# US 6,297,014 B1 · Genetic test to determine non-responsiveness to statin drug treatment

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- Number: US 6,297,014 B1
- Document ID: US6297014B1
- Status: Granted
- Kind: B1
- Application number: 09/347,114
- Filed: 1999-07-02
- Granted: 2001-10-02
- Earliest claimed: 1999-07-02
- Assignees: Cedars-Sinai Medical Center
- Inventors: Kent D. Taylor; Maren T. Scheuner; Jerome I. Rotter; Huiying Yang
- Web page: https://patentgrep.com/patent/US6297014B1

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

In a method for detecting a genetic predisposition in a human for non-responsiveness to statin drug treatment for coronary artery disease, nucleic acids comprising nucleotide sequences of the human lipoprotein lipase (LPL) gene are amplified and analyzed. Homozygosity for a variant allele in a non-coding or untranslated region of the 3′ end of LPL, for example, LPL HindIII 2/2 or (TTTA)n 4/4 genotypes, is linked to non-responsiveness to treatment with statin drugs, including lovastatin, pravastatin, simvastatin, atorvastatin, fluvastatin, or cerivastatin. Oligonucleotide primer sequences, primer sets, and genetic testing kits allow the practitioner to practice the method and thus better individualize the treatment and improve the care of patients with coronary artery disease.

## Claims

### Claim 1

A method of detecting a genetic predisposition in a human subject for non-responsiveness to treatment with a statin drug selected from the group consisting of lovastatin, pravastatin, and simvastatin, comprising:
a) collecting a tissue sample from a human subject;
b) amplifying nucleic acids that include the normal locus of the HindIII recognition site in intron 8 of the human lipoprotein lipase gene (LPL) from said tissue sample to obtain amplification products; and
c) analyzing the amplification products for the absence of a HindIII recognition site in intron 8 of the human lipoprotein lipase gene, homozygosity for an absence of said HindIII recognition site indicating a genetic predisposition for non-responsiveness to treatment with a statin drug selected from the group consisting of lovastatin, pravastatin, and simvastatin.

### Claim 2 (depends on 1)

The method of claim 1, wherein the tissue sample is a blood sample.

### Claim 3 (depends on 1)

The method of claim 1, further comprising restricting the amplification products with a restriction enzyme before analyzing the amplification products.

### Claim 4 (depends on 1)

The method of claim 1, wherein the restriction enzyme is HindIII.

### Claim 5 (depends on 1)

The method of claim 1, wherein an oligonucleotide primer is used in amplifying said nucleic acids.

### Claim 6 (depends on 1)

The method of claim 1, wherein an oligonucleotide primer comprising the sequence GCA TCT GCC TTC AGC TAG ACA TTG (SEQ. ID. NO. 1) is used in amplifying said nucleic acids.

### Claim 7 (depends on 1)

The method of claim 1, wherein an oligonucleotide primer comprising the sequence TCT TCC AGA AGG GTG AGA TTC CAA (SEQ. ID. NO. 2) is used in amplifying said nucleic acids.

### Claim 8 (depends on 1)

The method of claim 1, wherein an oligonucleotide primer having the sequence GCA TCT GCC TTC AGC TAG ACA TTG (SEQ. ID. NO. 1) or TCT TCC AGA AGG GTG AGA TTC CAA (SEQ. ID. NO. 2) is used in amplifying said nucleic acids.

### Claim 9 (depends on 1)

The method of claim 1, wherein a reverse oligonucleotide primer having the sequence 5′-GCA TCT GCC TTC AGC TAG ACA TTG-3′ (SEQ. ID. NO. 1) and a forward oligonucleotide primer having the sequence 5′-TCT TCC AGA AGG GTG AGA TTC CAA-3′ (SEQ. ID. NO. 2) are used in amplifying said nucleic acids.

### Claim 10 (depends on 1)

The method of claim 1, wherein an oligonucleotide primer is used in amplifying said nucleic acids, said primer comprising a sequence selected from the group essentially consisting of (SEQ. ID. NO.:1), (SEQ. ID. NO.:2), (SEQ. ID. NO.:3), (SEQ. ID. NO.:4), (SEQ. ID. NO.:5), (SEQ. ID. NO.:6), (SEQ. ID. NO.:7), (SEQ. ID. NO.:8), (SEQ. ID. NO.:9), (SEQ. ID. NO.:10), (SEQ. ID. NO.:10), (SEQ. ID. NO.:11), (SEQ. ID. NO.:12), (SEQ. ID. NO.:13), (SEQ. ID. NO.:14), (SEQ. ID. NO.:15), (SEQ. ID. NO.:16), (SEQ. ID. NO.:17), (SEQ. ID. NO.:18), (SEQ. ID. NO.:19), (SEQ. ID. NO.:20), (SEQ. ID. NO.:21), (SEQ. ID. NO.:22), (SEQ. ID. NO.:23), (SEQ. ID. NO.:24), (SEQ. ID. NO.:25), (SEQ. ID. NO.:26), (SEQ. ID. NO.:27), (SEQ. ID. NO.:28), (SEQ. ID. NO.:29), (SEQ. ID. NO.:30), (SEQ. ID. NO.:31), (SEQ. ID. NO.:32), (SEQ. ID. NO.:35), (SEQ. ID. NO.:36), (SEQ. ID. NO.:37), (SEQ. ID. NO.:38), (SEQ. ID. NO.:39), (SEQ. ID. NO.:40), (SEQ. ID. NO.:41), (SEQ. ID. NO.:42), (SEQ. ID. NO.:43), (SEQ. ID. NO.:44), (SEQ. ID. NO.:45),(SEQ. ID. NO.:46), (SEQ. ID. NO.:47), (SEQ. ID. NO.:48), (SEQ. ID. NO.:49), (SEQ. ID. NO.:50), (SEQ. ID. NO.:51), (SEQ. ID. NO.:52), (SEQ. ID. NO.:53), (SEQ. ID. NO.:54), (SEQ. ID. NO.:55), (SEQ. ID. NO.:56), (SEQ. ID. NO.:57), (SEQ. ID. NO.:58), (SEQ. ID. NO.:59), (SEQ. ID. NO.:60), (SEQ. ID. NO.:61), (SEQ. ID. NO.:62), (SEQ. ID. NO.:63), (SEQ. ID. NO.:64), (SEQ. ID. NO.:65), (SEQ. ID. NO.:66), (SEQ. ID. NO.:67), (SEQ. ID. NO.:68), (SEQ. ID. NO.:69), (SEQ. ID. NO.:70), (SEQ. ID. NO.:71), (SEQ. ID. NO.:72), (SEQ. ID. NO.:73), (SEQ. ID. NO.:74), (SEQ. ID. NO.:75), (SEQ. ID. NO.:76), (SEQ. ID. NO.:77), (SEQ. ID. NO.:78), or (SEQ. ID. NO.:79), or comprising a sequence overlapping the sequence of any of these with respect to its position on the Nickerson reference sequence.

### Claim 11 (depends on 5)

The method of claim 5, wherein said oligonucleotide primer is labeled with a fluorescent dye.

### Claim 12 (depends on 11)

The method of claim 11, wherein said dye is SYBR Green I, YO-PRO-1, thiazole orange, Hex, pico green, edans, fluorescein, FAM, or TET.

### Claim 13

A method of detecting a genetic predisposition for non-responsiveness to lovastatin, pravastatin, or simvastatin treatment in a human subject with coronary artery disease, comprising:
a) collecting a tissue sample from a human subject;
b) amplifying nucleic acids comprising the HindIII restriction site in intron 8 of the human lipoprotein lipase gene (LPL) from said tissue sample to obtain amplification products; and
c) analyzing the amplification products for the absence of a HindIII recognition site in intron 8 of the human lipoprotein lipase gene, homozygosity for an absence of said HindIII recognition site indicating a genetic predisposition in said human subject for non-responsiveness to lovastatin, pravastatin, or simvastatin treatment for coronary artery disease.

### Claim 14 (depends on 13)

The method of claim 13, wherein the tissue sample is a blood sample.

### Claim 15 (depends on 13)

The method of claim 13, further comprising restricting the amplification products with a restriction enzyme before analyzing the amplification products.

### Claim 16 (depends on 13)

The method of claim 13, wherein the restriction enzyme is HindIII.

### Claim 17 (depends on 13)

The method of claim 13, wherein an oligonucleotide primer is used in amplifying said nucleic acids.

### Claim 18 (depends on 13)

The method of claim 13, comprising the sequence GCA TCT GCC TTC AGC TAG ACA TTG (SEQ. ID. NO. 1) is used in amplifying said nucleic acids.

### Claim 19 (depends on 13)

The method of claim 13, wherein an oligonucleotide primer comprising the sequence TCT TCC AGA AGG GTG AGA TTC CAA (SEQ. ID. NO. 2) is used in amplifying said nucleic acids.

### Claim 20 (depends on 13)

The method of claim 13, wherein an oligonucleotide primer having the sequence GCA TCT GCC TTC AGC TAG ACA TTG (SEQ. ID. NO. 1) or TCT TCC AGA AGG GTG AGA TTC CAA (SEQ. ID. NO. 2) is used in amplifying said nucleic acids.

### Claim 21 (depends on 13)

The method of claim 13, wherein a reverse oligonucleotide primer having the sequence 5′-GCA TCT GCC TTC AGC TAG ACA TTG-3′ (SEQ. ID. NO. 1) and a forward oligonucleotide primer having the sequence 5′-TCT TCC AGA AGG GTG AGA TTC CAA-3′ (SEQ. ID. NO. 2) are used in amplifying said nucleic acids.

### Claim 22 (depends on 13)

The method of claim 13, wherein an oligonucleotide primer is used in amplifying said nucleic acids, said primer comprising a nucleotide sequence of (SEQ. ID. NO.:1), (SEQ. ID. NO.:2), (SEQ. ID. NO.:3), (SEQ. ID. NO.:4), (SEQ. ID. NO.:5), (SEQ. ID. NO.:6), (SEQ. ID. NO.:7), (SEQ. ID. NO.:8), (SEQ. ID. NO.:9), (SEQ. ID. NO.:10), (SEQ. ID. NO.:11), (SEQ. ID. NO.:12), (SEQ. ID. NO.:13), (SEQ. ID. NO.:14), (SEQ. ID. NO.:15), (SEQ. ID. NO.:16), (SEQ. ID. NO.:17), (SEQ. ID. NO.:18), (SEQ. ID. NO.:19), (SEQ. ID. NO.:20), (SEQ. ID. NO.:21), (SEQ. ID. NO.22), (SEQ. ID. NO.:23), (SEQ. ID. NO.:24), (SEQ. ID. NO.:25), (SEQ. ID. NO.:26), (SEQ. ID. NO.27), (SEQ. ID. NO.:28), (SEQ. ID. NO.:29), (SEQ. ID. NO.:30), (SEQ. ID. NO.:31), (SEQ. ID. NO.:32), (SEQ. ID. NO.:35), (SEQ. ID. NO.:36), (SEQ. ID. NO.:37), (SEQ. ID. NO.:38), (SEQ. ID. NO.:39), (SEQ. ID. NO.:40), (SEQ. ID. NO.:41), (SEQ. ID. NO.:42), (SEQ. ID. NO.:43), (SEQ. ID. NO.:44), (SEQ. ID. NO.:45),(SEQ. ID. NO.:46), (SEQ. ID. NO.:47), (SEQ. ID. NO.:48), (SEQ. ID. NO.:49), (SEQ. ID. NO.:50), (SEQ. ID. NO.:51), (SEQ. ID. NO.:52), (SEQ. ID. NO.:53), (SEQ. ID. NO.:54), (SEQ. ID. NO.:55), (SEQ. ID. NO.:56), (SEQ. ID. NO.:57), (SEQ. ID. NO.:58), (SEQ. ID. NO.:59), (SEQ. ID. NO.:60), (SEQ. ID. NO.:61), (SEQ. ID. NO.:62), (SEQ. ID. NO.:63), (SEQ. ID. NO.:64), (SEQ. ID. NO.:65), (SEQ. ID. NO.:66), (SEQ. ID. NO.:67), (SEQ. ID. NO.:68), (SEQ. ID. NO.:69), (SEQ. ID. NO.:70), (SEQ. ID. NO.:71), (SEQ. ID. NO.:72), (SEQ. ID. NO.:73), (SEQ. ID. NO.:74), (SEQ. ID. NO.:75), (SEQ. ID. NO.:76), (SEQ. ID. NO.:77), (SEQ. ID. NO.:78), or (SEQ. ID. NO.:79), or comprising a sequence overlapping the sequence of any of these with respect to its position on the Nickerson reference sequence.

### Claim 23 (depends on 13)

The method of claim 13, wherein said oligonucleotide primer is labeled with a fluorescent dye.

### Claim 24 (depends on 23)

The method of claim 23, wherein said dye is SYBR Green I, YO-PRO-1, thiazole orange, Hex, pico green, edans, fluorescein, FAM, or TET.

### Claim 25 (depends on 13)

The method of claim 13, wherein said human subject is a coronary artery bypass graft patient.

### Claim 26

A method of detecting genetic predisposition in a CABG patient for non-responsiveness to treatment with a statin drug selected from the group consisting of lovastatin, pravastatin, and simvastatin, comprising:
a) collecting a tissue sample from a CABG patient;
b) amplifying nucleic acids comprising the locus of the HindIII recognition site in intron 8 of the human lipoprotein lipase (LPL) gene from said blood sample to obtain amplification products; and
c) analyzing the amplification products for the absence of the HindIII recognition site in intron 8 of the human lipoprotein lipase gene, homozygosity for an absence of said HindIII recognition site indicating a genetic predisposition in said CABG patient for non-responsiveness to treatment for coronary artery disease with a statin drug selected from the group consisting of lovastatin, pravastatin, and simvastatin.

### Claim 27 (depends on 26)

The method of claim 26, wherein said tissue sample is a blood sample.

### Claim 28 (depends on 26)

The method of claim 26, further comprising restricting the amplification products with a restriction enzyme before analyzing the amplification products.

### Claim 29 (depends on 28)

The method of claim 28, wherein the restriction enzyme is HindIII.

### Claim 30 (depends on 26)

The method of claim 26, wherein an oligonucleotide primer is used in amplifying said nucleic acids.

### Claim 31 (depends on 26)

The method of claim 26, comprising the sequence GCA TCT GCC TTC AGC TAG ACA TTG (SEQ. ID. NO. 1) is used in amplifying said nucleic acids.

### Claim 32 (depends on 26)

The method of claim 26, wherein an oligonucleotide primer comprising the sequence TCT TCC AGA AGG GTG AGA TTC CAA (SEQ. ID. NO. 2) is used in amplifying said nucleic acids.

### Claim 33 (depends on 26)

The method of claim 26, wherein an oligonucleotide primer having the sequence GCA TCT GCC TTC AGC TAG ACA TTG (SEQ. ID. NO. 1) or TCT TCC AGA AGG GTG AGA TTC CAA (SEQ. ID. NO. 2) is used in amplifying said nucleic acids.

### Claim 34 (depends on 26)

The method of claim 26, wherein a reverse oligonucleotide primer having the sequence 5′-GCA TCT GCC TTC AGC TAG ACA TTG-3′ (SEQ. ID. NO. 1) and a forward oligonucleotide primer having the sequence 5′-TCT TCC AGA AGG GTG AGA TTC CAA-3′ (SEQ. ID. NO. 2) are used in amplifying said nucleic acids.

### Claim 35 (depends on 26)

The method of claim 26, wherein an oligonucleotide primer is used in amplifying said nucleic acids, said primer comprising a sequence of (SEQ. ID. NO.:1), (SEQ. ID. NO.:2), (SEQ. ID. NO.:3), (SEQ. ID. NO.:4), (SEQ. ID. NO.:5), (SEQ. ID. NO.:6), (SEQ. ID. NO.:7), (SEQ. ID. NO.:8), (SEQ. ID. NO.:9), (SEQ. ID. NO.:10), (SEQ. ID. NO.:11), (SEQ. ID. NO.:12), (SEQ. ID. NO.:13), (SEQ. ID. NO.:14), (SEQ. ID. NO.:15), (SEQ. ID. NO.:16), (SEQ. ID. NO.:17), (SEQ. ID. NO.:18), (SEQ. ID. NO.:19), (SEQ. ID. NO.:20), (SEQ. ID. NO.:21), (SEQ. ID. NO.:22), (SEQ. ID. NO.:23), (SEQ. ID. NO.:24), (SEQ. ID. NO.:25), (SEQ. ID. NO.:26), (SEQ. ID. NO.:27), (SEQ. ID. NO.:28), (SEQ. ID. NO.:29), (SEQ. ID. NO.:30), (SEQ. ID. NO.:31), (SEQ. ID. NO.:32), (SEQ. ID. NO.:35), (SEQ. ID. NO.:36), (SEQ. ID. NO.:37), (SEQ. ID. NO.:38), (SEQ. ID. NO.:39), (SEQ. ID. NO.:40), (SEQ. ID. NO.:41), (SEQ. ID. NO.:42), (SEQ. ID. NO.:43), (SEQ. ID. NO.:44), (SEQ. ID. NO.:45),(SEQ. ID. NO.:46), (SEQ. ID. NO.:47), (SEQ. ID. NO.:48), (SEQ. ID. NO.:49), (SEQ. ID. NO.:50), (SEQ. ID. NO.:51), (SEQ. ID. NO.:52), (SEQ. ID. NO.:53), (SEQ. ID. NO.:54), (SEQ. ID. NO.:55), (SEQ. ID. NO.:56), (SEQ. ID. NO.:57), (SEQ. ID. NO.:58), (SEQ. ID. NO.:59), (SEQ. ID. NO.:60), (SEQ. ID. NO.:61), (SEQ. ID. NO.:62), (SEQ. ID. NO.:63), (SEQ. ID. NO.:64), (SEQ. ID. NO.:65), (SEQ. ID. NO.:66), (SEQ. ID. NO.:67), (SEQ. ID. NO.:68), (SEQ. ID. NO.:69), (SEQ. ID. NO.:70), (SEQ. ID. NO.:71), (SEQ. ID. NO.:72), (SEQ. ID. NO.:73), (SEQ. ID. NO.:74), (SEQ. ID. NO.:75), (SEQ. ID. NO.:76), (SEQ. ID. NO.:77), (SEQ. ID. NO.:78), or (SEQ. ID. NO.:79), or comprising a sequence overlapping the sequence of any of these with respect to its position on the Nickerson reference sequence.

### Claim 36 (depends on 30)

The method of claim 30, wherein said oligonucleotide primer is labeled with a fluorescent dye.

### Claim 37 (depends on 36)

The method of claim 36, wherein said dye is SYBR Green I, YO-PRO-1, thiazole orange, Hex, pico green, edans, fluorescein, FAM, or TET.

### Claim 38

A method of detecting genetic predisposition in a CABG for non-responsiveness to treatment with lovastatin, pravastatin, or simvastatin, comprising:
a) collecting a tissue sample from a CABG patient,
b) amplifying nucleic acids comprising the normal locus of the HindIII recognition site in intron 8 of the human lipoprotein lipase (LPL) gene from said blood sample to obtain amplification products, by using a reverse oligonucleotide primer having the sequence 5′-GCA TCT GCC TTC AGC TAG ACA TTG-3′ (SEQ. ID. NO. 1) and a forward oligonucleotide primer having the sequence 5′-TCT TCC AGA AGG GTG AGA TTC CAA-3′ (SEQ. ID. NO. 2); and
c) analyzing the amplification products for the absence of the HindIII recognition site in intron 8 of the human lipoprotein lipase gene, homozygosity for an absence of said HindIII recognition site indicating a genetic predisposition in said CABG patient for non-responsiveness to treatment for coronary artery disease with lovastatin, pravastatin, or simvastatin.

### Claim 39 (depends on 38)

The method of claim 38, wherein said tissue sample is a blood sample.

### Claim 40 (depends on 38)

The method of claim 38, wherein said oligonucleotide primer is labeled with a fluorescent dye.

### Claim 41 (depends on 40)

The method of claim 40, wherein said dye is SYBR Green I, YO-PRO-1, thiazole orange, Hex, pico green, edans, fluorescein, FAM, or TET.

### Claim 42

A method of detecting in a human subject a genetic predisposition for non-responsiveness to treatment for coronary artery disease with a statin drug selected from the group consisting of lovastatin, pravastatin, or simvastatin, said method comprising:
a) collecting a tissue sample from a human subject;
b) amplifying nucleic acids comprising the normal locus of the HindIII recognition site in intron 8 of the human lipoprotein lipase (LPL) gene from said blood sample to obtain amplification products, by using a reverse oligonucleotide primer having the sequence 5′-GCA TCT GCC TTC AGC TAG ACA TTG-3′ (SEQ. ID. NO. 1) and a forward oligonucleotide primer having the sequence 5′-TCT TCC AGA AGG GTG AGA TTC CAA-3′ (SEQ. ID. NO. 2);
c) restricting said amplification products with HindIII; and
d) analyzing the restriction fragments for the absence of the HindIII recognition site in intron 8 of the human lipoprotein lipase gene,
wherein homozygosity for an absence of said HindIII recognition site indicates a genetic predisposition for non-responsiveness to treatment for coronary artery disease with a statin drug selected from the group consisting of lovastatin, pravastatin, or simvastatin.

### Claim 43 (depends on 42)

The method of claim 42, wherein the tissue sample is a blood sample.

### Claim 44 (depends on 42)

The method of claim 42, wherein said oligonucleotide primer is labeled with a fluorescent dye.

### Claim 45 (depends on 42)

The method of claim 42, wherein said dye is SYBR Green I, YO-PRO-1, thiazole orange, Hex, pico green, edans, fluorescein, FAM, or TET.

### Claim 46

A method of detecting a genetic predisposition in a human subject for non-responsiveness to treatment with a statin drug selected from the group consisting of lovastatin, pravastatin, or simvastatin, said method comprising:
a) collecting a tissue sample from a human subject;
b) amplifying nucleic acids comprising the normal locus of the (TTTA)n tetranucleotide repeat sequence in intron 6 of the human lipoprotein lipase gene (LPL) from said tissue sample to obtain amplification products; and
c) analyzing the amplification products for (TTTA)n tetranucleotide repeat alleles present in said nucleic acids, homozygosity for a (TTTA)n 4 allele indicating a genetic predisposition for non-responsiveness to treatment for coronary artery disease with a statin drug selected from the group consisting of lovastatin, pravastatin, or simvastatin.

### Claim 47 (depends on 46)

The method of claim 46, wherein the tissue sample is a blood sample.

### Claim 48 (depends on 46)

The method of claim 46, wherein an oligonucleotide primer is used in amplifying said nucleic acids.

### Claim 49 (depends on 46)

The method of claim 46, comprising the sequence 5′-CCT GGG TAA CTG AGC GAG ACT GTG TC-3′ (SEQ. ID. NO.:33) is used in amplifying said nucleic acids.

### Claim 50 (depends on 46)

The method of claim 46, wherein an oligonucleotide primer comprising the sequence 5′- ATC TGA CCA AGG ATA GTG GGA TAT A-3′ (SEQ. ID. NO.:34) is used in amplifying said nucleic acids.

### Claim 51 (depends on 46)

The method of claim 46, wherein an oligonucleotide primer having the sequence 5′-CCT GGG TAA CTG AGC GAG ACT GTG TC-3′ (SEQ. ID. NO.:33) or 5′-ATC TGA CCA AGG ATA GTG GGA TAT A-3′ (SEQ. ID. NO.:34) is used in amplifying said nucleic acids.

### Claim 52 (depends on 46)

The method of claim 46, wherein a reverse oligonucleotide primer having the sequence 5′-CCT GGG TAA CTG AGC GAG ACT GTG TC-3′ (SEQ. ID. NO.:33) and a forward oligonucleotide primer having the sequence 5′-ATC TGA CCA AGG ATA GTG GGA TAT A-3′ (SEQ. ID. NO.:34) are used in amplifying said nucleic acids.

### Claim 53 (depends on 46)

The method of claim 46, wherein amplifying said nucleic acids is done using an oligonucleotide primer comprising a nucleotide sequence of (SEQ. ID. NO.:33), (SEQ. ID. NO.:34), (SEQ. ID. NO.:82), (SEQ. ID. NO.:83), (SEQ. ID. NO.:84), (SEQ. ID. NO.:85), (SEQ. ID. NO.:86), (SEQ. ID. NO.:87), (SEQ. ID. NO.:88), (SEQ. ID. NO.:89), (SEQ. ID. NO.:90), (SEQ. ID. NO.:91), or (SEQ. ID. NO.:92), or comprising a sequence overlapping the sequence of any of these with respect to its position on the Nickerson reference sequence.

### Claim 54 (depends on 53)

The method of claim 53, wherein said oligonucleotide primer is labeled with a fluorescent dye.

### Claim 55 (depends on 54)

The method of claim 54, wherein said dye is SYBR Green I, YO-PRO-1, thiazole orange, Hex, pico green, edans, fluorescein, FAM, or TET.