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Technical Briefs |
a author for correspondence: fax 617-556-3103, e-mail dosgood{at}hnrc.tufts.edu
The scavenger receptor class B type 1 (SR-BI), a multiligand
receptor, appears to be a physiologically relevant HDL receptor in
rodents (1)(2). To determine its role in humans,
the human SRB1 gene has been characterized
(3)(4) and its genetic variation investigated in
a Caucasian population (4). We have reported three variants,
at exons 1 and 8 and intron 5, with allele frequencies >0.1 that have
significant associations with lipid and anthropometric variables
(4). The exon 1 variant (G
A) was associated with a
favorable, antiatherogenic lipid profile in men. Women carriers of the
intron 5 variant (CT) showed a higher body mass index
(P = 0.031) than those women homozygous for the common
(wild-type) allele. The exon 8 variant (CT) was associated with
lower LDL-cholesterol concentrations compared with those homozygous for
the common (wild-type) allele. All three variants were single
nucleotide polymorphisms (SNPs), and genotyping was carried out by
restriction digestion with AluI, HaeIII, and
ApaI for exon 1, exon 8, and intron 5, respectively. Because
these are the first published associations indicating that SR-BI may
play a role in lipid metabolism, these observations need to be examined
and confirmed in other populations.
Traditionally, genotyping for genetic mutations has been analyzed by restriction endonuclease digestion and visualized on agarose or polyacrylamide gels. Restriction digestion is an essential procedure in the detection of nucleotide mutations. However, its sensitivity depends on the quality of the amplification of the gene of interest and the staining intensity of the digested products. Furthermore, if no natural restriction site is created or lost, then a cut site must be generated by changing the primer sequence. This can be time-consuming and sometimes difficult to optimize.
Allelic discrimination using the 5' Nuclease Assay with fluorogenic
probes provides a rapid and sensitive method for detecting known
mutants or polymorphisms (5)(6). This method
combines PCR and mutation detection in a single step. A hybridization
probe is cleaved by the 5' nuclease activity of Taq DNA polymerase only
if the specific sequence is successfully amplified. Two TaqMan (PE
Applied Biosystems) probes are used, one for each allele. Each probe
consists of an oligonucleotide with a 5' reporter dye and a 3' quencher
dye. The reporter dyes used are 6-carboxy-fluorescein (FAM) and
VIC®, and 6-carboxy-tetramethylrhodamine (TAMRA)
is used as the quencher dye (7). Initially, the proximity of
the quencher suppresses the fluorescent signal given by the reporter
through Förster resonance energy transfer (7). The
TaqMan probe hybridizes to a smaller 20- to 24mer sequence, which
includes the SNP. AmpliTaq Gold (PE Applied Biosystems) enzyme then
cleaves the probe with its 5'-3' nuclease activity. Thus, the reporter
dye and quencher dye become separated, causing an increase in the
fluorescence intensity of the reporter dye. Our laboratory has reported
the implementation of this method using the Perkin-Elmer/Applied
Biosystems 7700 Sequence Detection Systems (SDS) and TaqMan reagents
with a point mutation in the intestinal fatty acid binding protein
(8). We now report the successful implementation of this
method in genotyping the SNPs in the human SRB1 gene locus.
The primer and probe sequences used are given in Table 1
. PCR was performed in a 10-µL final volume for each
individual SNP. The reaction mixture contained 5 µL of TaqMan 2x
Universal PCR Master Mix (AmpliTaq Gold polymerase, Amperase
uracil-N-glycosylase, dUTP, dGTP, dCTP, dATP, 6-carboxy-x-rhodamine
dye, Tris-HCl, KCl, MgCl2), 200 nmol/L
FAM-labeled probe, 150 nmol/L VIC-labeled probe, 900 nmol/L reverse
primer, 900 nmol/L forward primer, and 2–20 ng of genomic DNA. The
thermal cycler program includes one cycle at 50 °C for 2 min to
activate uracil-N-gylcosylase, which is added to prevent carryover
contamination; one cycle at 95 °C for 10 min to activate the
AmpliTaq Gold Polymerase; and then 40 cycles of 95 °C for 15 s
for denaturing and 62 °C (for exons 1 and 8) or 69 °C (for
intron 5; see Table 1
) for 60 s for annealing/extending.
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Allelic discrimination was performed on the post-PCR product. The 7700
SDS collects fluorescence data on the samples for ~5 s, and SDS
software analyzes the fluorescence, which can be visualized in graph
form (Fig. 1
for exon 8). Clusters of points, where each point represents a
sample, correspond to a particular genotype or no amplification. For
exon 8 genotyping, if there is fluorescence from the reporter (VIC) for
the wild-type allele, then the sample is typed as a CC. Fluorescence
from only the FAM reporter represents the homozygosity for the mutant
allele and is genotyped as TT. Intermediate fluorescence from both
reporters represents the heterozygous population (CT). Similar
genotyping was performed for the exon 1 and intron 5 SNPs (data not
shown), with the only difference being the reporters used (see Table 1
).
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We have genotyped 95 samples, using both fluorescent probes and the traditional enzyme restriction digestion for all three SNPs, and found no disagreement in the genotyping. This method allows for rapid screening for genotyping. Ninety-six samples can be amplified and genotyped in <3 h. Although the system is expensive, long-term savings can be substantial. We have estimated the cost at $1.00 per sample. In addition, there are no hazardous reagents such as ethidium bromide, which is commonly used to visualize restriction digestion products on agarose or acrylamide gels. Moreover, because this method is sensitive and tolerates a broad range of DNA concentrations, there is a high rate for successful genotyping.
Acknowledgments
This work was supported by Grant HL54776 from the National Heart, Lung, and Blood Institute, and Cooperative Agreement 58-1950-9-001 from the US Department of Agriculture Research Service. We would like to thank Dan Shaffer of Perkin-Elmer for help in designing the primers and probes.
Footnotes
JM-USDA Human Nutrition Research Center on Aging at Tufts University, Lipid Metabolism Laboratory, 711 Washington St., Boston, MA 02111
References
The following articles in journals at HighWire Press have cited this article:
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  P. Borel, M. Moussa, E. Reboul, B. Lyan, C. Defoort, S. Vincent-Baudry, M. Maillot, M. Gastaldi, M. Darmon, H. Portugal, et al. Human Plasma Levels of Vitamin E and Carotenoids Are Associated with Genetic Polymorphisms in Genes Involved in Lipid Metabolism J. Nutr., December 1, 2007; 137(12): 2653 - 2659. [Abstract] [Full Text] [PDF]
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J. Delgado-Lista, F. Perez-Jimenez, T. Tanaka, P. Perez-Martinez, Y. Jimenez-Gomez, C. Marin, J. Ruano, L. Parnell, J. M. Ordovas, and J. Lopez-Miranda An Apolipoprotein A-II Polymorphism (-265T/C, rs5082) Regulates Postprandial Response to a Saturated Fat Overload in Healthy Men J. Nutr., September 1, 2007; 137(9): 2024 - 2028. [Abstract] [Full Text] [PDF]
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 P. Perez-Martinez, F. Perez-Jimenez, C. Bellido, J. M. Ordovas, J. A. Moreno, C. Marin, P. Gomez, J. Delgado-Lista, F. Fuentes, and J. Lopez-Miranda A Polymorphism Exon 1 Variant at the Locus of the Scavenger Receptor Class B Type I (SCARB1) Gene Is Associated with Differences in Insulin Sensitivity in Healthy People during the Consumption of an Olive Oil-Rich Diet J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2297 - 2300. [Abstract] [Full Text] [PDF]
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 M. E. Brousseau, A. L. Goldkamp, D. Collins, S. Demissie, A. C. Connolly, L. A. Cupples, J. M. Ordovas, H. E. Bloomfield, S. J. Robins, and E. J. Schaefer Polymorphisms in the gene encoding lipoprotein lipase in men with low HDL-C and coronary heart disease: The Veterans Affairs HDL Intervention Trial J. Lipid Res., October 1, 2004; 45(10): 1885 - 1891. [Abstract] [Full Text] [PDF]
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D. Osgood, D. Corella, S. Demissie, L. A. Cupples, P. W. F. Wilson, J. B. Meigs, E. J. Schaefer, O. Coltell, and J. M. Ordovas Genetic Variation at the Scavenger Receptor Class B Type I Gene Locus Determines Plasma Lipoprotein Concentrations and Particle Size and Interacts with Type 2 Diabetes: The Framingham Study J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2869 - 2879. [Abstract] [Full Text] [PDF]
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 P. Perez-Martinez, J. M Ordovas, J. Lopez-Miranda, P. Gomez, C. Marin, J. Moreno, F. Fuentes, R. A. Fernandez de la Puebla, and F. Perez-Jimenez Polymorphism exon 1 variant at the locus of the scavenger receptor class B type I gene: influence on plasma LDL cholesterol in healthy subjects during the consumption of diets with different fat contents Am. J. Clinical Nutrition, April 1, 2003; 77(4): 809 - 813. [Abstract] [Full Text] [PDF]
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C. Vargas-Martinez, J. M. Ordovas, P. W. Wilson, and J. Selhub The Glutamate Carboxypeptidase Gene II (C>T) Polymorphism Does Not Affect Folate Status in The Framingham Offspring Cohort J. Nutr., June 1, 2002; 132(6): 1176 - 1179. [Abstract] [Full Text] [PDF]
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 E.S. Tai, S. Demissie, L.A. Cupples, D. Corella, P.W. Wilson, E.J. Schaefer, and J.M. Ordovas Association Between the PPARA L162V Polymorphism and Plasma Lipid Levels: The Framingham Offspring Study Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 805 - 810. [Abstract] [Full Text] [PDF]
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 E. Schutz, N. von Ahsen, and M. Oellerich Genotyping of Eight Thiopurine Methyltransferase Mutations: Three-Color Multiplexing, ""Two-Color/Shared"" Anchor, and Fluorescence-quenching Hybridization Probe Assays Based on Thermodynamic Nearest-Neighbor Probe Design Clin. Chem., November 1, 2000; 46(11): 1728 - 1737. [Abstract] [Full Text] [PDF]
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