Methods for detection of ataxia telangiectasia mutations

• US 20030082582A1
• Filed: 06/17/2002
• Published: 05/01/2003
• Est. Priority Date: 07/23/1999
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Gatti, Richard A.
• Granted Patent: US 6,951,724 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/131 Single/double strand conformational analysis, i.e. SSCP/DSCP
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention is directed to a method of screening large, complex, polyexonic eukaryotic genes such as the ATM gene for mutations and polymorphisms by an improved version of single strand conformation polymorphism (SSCP) electrophoresis that allows electrophoresis of two or three amplified segments in a single lane. The present invention also is directed to new mutations and polymorphisms in the ATM gene that are useful in performing more accurate screening of human DNA samples for mutations and in distinguishing mutations from polymorphisms, thereby improving the efficiency of automated screening methods.

Claims

1. An isolated and purified nucleic acid fragment comprising nucleic acid having complementarity or identity to a mutation in the ataxia-telangiectasia mutated (ATM) gene, the mutation being selected from the group consisting of:

(a) 10744A>

G;

(b) 11482G>

A;

(c) IVS3-558A>

T;

(d) 146C>

G;

(e) 381delA;

(f) IVS8-3delGT;

(g) 1028delAAAA;

(h) 1120C>

T;

(i) 1930ins16;

(j) IVS16+2T>

C;

(k) 2572T>

C;

(l) IVS21+1G>

A;

(m) 3085delA;

(n) 3381delTGAC;

(o) 3602delTT;

(p) 4052delT;

(q) 4396C>

T;

(r) 5188C>

T;

(s) 5290delC;

(t) 5546delT;

(u) 5791G>

CCT;

(v) 6047A>

G;

(w) IVS44-1G>

T;

(x) 6672delGC/6677delTACG;

(y) 6736dell 1/6749del7;

(z) 7159insAGCC;

(aa) 7671delGTTT;

(ab) 7705del14;

(ac) 7865C>

T;

(ad) 7979delTGT;

(ae) 8177C>

T;

(af) 8545C>

T;

(ag) 8565T>

A;

(ah) IVS64+1G>

T; and

(ai) 9010del28.

2. The fragment of claim 1 wherein the fragment has complementarity to the mutation in the ATM gene, is hairpin shaped, is covalently linked to a fluorophore and to a quencher, and has a structure such that the fluorophore is internally quenched by the quencher when the fragment is not base-paired and such that the internal quenching is relieved when the fragment is base-paired, thereby restoring fluorescence of the fluorophore.

3. The fragment of claim 1 wherein the fragment is DNA, has complementarity to the mutation in the ATM gene, and wherein the fragment further includes, covalently linked to either its 5′

-end or to its 3′

-end, a segment of about 40 bases, the segment of about 40 bases comprising a repeating unit of dCdG or dGdC.

4. A method for testing a DNA sample of a human for the presence or absence of a mutation in the ATM gene comprising the steps of:

(a) providing a sample of DNA from a human; and

(b) testing the sample for the presence of a mutation in the ATM gene, the mutation being selected from the group consisting of;

(i) 10744A>

g;

(ii) 11482G>

A;

(iii) IVS3-558A>

T;

(iv) 146C>

G;

(v) 381delA;

(vi) IVS8-3delGT;

(vii) 1028delAAAA;

(viii) 1120C>

T;

(ix) 1930ins16;

(x) IVS16+2T>

C;

(xi) 2572T>

C;

(xii) IVS21+1G>

A;

(xiii) 3085delA;

(xiv) 3381delTGAC;

(xv) 3602delTT;

(xvi) 4052delT;

(xvii) 4396C>

T;

(xviii) 5188C>

T;

(xix) 5290delC;

(xx) 5546delT;

(xxi) 5791G>

CCT;

(xxii) 6047A>

G;

(xxiii) IVS44-1G>

T;

(xxiv) 6672delGC/6677delTACG;

(xxv) 6736dell 1/6749del7;

(xxvi) 7159insAGCC;

(xxvii) 7671delGTTT;

(xxviii) 7705del14;

(xxix) 7865C>

T;

(xxx) 7979delTGT;

(xxxi) 8177C>

T;

(xxxii) 8545C>

T;

(xxxiii) 8565T>

A;

(xxxiv) IVS64+1G>

T; and

(xxxv) 9010del28.

5. An isolated and purified nucleic acid fragment comprising nucleic acid having complementarity or identity to a polymorphism in the ataxia-telangiectasia mutated (ATM) gene, the polymorphism being selected from the group consisting of:

(a) 10807A>

G;

(b) IVS3-122T>

C;

(c) IVS6+70delT;

(d) IVS16-34C>

A;

(e) IVS22-77T>

C;

(f) IVS24-9delT;

(g) IVS25-13delA;

(h) 5557G>

A;

(i) IVS48-69insATT; and

(j) IVS62-55T>

C.

6. The fragment of claim 5 wherein the fragment has complementarity to the polymorphism in the ATM gene, is hairpin shaped, is covalently linked to a fluorophore and to a quencher, and has a structure such that the fluorophore is internally quenched by the quencher when the fragment is not base-paired and such that the internal quenching is relieved when the fragment is base-paired, thereby restoring fluorescence of the fluorophore.

7. The fragment of claim 5 wherein the fragment is DNA, has complementarity to the polymorphism in the ATM gene, and wherein the fragment further includes, covalently linked to either its 5′

-end or to its 3′

-end, a segment of about 40 bases, the segment of about 40 bases comprising a repeating unit of dCdG or dGdC.

8. A method for testing a DNA sample of a human for the presence or absence of a polymorphism in the ATM gene comprising the steps of:

(a) providing a sample of DNA from a human; and

(b) testing the sample for the presence of a polymorphism in the ATM gene, the polymorphism being selected from the group consisting of;

(i) 108-7A>

G;

(ii) IVS3-122T>

C;

(iii) IVS6+70delT;

(iv) IVS16-34C>

A;

(v) IVS22-77T>

C;

(vi) IVS24-9delT;

(vii) IVS25-13delA;

(viii) 5557G>

A;

(ix) IVS48-69insATT; and

(x) IVS62-55T>

C.

9. An isolated and purified nucleic acid fragment comprising nucleic acid having complementarity or identity to a polymorphism in the ataxia-telangiectasia mutated (ATM) gene, the polymorphism being selected from the group consisting of:

(a) 10677G>

C;

(b) 10742G>

T;

(c) 10819G>

T;

(d) 10948A>

G;

(e) IVS3-300G>

A;

(f) IVS8>

24del5;

(g) IVS13-137T>

C;

(h) IVS14-55T>

G;

(i) 1986T>

C;

(j) IVS20+27delT;

(k) IVS23-76T>

C;

(l) IVS25-35T>

A;

(m) IVS27-65T>

C;

(n) IVS30-54T>

C;

(o) 4362A>

C;

(p) IVS38-8T>

C;

(q) 5793T>

C;

(r) IVS47-11G>

T;

(s) IVS49-16T>

A;

(t) IVS53+34insA;

(u) IVS60-50 delTTAGTT;

(v) IVS62+8A>

C;

(w) IVS62-65G>

A; and

(x) 9200C>

G.

10. The fragment of claim 9 wherein the fragment has complementarity to the polymorphism in the ATM gene, is hairpin shaped, is covalently linked to a fluorophore and to a quencher, and has a structure such that the fluorophore is internally quenched by the quencher when the fragment is not base-paired and such that the internal quenching is relieved when the fragment is base-paired, thereby restoring fluorescence of the fluorophore.

11. The fragment of claim 9 wherein the fragment is DNA, has complementarity to the polymorphism in the ATM gene, and wherein the fragment further includes, covalently linked to either its 5′

-end or to its 3′

-end, a segment of about 40 bases, the segment of about 40 bases comprising a repeating unit of dCdG or dGdC.

12. A method for testing a DNA sample of a human for the presence or absence of a polymorphism in the ATM gene comprising the steps of:

(a) providing a sample of DNA from a human; and

(b) testing the sample for the presence of a polymorphism in the ATM gene, the polymorphism being selected from the group consisting of;

(i) 10677G>

C;

(ii) 10742G>

T;

(iii) 10819G>

T;

(iv) 10948A>

G;

(v) IVS3-300G>

A;

(vi) IVS8>

24del5;

(vii) IVS13-137T>

C;

(viii) IVS14-55T>

G;

(ix) 1986T>

C;

(x) IVS20+27delT;

(xi) IVS23-76T>

C;

(xii) IVS25-35T>

A;

(xiii) IVS27-65T>

C;

(xiv) IVS30-54T>

C;

(xv) 4362A>

C;

(xvi) IVS38-8T>

C;

(xvii) 5793T>

C;

(xviii) IVS47-11G>

T;

(xix) IVS49-16T>

A;

(xx) IVS53+34insA;

(xxi) IVS60-50 delTTAGTT;

(xxii) IVS62+8A>

C;

(xxiii) IVS62-65G>

A; and

(xxiv) 9200C>

G.