Multiplex genomic DNA amplification for deletion detection

• US 5,582,989 A
• Filed: 09/30/1994
• Issued: 12/10/1996
• Est. Priority Date: 10/12/1988
• Status: Expired due to Term

• Current Assignee: Baylor College of Medicine (Baylor College)
• Original Assignee: Baylor College of Medicine (Baylor College)
• Inventors: Chamberlain, Jeffrey S., Ranier, Joel E., Nguyen, Phi N., Caskey, Charles T., Gibbs, Richard A. L.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/315,673
• Time in Patent Office: 802 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.1, 536/24.3-24.33, 935/77, 935/78, 935/88
• US Class Current: 435/6.18
• CPC Class Codes: C07K 14/4708 Duchenne dystrophy
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• 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 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention relates to a method for detecting multiple DNA sequences simultaneously. The method involves amplification of multiple sequences simultaneously by annealing a plurality of paired oligonucleotide primers to single stranded DNA. One member of each pair is complementary to the sense strand of a sequences and the other member is complementary to a different segment of the anti-sense strand of the same sequence. The amplification occurs by alternately annealing and extending the primers. The invention also includes oligonucleotide primer sequences helpful in detecting genetic diseases and/or exogenous DNA sequences.

Claims

1. A method for simultaneously detecting known deletions from at least three DNA sequences, comprising the steps of:

treating said DNA to form single-stranded complementary strands;

adding at least three pairs of oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense-strand and the other primer of each pair substantially complementary to a different part of the same sequence in the complementary anti-sense strand;

annealing the at least three pairs of primers to their complementary sequences, all primers being subjected to the same reaction conditions;

simultaneously extending said at least three pairs of annealed primers from each primer'"'"'s 3'"'"' terminus to synthesize an extension product complementary to the strands annealed to each primer, said extension products, after separation from their complement, being capable of serving as templates for the synthesis of an extension product from the other primer of each pair;

separating said extension products from said templates to produce single-stranded molecules;

amplifying said single stranded molecules by repeating, at least once, said annealing, extending and separating steps; and

identifying said amplified extension products from each different sequence.

2. The method of claim 1 for detecting deletions from at least genomic DNA sequences, wherein said sequences are selected from the group of known sequences on the X and Y chromosomes.

3. The method of claim 2 for the detection of X-linked disease, wherein said genomic DNA sequences contain a deletion that causes a genetic disease.

4. The method of claim 3 for the detection of said X-linked genetic diseases selected from the group consisting of ornithine transcarbamylase deficiency, hypoxanthine phosphoribosyltransferfase deficiency, steroid sulfatase deficiency and X-linked muscular dystrophy.

5. The method of claim 4 for the detection of X-linked muscular dystrophy, wherein each pair of said at least three pairs of primers are complementary to different sequences within the gene coding for the dystrophin protein.

6. The method of claim 5, wherein the at least three pairs of primers is selected from the group consisting of:

(1) 5'"'"'-GACTTTCGATGTTGAGATTACTTTCCC-3'"'"'

(2) 5'"'"'-AAGCTTGAGATGCTCTCACCTTTTCC-3'"'"',

(1) 5'"'"'-GTCCTTTACACACTTTACCTGTTGAG-3'"'"'

(2) 5'"'"'-GGCCTCATTCTCATGTTCTAATTAG-3'"'"',

(1) 5'"'"'-AAACATGGAACATCCTTGTGGGGAC-3'"'"'

(2) 5'"'"'-CATTCCTATTAGATCTGTCGCCCTAC-3'"'"',

(1) 5'"'"'-GATAGTGGGCTTTACTTACATCCTTC-3'"'"'

(2) 5'"'"'-GAAAGCACGCAACATAAGATACACCT-3'"'"',

(1) 5'"'"'-CTTGATCCATATGCTTTTACCTGCA-3'"'"'

(2) 5'"'"'-TCCATCACCCTTCAGAACCTGATCT-3'"'"',

(1) 5'"'"'-TTGAATACATTGGTTAAATCCCAACATG-3'"'"'

(2) 5'"'"'-CCTGAATAAAGTCTTCCTTACCACAC-3'"'"', and

(1) 5'"'"'-TTCTACCACATCCCATTTTCTTCCA-3'"'"'

(2) 5'"'"'-GATGGCAAAAGTGTTGAGAAAAAGTC-3'"'"'.

7. The method of claim 3, wherein said genomic DNA is from fetal tissue.

8. The method of claim 1 for detecting deletions from at least three genomic DNA sequences, wherein the at least three pairs of primers is selected from the group consisting of:

(1) 5'"'"'-GACTTTCGATGTTGAGATTACTTTCCC-3'"'"'

(2) 5'"'"'-AAGCTTGAGATGCTCTCACCTTTTCC-3'"'"',

(1) 5'"'"'-GTCCTTTACACACTTTACCTGTTGAG-3'"'"'

(2) 5'"'"'-GGCCTCATTCTCATGTTCTAATTAG-3'"'"',

(1) 5'"'"'-AAACATGGAACATCCTTGTGGGGAC-3'"'"'

(2) 5'"'"'-CATTCCTATTAGATCTGTCGCCCTAC-3'"'"',

(1) 5'"'"'-GATAGTGGGCTTTACTTACATCCTTC-3'"'"',

(2) 5'"'"'-GAAAGCACGCAACATAAGATACACCT-3'"'"',

(1) 5'"'"'-CTTGATCCATATGCTTTTACCTGCA-3'"'"'

(2) 5'"'"'-TCCATCACCCTTCAGAACCTGATCT-3'"'"',

(1) 5'"'"'-TTGAATACATTGGTTAAATCCCAACATG-3'"'"'

(2) 5'"'"'-CCTGAATAAAGTCTTCCTTACCACAC-3'"'"',

(1) 5'"'"'-TTCTACCACATCCCATTTTCTTCCA-3'"'"'

(2) 5'"'"'-GATGGCAAAAGTGTTGAGAAAAAGTC-3'"'"',

(1) 5'"'"'-TGGTCTCCTTAAACCTGTCTT-3'"'"'

(2) 5'"'"'-ACACAACTGTGTTCACTAG-3'"'"',

(1) 5'"'"'-ACGTGGAGTGACGATGCTCTTCCC-3'"'"'

(2) 5'"'"'-GTGGGATTCACCACTTTTCCC-3'"'"', and

(1) 5'"'"'-GAAGTCAAGGACACCGAGGAA-3'"'"'

(2) 5'"'"'-AGCCCTCTGGCCAGTCCTAGTG-3'"'"'.

9. The method of claim 1, wherein said at least three pairs of primers have Tms such that the lowest Tm and the highest Tm vary by no more than 8.3°

C.

10. The method of claim 1, wherein said at least three pairs of primers have Tms such that the lower Tm of each pair varies from the lower Tm of each other pair by no more than 4.4°

C.

11. A method for simultaneously detecting at least three DNA sequences, comprising the steps of:

adding to a common reaction vessel containing a sample mixture of at least three distinct, target sequences in single-stranded form, at least three pairs of oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense-strand and the other primer of each pair substantially complementary to a different part of the same sequence in the complementary anti-sense strand;

annealing the at least three pairs of primers to their complementary sequences, all primers being subject to the same reaction conditions;

simultaneously extending said at least three pairs of annealed primers from each primer'"'"'s 3'"'"' terminus to synthesize an extension product complementary to the strands annealed to each primer, said extension products, after separation from their complement, being capable of serving as templates for the synthesis of an extension product from the other primer of each pair;

separating said extension products from said templates to produce single-stranded molecules;

amplifying said single stranded target sequences by repeating, at least once, said annealing, extending and separating steps; and

identifying whether amplified extension products have been synthesized from each different sequence, as a result of the presence or absence of each target sequence.

12. A method for simultaneously detecting known deletions from at least three DNA sequences, comprising the steps of:

treating said DNA to form single-stranded complementary strands;

adding at least three pairs of oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense-strand and the other primer of each pair substantially complementary to a different part of the same sequence in the complementary anti-sense strand and each primer having a Tm such that the lowest Tm and highest Tm of all added primers varies by no more than 8.3°

C.;

annealing the at least three pairs of primers to their complementary sequences, all primers being subjected to the same reaction conditions;

simultaneously extending said at least three pairs of annealed primers from each primer'"'"'s 3'"'"' terminus to synthesize an extension product complementary to the strands annealed to each primer, said extension products, after separation from their complement, being capable of serving as templates for the synthesis of an extension product from the other primer of each pair;

separating said extension products from said templates to produce single-stranded molecules;

amplifying said single stranded molecules by repeating, at least once, said annealing, extending and separating steps; and

identifying said amplified extension products from each different sequence; and

analyzing said amplified extension products to detect known deletions.

13. A method for simultaneously detecting a presence or absence of at least three target DNA sequences, comprising the steps of:

adding to a common reaction vessel containing a sample mixture of at least three distinct, target sequences in single-stranded form, at least three pairs of oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense-strand and the other primer of each pair substantially complementary to a different part of the same sequence in the complementary anti-sense strand and each primer having a Tm such that the lowest Tm and highest Tm of all added primers varies by no more than 8.3°

C.;

annealing the at least three pairs of primers to their complementary sequences, all primers being subject to the same reaction conditions;

simultaneously extending said at least three pairs of annealed primers from each primer'"'"'s 3'"'"' terminus to synthesize an extension product complementary to the strands annealed to each primer, said extension products, after separation from their complement, being capable of serving as templates for the synthesis of an extension product from the other primer of each pair;

separating said extension products from said templates to produce single-stranded molecules;

amplifying said single stranded target sequences by repeating, at least once, said annealing, extending and separating steps; and

identifying whether amplified extension products have been synthesized from each different target sequence, wherein a presence of an extension product indicates the presence of a target sequence and an absence of an extension product indicates the absence of a target sequence.

14. A method for simultaneously detecting known deletions from at least three DNA sequences, comprising the steps of:

treating said DNA to form single-stranded complementary strands;

adding at least three pairs of oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense-strand and the other primer of each pair substantially complementary to a different part of the same sequence in the complementary anti-sense strand and each primer having a Tm such that the lowest Tm and highest Tm of all added primers varies by no more than 4.4°

C.;

annealing the at least three pairs of primers to their complementary sequences, all primers being subjected to the same reaction conditions;

simultaneously extending said at least three pairs of annealed primers from each primer'"'"'s 3'"'"' terminus to synthesize an extension product complementary to the strands annealed to each primer, said extension products, after separation from their complement, being capable of serving as templates for the synthesis of an extension product from the other primer of each pair;

separating said extension products from said templates to produce single-stranded molecules;

amplifying said single stranded molecules by repeating, at least once, said annealing, extending and separating steps; and

identifying said amplified extension products from each different sequence; and

analyzing said amplified extension products to detect known deletions.

15. A method for simultaneously detecting at least three target DNA sequences, comprising the steps of:

adding to a common reaction vessel containing a sample mixture of at least three distinct, target sequences in single-stranded form, at least three pairs of oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense-strand and the other primer of each pair substantially complementary to a different part of the same sequence in the complementary anti-sense strand and each primer having a Tm such that the lowest Tm and highest Tm of all added primers varies by no more than 4.4°

C.;

annealing the at least three pairs of primers to their complementary sequences, all primers being subject to the same reaction conditions;

simultaneously extending said at least three pairs of annealed primers from each primer'"'"'s 3'"'"' terminus to synthesize an extension product complementary to the strands annealed to each primer, said extension products, after separation from their complement, being capable of serving as templates for the synthesis of an extension product from the other primer of each pair;

separating said extension products from said templates to produce single-stranded molecules;

amplifying said single stranded target sequences by repeating, at least once, said annealing, extending and separating steps; and

identifying whether amplified extension products have been synthesized from each different target sequence, wherein a presence of an extension product indicates the presence of a target sequence and an absence of an extension product indicates the absence of a target sequence.