A high-throughput MS-PCR method on MADGE gels for ANG II type-1 receptor A1166C polymorphism

CLIVE C. J. HUNT, JODI E. BURLEY, CAROLINE M. L. CHAPMAN and JOHN P. BEILBY

The Western Australian Centre for Pathology and Medical Research (PathCentre), Perth, Australia


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hunt, Clive C. J., Jodi E. Burley, Caroline M. L. Chapman, and John P. Beilby. A high-throughput MS-PCR method on MADGE gels for ANG II type-1 receptor A1166C polymorphism. Physiol. Genomics 1: 71–73, 1999.—We have developed a highly accurate, low-cost, single-step, mutagenically separated polymerase chain reaction (MS-PCR) method for the determination of angiotensin II type-1 receptor (AT1) A1166C gene polymorphism. The genotypes are determined using the microtiter array diagonal gel electrophoresis (MADGE) system. We have compared the MS-PCR method with allele-specific oligonucleotide hybridization and Dde I digestion techniques for determining the AT1 A1166C genotype. The combination of MS-PCR and MADGE serves as a model for high-throughput single-nucleotide polymorphism genotyping in large population studies.

receptors; angiotensin II type-1 receptor; mutagenically separated polymerase chain reaction; microtiter array diagonal gel electrophoresis; polymorphism; genetics


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THIS STUDY describes the combination of two techniques, mutagenically separated polymerase chain reaction (MS-PCR) (10) and microtiter array diagonal gel electrophoresis (MADGE) (5), for large-scale, single-nucleotide polymorphism (SNP) genotyping studies. The A1166C polymorphism in the 3'-untranslated region of the angiotensin II type-1 receptor (AT1) is used as an example for demonstrating these combined techniques and discusses the advantages over the currently used allele-specific oligonucleotide hybridization (ASO) method (3, 13) and the Dde I restriction digest method (6, 8) for this polymorphism. The role of the A1166C polymorphism in cardiovascular disease is controversial (14, 8, 9, 1114), and this in part may be due to inaccurate results from the ASO method.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

MS-PCR.
Using the guidelines for the MS-PCR strategy described by Rust et al. (10), primers were designed to detect the A1166C polymorphism in the AT1 gene.


P1 and P2 are the forward primers; P3 is the reverse primer. The short primer P1 is specific for the A1166 allele, and the long primer P2 is specific for the C1166 allele. P3 is the common 3' complementary primer.

The bold underlined nucleotides of primers P1 and P2 are the introduced mutagenic positions. The italicized underlined nucleotide at the 3' end of primers P1 and P2 is the polymorphism.

A range of primers and mismatched nucleotide combinations were tested using the PCR simulation application program Amplify 1.2 (7) before deciding on the above primer sequences.

A 15-µl reaction volume contained 40 ng of genomic DNA, 1.5 µl 10x PCR buffer, 1 mmol/l MgCl2, 200 µmol/l for each of the dNTPs, 0.4 µmol/l (or 6 pmol) of primers P1 and P3, 0.3 µmol/l (or 4.5 pmol) of primer P2, and 0.5 U Taq (Fisher Biotech Perth, Australia). All PCR reactions were carried out in a PTC-100 (MJ Research), 96-well thermocycler and had an initial denaturation temperature of 94°C for 4 min, followed by 40 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 45 s, with a final extension temperature of 72°C for 3 min.

MADGE.
The MADGE system uses a horizontal polyacrylamide gel to separate the MS-PCR products in a 96-well format. It produces better separation for small products than agarose gel.

Five microliters of product were loaded per well onto a 10% polyacrylamide gel (19:1) (National Diagnostics) using the MADGE (MadgeBio, Grantham & Southamptom, UK) system and electrophoresed at 100 V for 100 min in 1x Tris-borate-EDTA (TBE). Gels were photographed using a Kodak DC 120 digital camera on a 302-nm ultraviolet transilluminator.

ASO genotyping.
A modification of the ASO method (3, 13) for genotyping the AT1 A1166C polymorphism was used for comparison of genotypes. Published primers (40 pmol) were end-labeled with fluorescein-11-dUTP (ECL 3'-oligolabeling detection system, Amersham UK, Little Chalfont, UK). To 47 µl of PCR product, 180 µl denaturing solution (1.5 mol/l NaCl, 0.5 mol/l NaOH) were added, and the mixture was dot-blotted (50 µl/dot) onto nucleic acid transfer membrane (Hybond N+, Amersham UK). Approximately 10 pmol of the labeled oligonucleotide were hybridized with the membrane for 2–16 h (A1166 at 32°C and C1166 at 38°C). Membrane blocking, antibody incubations, washes, and signal detection were performed according to manufacturer's specifications (ECL Amersham UK) and using Hyperfilm MP (Amersham UK).

Dde I digestion.
The forward primer 5'-GATAATTATGGCAATTGTGC-3' and reverse primer 5'-GCTTTTGTTCAGAGCTTTAG-3' were designed to amplify a 523-bp product with a Dde I cut site at the A1166C polymorphism for the C allele. A separate cut site was used as a digestion control at position 1023 in the coding sequence. This created two digestion fragments of 303 and 220 bp for the A1166 allele and three digestion fragments of 303, 143, and 77 bp for the C1166 allele.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The CC genotype using MS-PCR yields a 117-bp PCR product for the longer allele-specific primer (P2) and the common complementary primer (P3). The AA genotype produces a 97-bp product using the shorter allele-specific primer (P1) and P3; heterozygous individuals produce 97-bp and 117-bp products (Figs. 1 and 2A).



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Fig. 1. ANG II type-1 receptor (AT1) A1166C mutagenically separated polymerase chain reaction (MS-PCR) on microtiter array diagonal gel electrophoresis (MADGE) gel. Four microliters of PCR products were loaded onto a 10% polyacrylamide (19:1) MADGE gel, electrophoresed at 100 V for 100 min, and prestained with ethidium bromide in 1x TBE.

 


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Fig. 2. A: six discrepant genotypes by AT1 MS-PCR on a MADGE gel showing the A and C alleles. B: the same six discrepant genotypes in order, showing the A and C alleles by allele-specific oligonucleotide hybridization.

 
MS-PCR A1166C genotyping was performed on 1,109 individuals from a randomly selected Australian cross-sectional population consisting of an equal number of males and females. The genotype frequencies were 49% AA, 42% AC, and 9% CC; the allele frequencies were 0.70 A and 0.30 C and agree with other studies of mostly similar European and Caucasian studies (3, 6) and especially with the European ECTIM (Etude Cas-Temoins sur l'Infarctus du Myocarde) study (13).

We used the previously described ASO genotyping method (3, 13) for genotyping the A1166C polymorphism in 941 individuals of the 1,109-individual study group and found a discrepancy in 86 individuals, or a 9.1% genotyping error, compared with the MS-PCR method. Six discrepant genotypes were sequenced and confirmed the MS-PCR method genotyping results in all six cases (Fig. 2, A and B). The 86 individuals were also tested by the Dde I digestion method, and these results also confirmed the MS-PCR method was correct in all cases. The ASO genotyping errors did not change the allele or genotype frequencies because the errors were random, but nevertheless there was still a 6%, 10%, and 23% error between the AA, AC, and CC alleles, respectively.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The previously described ASO method (3, 13) often yielded unclear genotypes, with some specimens producing dots with various shades of gray. The CC allele had a 23% genotyping error using the ASO method, and because it has been suggested to be important for the development of cardiovascular disease, having a more accurate method for classifying AT1 A1166C genotypes may help to clarify the conclusions drawn between particular phenotype or multigene studies.

There are considerable savings in cost and time and improved accuracy by using the MS-PCR method instead of the ASO method. Genotyping two 96-well plates required up to 3 days using the ASO method and 1 day using the MS-PCR method. In addition, the ASO method is approximately double the cost. The Dde I restriction digest method for A1166C genotyping involves extra time and expense. The polyacrylamide used in MADGE is also considerably cheaper than high-resolving agaroses.

PCR simulation software, such as Amplify 1.2, was very useful to design the MS-PCR primers, because the primer interactions are complex and can be tested for likelihood of success. MS-PCR optimization was straightforward using the guidelines suggested by Rust et al. (10). We found it necessary to have a lower concentration of the longer primer P2 to equalize the band intensities for heterozygous individuals. Also, maximal relative separation of the PCR products on the MADGE gels was achieved when the MS-PCR primers were designed to produce products of ~100 bp in size.

The MS-PCR technique used in conjunction with the MADGE system is more reliable, less expensive, and simpler than the previous ASO method and faster than the Dde I digestion method. The MS-PCR method may help clarify any associations that exist between the AT1 A1166C gene polymorphism with hypertension and cardiovascular disease. This is the first report that combines MS-PCR with MADGE and serves as a model for high-throughput genotyping in large studies. We have found combining the two techniques useful in genotyping several SNPs in different genes in well over 10,000 samples.


    ACKNOWLEDGMENTS
 
Address for reprint requests and other correspondence: J. P. Beilby, Dept. of Clinical Biochemistry, PathCentre, Locked Bag 2009, Nedlands, Western Australia 6009, Australia (E-mail: john.beilby{at}health.wa.gov.au).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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