Amplification karyotyping

• US 5,869,237 A
• Filed: 11/22/1994
• Issued: 02/09/1999
• Est. Priority Date: 11/15/1988
• Status: Expired due to Term

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Ward, David C., Lichter, Peter
• Primary Examiner(s): Elliott, George C.
• Assistant Examiner(s): LARSON, THOMAS GEORGE
• Application Number:
  US08/343,358
• Time in Patent Office: 1,540 Days
• Field of Search: 435/91.2, 435/6, 935/77, 935/78, 536/24.31, 536/24.33
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2543/10 the purpose being "in situ" analysis
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

A method is disclosed for determining the chromosomal identity of a sample of genomic DNA. The genomic DNA is amplified and labeled by polymerase chain reaction using primers substantially complementary to interspersed repetitive DNA sequences. The amplified and labeled genomic DNA fragments are then contacted with chromosomal DNA of known identity under conditions in which the chromosome-specific, but not the interspersed repetitive DNA sequences, of the amplified and labeled genomic DNA fragments are available for hybridization. Specific hybridization to chromosomal DNA of known identity determines the chromosomal identity of the sample of genomic DNA.

Claims

1. A method for determining the chromosomal identity of mammalian-specific genomic DNA containing interspersed repetitive DNA sequences present in a nucleic acid sample, comprising the steps of:

a) subjecting a nucleic acid sample containing mammalian-specific genomic DNA containing interspersed repetitive DNA sequences to polymerase chain reaction conditions using primers which are substantially complementary to at least one of the interspersed repetitive DNA sequences present in the mammalian-specified genomic DNA, to produce labeled amplified mammalian-specified genomic DNA fragments containing interspersed repetitive DNA sequences;

b) combining

i) a sample of the labeled amplified mammalian-specific genomic DNA fragments of step a);

ii) chromosomal DNA of known identity; and

iii) competitor DNA containing DNA sequences which are substantially complementary to at least one interspersed repetitive DNA sequence present in the mammalian-specific genomic DNA.

under conditions appropriate for intermolecular hybridization, wherein hybridization between repetitive sequences in the labeled amplified mammalian-specific genomic DNA fragments and the chromosomal DNA of known identity is suppressed, thereby allowing hybridization between chromosome-specific sequences in the labeled amplified genomic mammalian-specific DNA fragments and the chromosomal DNA of know identity to occur; and

c) detecting specific hybridization signals of labeled mammalian-specific genomic DNA fragments of the chromosomal DNA of known identity, wherein each detected signal is distinguishable and identified a specific chromosomal sequence present in the mammalian-specific genomic DNA containing interspersed repetitive DNA sequences, such that the chromosomal identity of the mammalian-specific genomic DNA containing interspersed repetitive DNA sequences present in the nucleic acid sample is determined.

2. A method of claim 1, wherein the labeled amplified mammalian-specific genomic DNA fragments and the competitor DNA are combined in step b), before the combination with the chromosomal DNA of known identity, under conditions which allow the repetitive DNA sequences in the labeled amplified mammalian-specific genomic DNA fragments and in the competitor DNA to preanneal.

3. A method of claim 1, wherein the interspersed repetitive DNA sequences comprise short interspersed repetitive DNA sequences.

4. A method of claim 3, wherein the short interspersed repetitive DNA sequences are Alu sequences.

5. A method of claim 1, wherein the interspersed repetitive DNA sequences comprise long interspersed repetitive DNA sequences.

6. A method of claim 5, wherein the long interspersed repetitive DNA sequences are human L1 elements.

7. A method of claim 1, wherein the chromosomal DNA of known identity comprises chromosomes fixed in situ.

8. A method of claim 1, wherein the chromosomal DNA of known identity comprises chromosomal libraries.

9. A method of claim 1, wherein the chromosomal DNA of known identity comprises segregated chromosomal DNA or fragments thereof.

10. A method of claim 1, wherein the chromosomal DNA of known identity comprises chromosomal DNA or fragments thereof are immobilized on a solid support.

11. A method of claim 1, wherein said nucleic acid sample is obtained from a hybird cell containing DNA from at least two different species of mammals and the chromosomal DNA of known identity comprises chromosomes or fragments thereof from one of said mammalian species.

12. A method of claim 1, wherein the competitor DNA is present at a concentration such that the repetitive sequences of the competitor DNA are present in molar excess relative to the repetitive sequences in the labeled mammalian-specific genomic DNA fragments.

13. A method of claim 1, wherein said mammalian-specific genomic DNA containing interspersed repetitive DNA sequences is human genomic DNA.

14. A method of claim 1, wherein said nucleic acid sample comprises total genomic DNA.

15. A method of claim 1, wherein said polymerase chain reaction conditions comprise a detectable reporter group.

16. A method for determining the chromosomal identity of human genomic DNA containing interspersed repetitive DNA sequences present in a somatic cell hybird, comprising the steps of:

a) subjecting a human genomic DNA sample containing human genomic DNA containing interspersed repetitive DNA present in a somatic cell hybird to polymerase chain reaction conditions using primers which are substantially complementary to at least one of the interspersed repetitive DNA sequences in the human genomic DNA, to produce labeled amplified human genomic DNA fragments containing interspersed repetitive DNA sequences;

b) combining a sample of the labeled amplified genomic DNA fragments of step a) with competitor DNA containing sequences which are substantially complementary to at least one interspersed repetitive DNA sequence present in the human genomic DNA, thereby rendering the chromosome-specific sequences but not the interspersed repetitive DNA sequences of the labeled amplified genomic DNA fragments available for further hybridization;

c) combining the product of step b) with human chromosomal DNA of known identity, under conditions appropriate for intermolecular hybridization, wherein hybridization between repetitive sequences in the labeled amplified human genomic DNA fragments and the human chromosomal DNA of known identity is suppressed, thereby allowing hybridization between chromosome-specific sequences in the labeled amplified human genomic DNA fragments and the human chromosomal DNA of known identity to occur; and

d) detecting specific hybridization signals of labeled amplified human genomic DNA fragments to the chromosomal DNA of known identity, wherein each detected signal is distinguishable and identifies a specific chromosomal sequences present in the human genomic DNA containing interspersed repetitive DNA sequences in a somatic cell hybird, such that the chromosomal identity of the human genomic DNA containing interspersed repetitive DNA sequences present in a somatic cell hybird is determined.