U.S. patent application number 11/358570 was filed with the patent office on 2006-06-22 for real-time polymerase chain reaction-based genotyping assay for single nucleotide polymorphism.
This patent application is currently assigned to The University of Tennessee Research Foundation. Invention is credited to Dick Gourley, Duane Miller, Pengfei Song, Charles R. Yates.
Application Number | 20060134689 11/358570 |
Document ID | / |
Family ID | 33299622 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134689 |
Kind Code |
A1 |
Yates; Charles R. ; et
al. |
June 22, 2006 |
Real-time polymerase chain reaction-based genotyping assay for
single nucleotide polymorphism
Abstract
The present invention provides fluorescence-based real-time PCR
assays for the rapid detection of single nucleotide polymorphisms
(SNPs). The genotyping assay can be used to detect SNPs of a number
of genes of interest that include, but are not limited to, the
human multidrug resistance gene (MDR1) single nucleotide
polymorphisms C3435T and G2677T, and cytochrome P-450 3A5 single
nucleotide polymorphisms CYP3A5*3 (A22893G) and CYP3A5*6
(G30597A).
Inventors: |
Yates; Charles R.; (Memphis,
TN) ; Miller; Duane; (Memphis, TN) ; Gourley;
Dick; (Memphis, TN) ; Song; Pengfei; (Memphis,
TN) |
Correspondence
Address: |
Benjamin Aaron Adler, Ph.D., J.D.;Adler & Associates
8011 Candle Lane
Houston
TX
77071
US
|
Assignee: |
The University of Tennessee
Research Foundation
|
Family ID: |
33299622 |
Appl. No.: |
11/358570 |
Filed: |
February 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10809757 |
Mar 25, 2004 |
7018816 |
|
|
11358570 |
Feb 21, 2006 |
|
|
|
60457512 |
Mar 25, 2003 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
536/23.2 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/156 20130101 |
Class at
Publication: |
435/006 ;
536/023.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04 |
Claims
1. An isolated DNA molecule useful as a primer for genotyping human
multidrug resistance gene (MDR1) single nucleotide polymorphism
G2677T, said DNA is selected from the group consisting of SEQ ID
NOs: 4-6.
2. A kit for genotyping human multidrug resistance gene (MDR1)
single nucleotide polymorphism G2677T, comprising primers having
the DNA sequences of claim 1.
3. A method of genotyping human multidrug resistance gene (MDR1)
single nucleotide polymorphism G2677T, comprising the steps of:
preparing DNA samples from an individual; amplifying said DNA with
primers SEQ ID NOs: 4 and 6 or SEQ ID NOs: 5 and 6 of claim 1; and
identifying the products of said DNA amplification, wherein the
presence of products amplified by primers SEQ ID NOs: 5 and 6
indicate said individual has the genotype G2677T.
4. The method of claim 3, wherein said products of DNA
amplification are identified by a method selected from the group
consisting of real-time fluorescence-based analysis, melt curve
analysis and gel electrophoresis.
5. The method of claim 4, wherein said gel electrophoresis
identifies a product of 216 base pairs that correspond to genotype
G2677T.
6. An isolated DNA molecule useful as a primer for genotyping human
cytochrome P-450 3A5 single nucleotide polymorphism CYP3A5*3, said
DNA is selected from the group consisting of SEQ ID NOs: 11-13.
7. A kit for genotyping human cytochrome P-450 3A5 single
nucleotide polymorphism CYP3A5*3, comprising primers having the DNA
sequences of claim 6.
8. A method of genotyping human cytochrome P-450 3A5 single
nucleotide polymorphism CYP3A5*3, comprising the steps of:
preparing DNA samples from an individual; amplifying said DNA with
primers SEQ ID NOs: 11 and 13 or SEQ ID NOs: 12 and 13 of claim 6;
and identifying the products of said DNA amplification, wherein the
presence of products amplified by primers SEQ ID NOs: 12 and 13
indicate said individual has the genotype CYP3A5*3.
9. The method of claim 8, wherein said products of DNA
amplification are identified by a method selected from the group
consisting of real-time fluorescence-based analysis, melt curve
analysis and gel electrophoresis.
10. The method of claim 9, wherein said gel electrophoresis
identifies a product of 238 base pairs that correspond to genotype
CYP3A5*3.
11. An isolated DNA molecule useful as a primer for genotyping
human cytochrome P-450 3A5 single nucleotide polymorphism CYP3A5*6,
said DNA is selected from the group consisting of SEQ ID NOs:
14-16.
12. A kit for genotyping human cytochrome P-450 3A5 single
nucleotide polymorphism CYP3A5*6, comprising primers having the DNA
sequences of claim 11.
13. A method of genotyping human cytochrome P-450 3A5 single
nucleotide polymorphism CYP3A5*6, comprising the steps of:
preparing DNA samples from an individual; amplifying said DNA with
primers SEQ ID NOs: 14 and 16 or SEQ ID NOs: 15 and 16 of claim 11;
and identifying the products of said DNA amplification, wherein the
presence of products amplified by primers SEQ ID NOs: 15 and 16
indicate said individual has the genotype CYP3A5*6.
14. The method of claim 13, wherein said products of DNA
amplification are identified by a method selected from the group
consisting of real-time fluorescence-based analysis, melt curve
analysis and gel electrophoresis.
15. The method of claim 14, wherein said gel electrophoresis
identifies a product of 273 base pairs that correspond to genotype
CYP3A5*6.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of non-provisional
application U.S. Ser. No. 10/809,757, filed Mar. 25, 2004 which
claims benefit of provisional U.S. Ser. No. 60/457,512, filed Mar.
25, 2003, now abandoned.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the field of
single nucleotide polymorphism genotyping. More specifically, the
present invention provides a real-time polymerase chain
reaction-based genotyping assay for the detection of single
nucleotide polymorphisms.
[0004] 2. Description of the Related Art
[0005] P-glycoprotein (P-gp), a member of the large adenosine
triphosphate-binding (ATP-binding) cassette superfamily of
transport proteins also called traffic ATPases, is the product of
the human multidrug resistance gene (MDR1). P-glycoprotein is
highly expressed on the apical (luminal) surface of organs that
have excretory functions, such as the bile canalicular membrane of
hepatocytes and the renal proximal tubule. Moreover, P-gp is
significantly expressed on the luminal surface of tissues that
serve as barriers, such as the brush border of the small intestine
and the capillary endothelial cells of the blood-brain barrier.
[0006] Tissue distribution suggests that P-gp protects the body
from toxic xenobiotics by secreting them into the bile, urine, and
intestinal lumen and by reducing their accumulation in the brain
and testes. As a result, interindividual variability in the
disposition of numerous drugs has been ascribed to differences in
P-gp expression. It has been reported that intestinal P-gp
expression accounted for approximately 30% of interindividual
variability in the maximal plasma concentration after oral
administration of cyclosporine.
[0007] A novel P-gp aberrant allele, MDR1*2, linked to 2 synonymous
single nucleotide polymorphisms (SNPs) (C1236T in exon 12 and
C3435T in exon 26) and a nonsynonymous single nucleotide
polymorphism in exon 21 (G2677T, Ala893Ser) was recently described
(Kim et al., 2001). The single nucleotide polymorphisms found on
exons 12, 21, and 26 are not strictly allelic; however, they
exhibit strong linkage disequilibrium and account for a majority of
the described haplotypes (Kim et al., 2001; Tang et al., 2002).
MDR1*2 was found to be associated with altered fexofenadine
disposition. Individuals carrying 2 wild type alleles (*1/*1) had a
40% greater fexofenadine systemic exposure after oral
administration compared with individuals heterozygous or homozygous
for MDR1*2. Reduced fexofenadine systemic exposure in carriers of
the MDR1*2 allele potentially results in reduced therapeutic
benefit after oral administration of fexofenadine.
[0008] Kim et al. (2001) reported significant ethnic differences in
MDR1*2 allelic frequency, with 62% and 13% of European Americans
and African Americans, respectively, carrying at least one MDR1*2
allele. Thus, polymorphic MDR1 expression may contribute to
interracial variability in drug disposition. Unfortunately,
attempts to determine the association between polymorphic P-gp
expression and drug disposition have yielded equivocal results.
[0009] To facilitate clarification of the significance of commonly
occurring MDR1 single nucleotide polymorphisms and their ethnic
frequency on drug disposition, a rapid and robust polymerase chain
reaction-based (PCR-based) screening method for the single
nucleotide polymorphisms C3435T and G2677T would be highly
desirable. The present invention fulfills this longstanding need
and desire in the art.
SUMMARY OF THE INVENTION
[0010] The present invention provides a real-time polymerase chain
reaction (PCR)-based method to detect single nucleotide
polymorphisms. Discrimination between wild type and mutant alleles
was achieved using PCR amplification of specific alleles modified
to prevent non-Watson Crick base pairing (Okimoto & Dodgson,
1996; Sommer et al., 1992; Bottema et al., 1993; Newton et al.,
1989). Two key nucleotide mismatches are required for alleleic
discrimination. The first nucleotide difference between primers
used to discriminate between wild type and mutant alleles was
located at the 3' terminal base. However, a single base pair
difference at the 3' end of the primer is insufficient, in most
cases, to achieve allelic discrimination. An additional internal
nucleotide mismatch (typically within 5 base pairs of the 3' end)
is required for specific amplification of either the wild-type or
mutant allele. Thus, a second nucleotide mismatch located three
bases from the 3' end for both the wild-type and mutant-specific
primers was included to generate an internal primer/template
mismatch that prevents amplification of the nonmatching primer.
[0011] In one embodiment, the present invention provides a
genotyping assay to detect MDR1 (human multidrug resistance gene)
single nucleotide polymorphisms (SNPs) C3435T and G2677T. C3435T
and G2677T are linked to MDR1*2, which is associated with enhanced
efflux activity in vitro and in vivo. PCR reactions for genotyping
C3435T and G2677T using allele-specific primers were conducted in
separate tubes. PCR amplification was monitored by Smart Cycler
(Cepheid, Sunnyvale, Calif.) using SYBR.TM. Green I, a non-specific
double stranded DNA intercalating fluorescent dye. PCR growth
curves exceeding the threshold cycle were considered positive.
Fluorescence melt-curve analysis was used to corroborate results
from PCR growth curves.
[0012] Using PCR growth curves, the assay disclosed herein
accurately determined hetero- and homozygosity for C3435T and
G2677T. Genotype assignments based on PCR growth curve, melt-curve
analysis, agarose gel electrophoresis, and direct DNA sequencing
results of PCR products were in perfect agreement. Thus, the
present invention provides a rapid MDR1 genotyping method that can
be used to assess the contribution of MDR1*2 to pharmacokinetic and
pharmacodynamic variability of P-gp substrates.
[0013] In another embodiment, the above described real-time
PCR-based method was used to detect cytochrome P-450 3A5 (CYP3A5)
single nucleotide polymorphisms CYP3A5*3 (A22893G) and CYP3A5*6
(G30597A).
[0014] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention. These
embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-1D show MDR1 C3435T allelic discrimination by
real-time analysis using the Smart Cycler. Plot of fluorescence
versus cycle number using human genomic DNA obtained from
individuals with CC (FIG. 1A), TT (FIG. 1B), or CT (FIG. 1C)
genotypes and nontemplate control (FIG. 1D). Interrogation for the
presence of either the C or T allele was conducted in physically
separate tubes using the common reverse primer 3435R coupled with
either the wild type specific primer 3435W (-.diamond-solid.-,
blue) or the mutant-specific primer 3435M (-.circle-solid.-, pink).
PCR growth curves that exceed the threshold fluorescence indicate
specific PCR product formation.
[0016] FIGS. 2A-2D show melt curve analysis of PCR products using
SYBR.TM. Green I. Melt curves were converted to melt peaks by
plotting the negative first derivative of the fluorescence versus
temperature ([-dF/dt]). Plot of [-dF/dt] versus temperature
obtained after amplification of CC (FIG. 2A), TT (FIG. 2B), CT
(FIG. 2C) genomic DNA and nontemplate control (FIG. 2D) using the
common reverse primer 3435R coupled with either the wild type
specific primer 3435W or the mutant-specific primer 3435M. The melt
temperature (T.sub.m=84.degree. C.) was identical for PCR products
formed using either the wild type or mutant-specific primers.
[0017] FIG. 3 shows MDR1 C3435T allelic discrimination by
conventional modified allele-specific PCR. An ethidium
bromide-stained 2% agarose gel containing PCR fragments (134 bp)
was run to confirm real-time PCR results. Odd-numbered lanes
contain PCR fragments after amplification with 3435R and 3435W.
Even-numbered lanes contain PCR fragments after amplification with
3435R and 3435M. PCR products amplified from genomic DNA with
different 3435 genotypes were loaded as follows: CC (lanes 1 and
2), TT (lanes 3 and 4), and CT (lanes 5 and 6). Lanes 7 and 8
represent nontemplate controls. Lane marker contains a 100-bp DNA
ladder.
[0018] FIGS. 4A-4C show CYP3A5 allelic discrimination using human
genomic DNA as template. Human genomic DNA obtained from
individuals with *1/*1 (FIG. 4A), *3/*3 (FIG. 4B), or *1/*3 (FIG.
4C) genotype interrogated for the presence of either the *1 or *3
allele using wild-type (-.diamond-solid.-, blue) and
mutant-specific (-.diamond-solid.-, orange) primers.
[0019] FIGS. 5A-5C show melting curve analysis of PCR products
using SYBR.TM. Green I. Negative first derivative of fluorescence
versus temperature curves for PCR products obtained after
amplification of *1/*1 (FIG. 5A), *3/*3 (FIG. 5B), or *1/*3 (FIG.
5C) genomic DNA using wild-type (-.diamond-solid.-, blue) and
mutant-specific (-.diamond-solid.-, orange) primers.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Fluorescence-based single nucleotide polymorphism detection
assays offer several important advantages over traditional PCR
approaches used to determine genotype (e.g. sequencing of PCR
products and restriction fragment length polymorphism, RFLP).
First, RFLP can in some instances result in significant false
positive rates as a result of incomplete restriction enzyme
digestion or the presence of other mutations close to the mutation
of interest. Second, fluorescence-based genotyping assays are more
amenable to high-throughput screening, as they do not require
extensive post-amplification manipulation.
[0021] Commonly used fluorescence-based PCR techniques for single
nucleotide polymorphism detection include the use of either the
nonspecific DNA intercalating dye SYBR.TM. Green I or an
allele-specific fluorogenic probe (i.e. Taqman). In many instances,
the use of SYBR.TM. Green I is more cost-effective when applied to
haplotype analysis of genes with multiple allelic variants since it
does not require the synthesis of numerous allele-specific
fluorogenic probes.
[0022] In the present invention, the use of allele-specific primers
containing an additional internal mismatch obviates the need for
extensive optimization of PCR amplification conditions associated
with traditional PCR amplification of specific alleles. The
inventors have successfully applied the approach described here to
genotype 11 other single nucleotide polymorphisms using, in most
instances, identical PCR amplification conditions.
[0023] Current methods for genotyping MDR1 or CYP3A5 include PCR
amplification followed by sequencing and fluorogenic probe-based
PCR assays. The simple, rapid, inexpensive, reproducible, and
reliable real-time PCR genotyping methods presented here constitute
a significant improvement over current techniques. Using this
approach, genotyping results can be obtained within 2 hours of
whole blood or tissue procurement. Importantly, these techniques
are also applicable in laboratories lacking access to real-time PCR
equipment since allelic discrimination can be determined using
traditional PCR and agarose gel electrophoresis.
[0024] Thus, the present invention is directed to a method of
genotyping MDR1 single nucleotide polymorphism C3435T, comprising
the step of amplifying DNA samples with primers SEQ ID NOs: 1 and 3
or SEQ ID NOs: 2 and 3. The presence of DNA products amplified by
primers SEQ ID NOs: 2 and 3 would indicate the individual has the
genotype C3435T. In general, the amplified DNA products can be
identified by real-time fluorescence-based analysis, melt curve
analysis or gel electrophoresis. In the case of gel
electrophoresis, the presence of a 134 base pairs product
corresponds to genotype C3435T.
[0025] In another embodiment, there is provided a method of
genotyping MDR1 single nucleotide polymorphism G2677T, comprising
the step of amplifying DNA samples with primers SEQ ID NOs: 4 and 6
or SEQ ID NOs: 5 and 6. The presence of DNA products amplified by
primers SEQ ID NOs: 5 and 6 would indicate the individual has the
genotype G2677T. In general, the amplified DNA products can be
identified by real-time fluorescence-based analysis, melt. curve
analysis or gel electrophoresis. In the case of gel
electrophoresis, the presence of a 216 base pairs product
corresponds to genotype G2677T.
[0026] In yet another embodiment, there is provided a method of
genotyping human cytochrome P-450 3A5 single nucleotide
polymorphism CYP3A5*3, comprising the step of amplifying DNA
samples with primers SEQ ID NOs: 11 and 13 or SEQ ID NOs: 12 and
13. The presence of DNA products amplified by primers SEQ ID NOs:
12 and 13 would indicate the individual has the genotype CYP3A5*3.
In general, the amplified DNA products can be identified by
real-time fluorescence-based analysis, melt curve analysis or gel
electrophoresis. In the case of gel electrophoresis, the presence
of a 238 base pairs product corresponds to genotype CYP3A5*3.
[0027] In still yet another embodiment, there is provided a method
of genotyping human cytochrome P-450 3A5 single nucleotide
polymorphism CYP3A5*6, comprising the step of amplifying DNA
samples with primers SEQ ID NOs: 14 and 16 or SEQ ID NOs: 15 and
16. The presence of DNA products amplified by primers SEQ ID NOs:
15 and 16 would indicate the individual has the genotype CYP3A5*6.
In general, the amplified DNA products can be identified by
real-time fluorescence-based analysis, melt curve analysis or gel
electrophoresis. In the case of gel electrophoresis, the presence
of a 273 base pairs product corresponds to genotype CYP3A5*6.
[0028] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion. One skilled in the
art will appreciate readily that the present invention is well
adapted to carry out the objects and obtain the ends and advantages
mentioned, as well as those objects, ends and advantages inherent
herein. Changes therein and other uses which are encompassed within
the spirit of the invention as defined by the scope of the claims
will occur to those skilled in the art.
EXAMPLE 1
MDR1 Single Nucleotide Polymorphisms Genotyping
[0029] The present example describes a real-time PCR assays for the
rapid detection of the MDR1 single nucleotide polymorphisms C3435T
and G2677T. These methods can be readily applied to investigate the
effect of MDR1 polymorphic expression on pharmacokinetic and
pharmacodynamic variability of P-gp substrates.
EXAMPLE 2
Primer Design
[0030] PCR primers are listed in Table 1. Oligonucleotide primers
were designed based on the published MDR1 sequence (Genbank
#AC005068) using the online program Primer3. Hairpin structures and
primer-dimers were predicted with Oligo Toolkit.TM.. The primers
were synthesized by Integrated DNA Technologies (Coralville, Iowa).
Expected amplicon lengths were 216 base pairs (bp) and 134 bp for
G2677T and C3435T, respectively. Discrimination between wild type
and mutant alleles was achieved using PCR amplification of specific
alleles modified to prevent non-Watson Crick base pairing (Okimoto
& Dodgson, 1996; Sommer et al., 1992; Bottema et al., 1993;
Newton et al., 1989). Briefly, the first nucleotide difference (C
or T) between sense primers (3435W and 3435M) used to discriminate
between wild type and mutant 3435 alleles is located at the 3'
terminal base. The second primer base change (A to G) located 3
bases from the 3' end generates an internal primer/template
mismatch, and this prevents amplification of the nonmatching
primer. These changes were made to prevent the generation of
possible spurious products, which could otherwise occur by the
annealing and extension of the 3435W primer to the first-round
product of 3435M. A similar strategy was used to achieve allelic
discrimination for G2677T (Table 1).
EXAMPLE 3
Real-Time PCR Amplification
[0031] The Smart Cycler (Cepheid, Sunnyvale, Calif.) was used to
monitor PCR amplification using SYBR.TM. Green I (Molecular Probes,
Eugene, Oreg.), a nonspecific double-stranded DNA intercalating
fluorescent dye. Thus, to achieve allelic discrimination between
wild type and mutant alleles, two physically separate PCR reactions
containing either wild type or mutant-specific primers were
performed. All reactions were carried out in a total volume of 25
.mu.L. Reaction conditions were identical for G2677T and C3435T
except where noted. Each reaction mixture contained a 1:12,500
dilution of SYBR.TM. Green I nucleic acid gel stain 10,000.times.
in dimethyl sulfoxide (DMSO) (Molecular Probes); 0.2 mM of dATP,
dCTP, dGTP, and dTTP; 200 nM of both forward and reverse primers;
1.0 U of Taq DNA polymerase (Promega, Madison, Wis.); 6% DMSO; and
20 to 120 ng of genomic DNA in 1.times.PCR buffer (pH 8.3,
10.times. solution containing 100 mM Tris-HCl, 500 mM KCl, 15 mM
MgCl2 and 0.01% gelatin) (Sigma, St. Louis, Mo.). Genomic DNA was
obtained from the Human Genetic Cell Repository, sponsored by the
National Institute of General Medical Sciences.
[0032] The amplification program for both G2677T and C3435T
consisted of 1 cycle of 95.degree. C. with 120-second hold followed
by 27 cycles of 95.degree. C. with 6-second hold, specified
annealing temperature of 62.degree. C. with 15-second hold, and
72.degree. C. with 20-second hold. After amplification, melt
analysis was performed by heating the reaction mixture from
60.degree. C. to 95.degree. C. at the rate of 0.2.degree. C./s. A
negative control without DNA template was run with every assay to
assess the overall specificity. PCR products for sequencing the
2677 locus were generated using the sense primer
(5'-AAGATTGCTTTGAGGAATGGT-3', SEQ ID NO:7) and the antisense primer
(5'-GCTATAGGTTCCAGGCTTGCT-3', SEQ ID NO:8). PCR products for
sequencing the 3435 locus were generated using the sense primer
(5'-GAGCCCATCCTGTTGACTG-3', SEQ ID NO:9) and the antisense primer
(5'-ACTATAGGCCAGAGAGGCTGC-3', SEQ ID NO:10).
EXAMPLE 4
PCR Product Analysis
[0033] The real-time fluorescence signal generated by the
nonspecific double-stranded DNA binding dye SYBR.TM. Green I was
analyzed using the Smart Cycler software. A threshold cycle (Ct)
was determined for each sample using the exponential growth phase
and the baseline signal from fluorescence versus cycle number
plots. A sample was deemed positive if fluorescence exceeded the
threshold. Threshold fluorescence level was automatically set by
the Smart Cycler software. Melt curves were transformed to the
negative first derivative melt curves ([-dF/dt] vs temperature). In
the melt analysis, the negative first derivative peaks, which are
characteristic of the PCR product melt temperature, were used to
identify specific PCR products. Amplification reactions were
routinely checked for the presence of nonspecific products by
agarose gel electrophoresis. PCR products were isolated by QIAquick
(Qiagen, Valencia, Calif.) after separation by agarose gel
electrophoresis and subjected to direct sequencing using the ABI
Prism Model 3100 (Applied Biosystems, Foster City, Calif.). Genomic
DNA obtained from individuals determined by sequencing to be homo-,
hetero-, and nullizygous for the 2677T and 3435T alleles was used
for genotyping assay development and validation.
EXAMPLE 5
MDR1 Genotyping Results
[0034] Allele-specific primers containing an additional nucleotide
mismatch 3 bases from their 3' termini had little effect on
specific PCR product yield. However, nonspecific PCR product yield
was drastically reduced to undetectable levels. Consequently, PCR
conditions were optimized such that the threshold cycle (Ct) was
exceeded only when specific amplification occurred (i.e., only in
the presence of a primer:template match). FIG. 1A illustrates the
results of the MDR1 C3435T allelic discrimination assay using
homozygous 3435C genomic DNA amplified with a common primer 3435R
and either the wild-type specific primer 3435W or the
mutant-specific primer 3435M. When primers 3435R and 3435W were
used to amplify homozygous 3435C genomic DNA, the PCR growth curve
exceeded the Ct value at approximately 21 cycles (FIG. 1A), and the
melt analysis (negative first derivative) yielded a characteristic
sharp peak at approximately 84.degree. C. for the product (FIG.
2A).
[0035] PCR growth curves remained at approximately background
fluorescence, and no distinct melt analysis peak was noted when
primers 3435R and 3435M were used to amplify homozygous 3435C
genomic DNA (FIGS. 1A and 2A). Agarose gel electrophoresis yielded
the expected 134-bp fragment when homozygous 3435C DNA was
amplified with primers 3435R and 3435W (FIG. 3). However, no bands
were visualized after homozygous 3435C DNA was amplified using
primers 3435R and 3435M (FIG. 3). Similarly, allelic discrimination
was achieved after amplification of homozygous 3435T DNA using
primers 3435R, 3435M, and 3435W (FIGS. 1B, 2B, and 3).
[0036] Overlapping PCR growth curves yielding similar Ct values
were obtained when CT genomic DNA was amplified using wild-type and
mutant-specific primers (FIG. 1C). In addition, a distinct melt
analysis peak was present after amplification with both wild-type
and mutant-specific primers (FIG. 2C). Results from real-time PCR
corroborate conventional PCR results (FIG. 3) and accurately
predict the presence of both wild-type and mutant 3435 alleles in
the heterozygote control. FIGS. 1D and 2D illustrate results from
nontemplate control reaction. Results obtained from optimization
and application of the G2677T genotyping assay to individuals with
GG, GT, and TT genotypes were similar to those reported for C3435T
(data not shown). Melt analysis yielded a characteristic sharp peak
at approximately 80.degree. C. (Table 1).
[0037] The validity of the present methods was verified by testing
20 individuals (10 Caucasians and 10 African Americans) comprising
all 3 G2677T and C3435T genotypes. The genotype distribution was in
Hardy-Weinberg equilibrium. The allele frequency for 2677T was 0.50
and 0.15 for Caucasians and African Americans, respectively. The
allele frequency for 3435T was 0.55 and 0.20 for Caucasians and
African Americans. The allele frequencies for 2677T and 3435T were
similar to those previously reported for Caucasians and African
Americans. Eighteen samples (3 individuals homo-, hetero-, and
nullizygous for either 2677T or 3435T) were sequenced. Sequencing
results were in perfect agreement with real-time PCR results.
TABLE-US-00001 TABLE 1 Primer Sequences For MDR1 Genotyping Primer
.sup.a Sequence .sup.b T.sub.m .sup.c C3435T 84.degree. C. 3435W 5'
43288 GTGGTGTCACAGGAAGAGGTC 3' 43268 (SEQ ID NO:1) 3435M 5' 43288
GTGGTGTCACAGGAAGAGGTT 3' 43268 (SEQ ID NO:2) 3435R 5' 43155
ACTATAGGCCAGAGAGGCTGC 3' 43175 (SEQ ID NO:3) G2677T 80.degree. C.
2677W 5' 65221 AGTTTGACTCACCTTCCCTGC 3' 65241 (SEQ ID NO:4) 2677M
5' 65221 AGTTTGACTCACCTTCCCTGA 3' 65241 (SEQ ID NO:5) 2677C 5'
65436 GCTATAGGTTCCAGGCTTGCT 3' 65416 (SEQ ID NO:6) .sup.a W
indicates wild type specific primer; M, mutant-specific primer; R
and C, common primers used in allelic discrimination assays. .sup.b
Nucleotides shown in bold indicate nucleotides mismatches from
published wild type sequence (Genbank #AC005068). .sup.c Amplicon
melt temperature (T.sub.m) obtained from melt curve analysis.
EXAMPLE 6
CYP3A5 Single Nucleotide Polymorphisms Genotyping
[0038] The present example describes a real-time PCR assays for the
rapid detection of the most prevalent inactivating alleles of the
CYP3A5 gene, CYP3A5*3 and CYP3A5*6. These methods can be readily
applied to determine the effect of CYP3A5 genotype on
inter-individual variability in the pharmacokinetics of CYP3A
substrates.
[0039] The cytochrome P-450 3A subfamily (CYP3A) is considered the
principal isoform of the CYP superfamily. CYP3A is abundantly
expressed in the liver and gut epithelium and thus contributes to
the high first pass extraction of a large number of orally
administered drugs. Establishment of a causal link between highly
variable CYP3A activity, exceeding 40% in some populations, and
inter-individual variability in the bioavailability of orally
administered medications has been the focal point of many studies.
However, these research efforts have been limited by a lack of
information regarding mechanisms controlling basal expression of
CYP3A.
[0040] CYP3A4 and CYP3A5 constitute the majority of CYP3A activity
in the adult. Polymorphic expression of CYP3A4 and/or CYP3A5 could
conceivably contribute to differences in basal CYP3A activity. To
date, there have been no polymorphic CYP3A4 alleles linked to
clinically significant differences in drug pharmacokinetics.
[0041] Recently, Kuehl et al. (2001) described two non-functional
allelic variants of the CYP3A5 gene, CYP3A5 *3 and *6. The
molecular defect in CYP3A5*3 is a single nucleotide polymorphism
(SNP; A22893G) located in intron 3. The molecular defect in
CYP3A5*6 is a SNP (G30597A) located in exon 7. More than 50% of
African Americans express CYP3A5 compared to 33% of Caucasians.
This led Kuehl et al. to speculate that polymorphic CYP3A5
expression may be an important genetic contributor to
inter-individual and interracial differences in CYP3A-mediated drug
disposition.
EXAMPLE 7
Genomic DNA
[0042] Genomic DNA can be extracted from 2 ml whole blood
anti-coagulated with trisodium citrate using the QIAmp DNA Blood
Midi Kit (Qiagen, Valencia, Calif.) according to manufacturer's
instructions. All blood samples were kept at -80.degree. C. until
DNA isolation.
EXAMPLE 8
Primer Design
[0043] Discrimination between wild-type and mutant alleles was
achieved using modified PCR amplification of specific alleles
described above. The modification is made to improve amplification
specificity by incorporating an additional internal nucleotide
mismatch near the 3' termini of the allele-specific primers. PCR
primers are listed in Table 2. Oligonucleotide primers were
designed based upon the published CYP3A5 sequence (Genbank
#AC005020) using the online program Primer3. Oligo Toolkit.TM. was
used to predict hairpin structures and primer-dimers. Primers were
synthesized by Integrated DNA Technologies, INC. (Coralville,
Iowa). Expected amplicon lengths were 238 bp and 273 bp for the
A22893G and G30597A alleles, respectively.
EXAMPLE 9
Real-Time PCR Amplification
[0044] All reactions were carried out in a total volume of 25
.mu.L. Reaction conditions were identical for A22893G and G30597A
except where noted. Each reaction mixture contained 0.2 mmol/l of
each primer and a 1:12,500 dilution of SYBR.TM. Green I nucleic
acid gel stain 10,000.times. in dimethyl sulfoxide (DMSO)
(Molecular Probes); 0.2 mM of dATP, dCTP, dGTP, and dTTP; 1.0 U of
Taq DNA polymerase (Promega, Madison, Wis.); 6% DMSO; and 20 to 120
ng of genomic DNA in 1.times.PCR buffer (pH 8.3, 10.times. solution
containing 100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl2 and 0.01%
gelatin) (Sigma, St. Louis, Mo.).
[0045] The amplification program for both A22893G and G30597A
consisted of 1 cycle of 95.degree. C. with 120-second hold
("Hotstart") followed by 30 cycles of 95.degree. C. with 15-second
hold, specified annealing temperature of 58.degree. C. with
30-second hold, and 72.degree. C. with 30-second hold. After
amplification, melt analysis was performed by heating the reaction
mixture from 60.degree. C. to 95.degree. C. at the rate of
0.2.degree. C./s. A negative control without DNA template was run
with every assay to assess the overall specificity.
EXAMPLE 10
PCR Product Analysis
[0046] PCR products were analyzed as described above. Genomic DNA
obtained from individuals determined by sequencing to be homo-,
hetero-, and nullizygous for the *3 allele were used to develop the
CYP3A5*3 genotyping assay. The CYP3A5*6 allele has a frequency of
approximately 6% in African-Americans, but the *6 allele was not
found in Caucasians. Due to the low prevalence of the *6 allele,
the inventors were unable to identify an individual with the *6/*6
genotype. Thus, genomic DNA obtained from individuals determined by
sequencing to be hetero- and nullizygous for the *6 allele were
used to develop the CYP3A5*6 genotyping assay.
EXAMPLE 11
Genotyping Results
[0047] Allele-specific primers containing an additional nucleotide
mismatch at the third base from the 3' termini had little effect on
specific PCR product yield. However, non-specific PCR product yield
was drastically reduced to undetectable levels. Consequently, PCR
conditions were optimized such that the threshold cycle (Ct) was
exceeded only when specific amplification occurred (i.e., only in
the presence of a primer: template match).
[0048] FIG. 4A illustrates the results of the CYP3A5*3 allelic
discrimination assay using CYP3A5*1/*1 genomic DNA amplified with
the common primer 3A5*3C and either the wild-type specific primer
3A5*3W or the mutant-specific primer 3A5*3M. When primers 3A5*3C
and 3A5*3W were used to amplify *1/*1 genomic DNA, the PCR growth
curve exceeded the Ct value of approximately 25 cycles (FIG. 4A),
and the melting analysis (negative first derivative) yielded a
characteristic sharp peak at approximately 80.degree. C. for the
product (FIG. 5A). PCR growth curves remained at approximately
background fluorescence (FIG. 4A) when 3A5*3C and 3A5*3M were used
to amplify *1/*1 genomic DNA and no distinct melting peaks were
noted (FIGS. 4A and 5A).
[0049] Agarose gel electrophoresis yielded the expected 238 bp
fragment when *1/*1 DNA was amplified with primers 3A5*3C and
3A5*3W. However, no bands were visualized after *1/*1 genomic DNA
was amplified using primers 3A5*3C and 3A5*3M (data not shown).
Similarly, allelic discrimination was achieved after amplification
of *3/*3 DNA using primers 3A5*3C, 3A5*3M, and 3A5*3W (FIGS. 4B and
5B).
[0050] Overlapping PCR growth curves yielding similar Ct values
were obtained when *1/*3 genomic DNA was amplified using wildtype
and mutant-specific primers (FIG. 4C). In addition, a distinct melt
analysis peak was present after amplification with both wild-type
and mutant-specific primers (FIG. 5C). These results accurately
predict the presence of both wild-type and mutant CYP3A5 alleles in
the heterozygote control. Sequencing of genomic DNA and individual
PCR products obtained from individuals homozygous, heterozygous,
and nullizygous for *3 were in perfect agreement with real-time PCR
assay results. Results obtained from optimization and application
of the CYP3A5*6 genotyping assay to individuals nullizygous and
heterozygous for the *6 allele were similar to those reported for
CYP3A5*3 (data not shown). Melt curve analysis yielded a
characteristic sharp peak at approximately 77.degree. C. (Table 2).
TABLE-US-00002 TABLE 2 Primer Sequences For CYP3A5*3 And CYP3A5*6
Genotyping Primer .sup.a Sequence .sup.b T.sub.m .sup.c CYP3A5*3
80.degree. C. 3A5*3W 5' 22912 TCCAAACAGGGAAGAGAAAT 3' 22893 (SEQ ID
NO:11) 3A5*3M 5' 22912 TCCAAACAGGGAAGAGAAAC 3' 22893 (SEQ ID NO:12)
3A5*3C 5' 22675 ACTGCCCTTGCAGCATTTAG 3' 22694 (SEQ ID NO:13)
CYP3A5*6 77.degree. C. 3A5*6W 5' 30578 CCTTTGTGGAGAGCACTGAG 3'
30597 (SEQ ID NO:14) 3A5*6M 5' 30578 CCTTTGTGGAGAGCACTGAA 3' 30597
(SEQ ID NO:15) 3A5*6C 5' 30850 TGGTGGGGTGTTGACAGCTA 3' 30831 (SEQ
ID NO:16) .sup.a W indicates wild type specific primer; M,
mutant-specific primer; C, common primer used in allelic
discrimination assays. .sup.b Nucleotides shown in bold indicate
nucleotides mismatches from published wild type sequence (Genbank
#AC005020). .sup.c Amplicon melt temperature (T.sub.m) obtained
from melt curve analysis.
[0051] The following references were cited herein: [0052] Bottema
et al., Polymerase chain reaction amplification of specific
alleles: a general method of detection of mutations, polymorphisms,
and haplotypes. Methods Enzymol. 218:388-402 (1993). [0053] Kim et
al., Identification of functionally variant MDR1 alleles among
European Americans and African Americans. Clin. Pharmacol. Ther.
70:189-199 (2001). [0054] Kuehl et al., Sequence diversity in CYP3A
promoters and characterization of the genetic basis of polymorphic
CYP3A5 expression. Nat. Genet. 27:383-91 (2001). [0055] Newton et
al., Analysis of any point mutation in DNA: the amplification
refractory mutation system (ARMS). Nucleic Acids Res. 17:2503-2516
(1989). [0056] Okimoto & Dodgson, Improved PCR amplification of
multiple specific alleles (PAMSA) using internally mismatched
primers. Biotechniques 21:20-26 (1996). [0057] Sommer et al., PCR
amplification of specific alleles (PASA) is a general method for
rapidly detecting known single-base changes. Biotechniques 12:82-87
(1992). [0058] Tang et al., Distinct haplotype profiles and strong
linkage disequilibrium at the MDR1 multidrug transporter gene locus
in three ethnic Asian populations. Pharmacogenetics 12:437-450
(2002).
[0059] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these patents and publications are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually
incorporated by reference.
Sequence CWU 1
1
16 1 21 DNA artificial sequence primer_bind 3435W primer sequence
for MDR1 genotyping 1 gtggtgtcac aggaagaggt c 21 2 21 DNA
artificial sequence primer_bind 3435M primer sequence for MDR1
genotyping 2 gtggtgtcac aggaagaggt t 21 3 21 DNA artificial
sequence primer_bind 3435R primer sequence for MDR1 genotyping 3
actataggcc agagaggctg c 21 4 21 DNA artificial sequence primer_bind
2677W primer sequence for MDR1 genotyping 4 agtttgactc accttccctg c
21 5 21 DNA artificial sequence primer_bind 2677M primer sequence
for MDR1 genotyping 5 agtttgactc accttccctg a 21 6 21 DNA
artificial sequence primer_bind 2677C primer sequence for MDR1
genotyping 6 gctataggtt ccaggcttgc t 21 7 21 DNA artificial
sequence primer_bind sense primer for sequencing the G2677T locus 7
aagattgctt tgaggaatgg t 21 8 21 DNA artificial sequence primer_bind
antisense primer for sequencing the G2677T locus 8 gctataggtt
ccaggcttgc t 21 9 19 DNA Artificial sequence primer_bind sense
primer for sequencing the C3435T locus 9 gagcccatcc tgttgactg 19 10
21 DNA Artificial sequence primer_bind antisense primer for
sequencing the C3435T locus 10 actataggcc agagaggctg c 21 11 20 DNA
Artificial sequence primer_bind 3A5*3W primer sequence for CYP3A5*3
genotyping 11 tccaaacagg gaagagaaat 20 12 20 DNA Artificial
sequence primer_bind 3A5*3M primer sequence for CYP3A5*3 genotyping
12 tccaaacagg gaagagaaac 20 13 20 DNA Artificial sequence
primer_bind 3A5*3C primer sequence for CYP3A5*3 genotyping 13
actgcccttg cagcatttag 20 14 20 DNA Artificial sequence primer_bind
3A5*6W primer sequence for CYP3A5*6 genotyping 14 cctttgtgga
gagcactgag 20 15 20 DNA Artificial sequence primer_bind 3A5*6M
primer sequence for CYP3A5*6 genotyping 15 cctttgtgga gagcactgaa 20
16 20 DNA Artificial sequence primer_bind 3A5*6C primer sequence
for CYP3A5*6 genotyping 16 tggtggggtg ttgacagcta 20
* * * * *