Representational approach to DNA analysis

• US 6,277,606 B1
• Filed: 03/02/1999
• Issued: 08/21/2001
• Est. Priority Date: 11/09/1993
• Status: Expired due to Fees

• Current Assignee: Cold Spring Harbor Laboratory
• Original Assignee: Cold Spring Harbor Laboratory
• Inventors: Wigler, Michael, Lisitsyn, Nikolai
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US09/261,079
• Time in Patent Office: 903 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/23.1
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/155 Cyclic reactions
• CPC Class Codes: C12Q 2539/107 Representational Difference Analysis [RDA]

Abstract

Methodology is provided for developing probes for identifying sequence differences between two related DNA populations, sets of DNA fragments or collections of restriction-endonuclease-cleaved DNA or cDNA. The method employs an initial stage to obtain a representation of both DNA populations, namely using the PCR to produce relatively short fragments, referred to as amplicons. Tester amplicons containing target DNA, sequences of interest, are ligated to adaptors and mixed with excess driver amplicons under melting and annealing conditions, followed by PCR amplification. The process may be repeated so as to greatly enrich the target DNA. Optionally, the target DNA may then be cloned and the DNA used as probes.

Claims

1. A kit comprising a panel of at least two probes distinguishing between a human normal cell and a human neoplastic cell, wherein said probes are prepared by the method comprising:

substantially completely digesting separately the DNA from a human neoplastic cell source and a related normal human cell source with a restriction endonuclease having a nucleotide recognition sequence of at least 4 nucleotides, wherein said normal cell source is driver DNA and said neoplastic cell source is tester DNA, wherein said tester DNA comprises target DNA, wherein said target DNA comprises sequence differences between the genomes of said neoplastic and normal cell sources comprising at least one of an insertion, deletion, rearrangement or DNA amplification to define target DNA;

ligating a first set of adaptors to said digested fragments and amplifying said fragments using primers to one of the strands of said first set of adaptors to provide amplified amounts of fragments of said digested sequences of less than about 2 kbp as amplicons;

carrying out a first round of the following steps for enrichment of target DNA;

removing said first set of adaptors from said amplicons and ligatiang a second set of adapter to 5′

ends of amplicons of tester DNA;

combining under melting and annealing conditions said tester amplicons with a large excess of driver amplicons, whereby a portion of the resulting dsDNA comprises self-annealed tester DNA including target DNA;

filling in the 3′

ends of overhangs;

amplifying said dsDNA with primers to one of said strands of said second set of adaptors to enrich for target DNA;

repeating said first round of steps for at least 1 additional round to provide a DNA composition comprising a predominant amount of target DNA; and

cloning said DNA composition to provide clones having a substantially homogeneous probe of target DNA.

2. A kit according to claim 1, wherein said method further comprises combining said substantially homogeneous probes under hybridizing conditions with driver DNA and tester DNA, and discarding said substantially homogeneous probes that bind to both driver DNA and tester DNA.

3. A kit according to claim 2, wherein said probes are labeled.

4. A kit comprising a panel of at least two probes distinguishing between a human normal cell and a human neoplastic cell, wherein said probes are prepared by the method comprising:

substantially completely digesting separately the DNA from a human neoplastic cell source and a related normal human cell source with a restriction endonuclease having a nucleotide recognition sequence of at least 4 nucleotides, wherein said normal cell source is driver DNA, and said neoplastic cell source is tester DNA, wherein said tester DNA comprises target DNA, wherein said target DNA comprises sequence differences between the genomes of said neoplastic and normal cell sources comprising loss of heterozygosity, homozygosity or hemizygous loss to define target DNA;

ligating a first set of adaptors to said digested fragments and amplifying said fragments using primers to one of the strands of said first set of adaptors to provide amplified amounts of fragments of said digested sequences of less than about 2 kbp as amplicons;

carrying out a first round of the following steps for enrichment of target DNA;

removing said first set of adaptors from said amplicons and ligatiang a second set of adaptors to 5′

ends of amplicons of tester DNA;

combining under melting and annealing conditions said tester amplicons with a large excess of driver amplicons, whereby a portion of the resulting dsDNA comprises self-annealed tester DNA including target DNA;

filling in the 3′

ends of overhangs;

amplifying said dsDNA with primers to one of said strands of said second set of adaptors to enrich for target DNA;

repeating said first round of steps for at least 1 additional round to provide a DNA composition comprising a predominant amount of target DNA; and

cloning said DNA composition to provide clones having a substantially homogeneous probe of target DNA.

5. A kit according to claim 4, wherein said method further comprises combining said substantially homogeneous probes under hybridizing conditions with driver DNA and tester DNA, and discarding said substantially homogeneous probes that bind to both driver DNA and tester DNA.

6. A kit according to claim 5, wherein said probes are labeled.