Consensus sequence primed polymerase chain reaction method for fingerprinting genomes

• US 5,437,975 A
• Filed: 02/25/1991
• Issued: 08/01/1995
• Est. Priority Date: 02/25/1991
• Status: Expired due to Term

• Current Assignee: California Institute Of Bio. Research
• Original Assignee: California Institute Of Bio. Research
• Inventors: McClelland, Michael, Welsh, John T.
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Horlick, Kenneth R.
• Application Number:
  US07/661,591
• Time in Patent Office: 1,618 Days
• Field of Search: 435/91, 435/16, 435/91.2, 536/27, 536/124.33, 935/78
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

A rapid method for generating a set of discrete DNA amplification products characteristic of a genome as a "fingerprint" for typing the genome comprises the steps of: forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, genomic DNA and at least one structural RNA consensus primer, and subjecting the PCR admixture to a plurality of PCR thermocycles to produce a plurality of DNA segments, thereby forming a set of discrete DNA amplification products. The method is known as the consensus sequence primed polymerase chain reaction (CP-PCR) method and is suitable for the identification of bacterial species and strains, including Staphylococcus and Streptococcus species, mammals and plants. The method of the present invention can identify species rapidly, using only a small amount of biological material, and does not require knowledge of the nucleotide sequence or other molecular biology of the nucleic acids of the organisms to be identified. Only one primer sequence is required for amplification and/or identification. The method can also be used to generate detectable polymorphisms for use in genetic mapping of animals and humans.

Claims

1. A method of generating a set of discrete DNA segments characteristic of a genome comprising:

(a) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, genomic DNA and at least one structural RNA consensus primer from about 10 to about 50 nucleotide bases in length;

(b) subjecting said PCR admixture of step (a) to a plurality of PCR thermocycles to produce a plurality of DNA segments, thereby forming a set of discrete DNA segments, wherein said consensus primer is a tRNA consensus primer selected from the group consisting of;

(T5A) 5'"'"'-AGTCCGGTGCTCTAACCAACTGAG-3'"'"' (SEQ ID NO;

1),

(T5B) 5'"'"'-AATGCTCTACCAACTGAACT-3'"'"' (SEQ ID NO;

2),

(T3A) 5'"'"'-GGGGGTTCGAATTCCCGCCGGCCCCA-3'"'"' (SEQ ID NO;

3), and

(T3B) 5'"'"'-AGGTCGCGGGTTCGAATCC-3'"'"' (SEQ ID NO;

4).

2. A method for typing an organism having genomic DNA, which method comprises:

(a) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, said genomic DNA and at least one structural RNA consensus primer from about 10 to about 50 nucleotide bases in length;

(b) subjecting said PCR admixture of step (a) to a plurality of PCR thermocycles to produce a plurality of DNA segments, thereby forming a set of discrete DNA segments,

(c) applying the set of discrete DNA segments produced in step (b) to a channel of a separating apparatus;

(d) size-separating the applied segments into bands within the channel to form a fingerprint of segments characteristic of said genome,

(e) comparing the fingerprint of step (d) with the fingerprints for control samples of genomic DNA from a panel of predetermined species of organisms prepared in accordance with steps (a)-(d), and recording the results of the comparison, wherein said consensus primer is a tRNA consensus primer selected from the group consisting of;

(T5A) 5'"'"'-AGTCCGGTGCTCTAACCAACTGAG-3'"'"' (SEQ ID NO;

1),

(T5B) 5'"'"'-AATGCTCTACCAACTGAACT-3'"'"' (SEQ ID NO;

2),

(T3A) 5'"'"'-GGGGGTTCGAATTCCCGCCGGCCCCA-3'"'"' (SEQ ID NO;

3), and

(T3B) 5'"'"'-AGGTCGCGGGTTCGAATCC-3'"'"' (SEQ ID NO;

4).

3. A consensus primer wherein said primer has a nucleotide sequence selected from the group consisting of:

(T5A) 5'"'"'-AGTCCGGTGCTCTAACCAACTGAG-3'"'"' (SEQ ID NO;

1),

(T5B) 5'"'"'-AATGCTCTACCAACTGAACT-3'"'"' (SEQ ID NO;

2),

(T3A) 5'"'"'-GGGGGTTCGAATTCCCGCCGGCCCCA-3'"'"' (SEQ ID NO;

3), and

(T3B) 5'"'"'-AGGTCGCGGGTTCGAATCC-3'"'"' (SEQ ID NO;

4).

4. A kit for typing an organism, said kit comprising an enclosure containing, in separate containers, at least one structural RNA consensus primer and at least a sample of isolated genomic DNA from a panel of species of organisms wherein said structural RNA consensus primer is a tRNA consensus primer selected from the group consisting of:

(T5A) 5'"'"'-AGTCCGGTGCTCTAACCAACTGAG-3'"'"' (SEQ ID NO;

1),

(T5B) 5'"'"'-AATGCTCTACCAACTGAACT-3'"'"' (SEQ ID NO;

2),

(T3A) 5'"'"'-GGGGGTTCGAATTCCCGCCGGCCCCA-3'"'"' (SEQ ID NO;

3),

and

(T3B) 5'"'"'-AGGTCGCGGGTTCGAATCC-3'"'"' (SEQ ID NO;

4).