pathogenic mutation in an enzyme gene of a human patient suffering from a disease, comprising the steps of:

(a) deleting at least one segment of a coding region of a normal enzyme cDNA in a starting expression vector to produce a vector containing an inactive enzyme sequence, wherein the segment deletion is within a subregion of the coding region of the normal cDNA corresponding to a subregion of a coding region of an enzyme cDNA from the patient;

(b) replacing in the vector containing the inactive enzymese the subregion of the normal cDNA having undergone the segment deletion with the inserted corresponding subregion of the patient enzyme cDNA to produce a hybrid enzyme;

(c) generating the hybrid enzyme from an expression system containing the expression vector, wherein the hybrid enzyme contains a portion encoded by at least one inserted subregion of the patient enzyme cDNA;

(d) assaying the hybrid enzyme for activity, an absence of enzyme activity or lower than normal enzyme activity of an enzyme generated from the normal enzyme cDNA indicating the presence of the pathogenic mutation in the inserted subregion of the patient enzyme cDNA;

(e) sequencing the inserted subregion of the patient enzyme cDNA in a total cDNA from the hybrid enzyme lacking activity or having lower than normal activity of the enzyme generated from the normal enzyme cDNA; and

(f) comparing the inserted subregion of the patient enzyme cDNA with a corresponding subregion of the normal enzyme cDNA or a normal enzyme genomic DNA to locate the position of the pathogenic mutation.

2. The method of claim 1, wherein the disease is premature occlusive arterial disease.

3. The method of claim 1, wherein the disease is genetically inherited.

4. The method of claim 3, wherein the genetically inherited disease is homocystinuria.

5. The method of claim 1, wherein the enzyme is cystathionine β

-synthase.

6. The method of claim 1, wherein the expression vector is a plasmid.

7. The method of claim 1, wherein the expression system is a prokaryotic or eukaryotic system.

8. The method of claim 7, wherein the prokaryotic system is Escherichia coli.

9. The method of claim 1, further comprises the screening of expression vectors from step (b) by PCR to confirm the presence 6f the patient'"'"'s cDNA insert by comparing the difference in size of the patient'"'"'s cDNA insert to a corresponding coding subregion having undergone segment deletion in the starting expression vector.

10. The method of claim 1, which further comprises identifying an enzyme allele in the expression vector by detecting a polymorphic marker for an allele.