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Technical Briefs |
2-Macroglobulin Gene (A2M-2) by a Semi-Automated PCR-Single-Stranded Conformational Polymorphism Method
1
Clinical Pathology Department, Warren G. Magnuson Clinical Center, and
2
Geriatric Psychiatry Branch, National Institute of Mental Health, NIH, Bethesda, MD 20892;
a address correspondence to this author at: Clinical Pathology Department, NIH, Bldg. 10, Rm. 2C-407, Bethesda, MD 20892-1508
Several genes such as the genes that code for apolipoprotein E4,
amyloid ß-precursor protein, and presenilin 1 and 2 have been
linked to the development of Alzheimer disease (AD)
(1). Recently, a polymorphism of the human
2-macroglobulin gene (A2M) was also
identified as a risk factor for AD (2). The A2M
gene is located on chromosome 12p13.3–p12.3 (3), and its
product (2-macroglobulin) is a serum
pan-proteinase inhibitor occurring in many organs and tissues
(4). Several studies have suggested that
2-macroglobulin may have protective effect
against the deposition of ß-amyloids (5)(6)
and may stimulate their degradation (7)(8). A
5-nucleotide deletion mutation at the 5' splice site of "exon II"
of the bait region (exon 18; A2M-2) may lead to loss of this
protection because of its location at the functional domain, and this
loss confers increased risk for AD (2). Currently available
methods based on DNA sequencing (2)(9),
heteroduplex formation in nondenaturing gel (9), and
PCR-restriction fragment length polymorphism (10) for
detection of this deletion mutation are all relatively cumbersome for
routine use in clinical laboratories. Here, we describe a
semi-automated PCR-single-stranded conformational
polymorphism (PCR-SSCP) detection method that is amenable to use for
large-scale screening.
Genomic DNA was extracted from EDTA-anticoagulated whole blood using the QIAamp blood kit (Qiagen). The A2M target DNA sequence was PCR amplified by modifying a previously described technique (9). The 5' upstream primer was 5'-CTT TCC TTG ATG ACC CAA GCG CC-3', and the 3' downstream primer was 5'-GTT GAA AAT AGT CAG CGA CCT C-3'. The PCR conditions were as follows: 37 °C for 10 min; 95 °C for 9 min; 30 cycles of 94 °C for 1 min, 59 °C for 40 s, and 72 °C for 40 s. The PCR reaction mixture of 100 µL contained 1 µg of human genomic DNA, 20 pmol of each primer, 200 µmol/L dNTPs, 2.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 0.1 g/L gelatin, and 3 U of Gold AmpliTaq polymerase (Perkin-Elmer). In addition, 5 µmol/L dUTP (Perkin-Elmer) and 10 kU/L uracil glycosylase (Perkin-Elmer) were added to the PCR mixtures to prevent contamination.
The PCR products were checked for purity by electrophoresis in 1% agarose gel followed by ethidium bromide staining. Images of the patterns were captured with a CCD camera (COHU) coupled with NIH image 1.62 software (NIH). The 326-bp PCR band was quantified by comparison to a DNA gel marker (Research Genetics).
SSCP analysis was performed essentially as described previously (11). In short, the PCR products were mixed with an equal volume of denaturing/loading solution containing 980 mL/L formamide (Amresco), incubated at 95 °C for 3 min, then immediately chilled on ice. Precast minigels with 20% homogeneous polyacrylamide and native buffer strips (Pharmacia Biotechnology) were used for electrophoresis. Gel electrophoresis and band development were carried out in the semi-automated PhastSystemTM (Pharmacia). The gels were prerun at 400 V, 10 mA, 1.0 W for 100 V·h at 4 °C. Sample (1µL) was applied at 25 V, 10 mA, 1.0 W for 2 V·h at 4 °C, and then electrophoresed at 200 V, 5 mA, 1.0 W for 800 V·h at 4 °C. The gels were silver stained according to the manufacturer's protocol.
To confirm the PCR-SSCP results, all PCR products were also analyzed by sequencing with the primer 5'-GTT GAA AAT AGT CAG CGA CCT C-3', using an ABI Prism 310 genetic analyzer (PE Applied Biosystems) and Big Dye Terminator cycle sequencing kit (PE Applied Biosystems). Before sequencing, PCR products were purified either with a Gel Extraction kit (Qiagen) or a PCR Product Purification kit (Roche/Boehringer Mannheim).
Representative PCR-SSCP patterns for the three different A2M
genotypes are shown in Fig. 1
. The same patterns were reproducible over a wide range of PCR
products (2.5–30.0 ng), as quantified by the above-described image
analysis method. When 30 human blood specimens were analyzed, a
complete match was found between the PCR-SSCP and DNA sequencing
results that included 16 cases of homozygous wild type
(A2M/A2M), 11 cases of heterozygous type
(A2M/A2M-2), and 3 cases of homozygous deletion
type (A2M-2/A2M-2).
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In summary, we have developed a semi-automated PCR-SSCP method that is simpler to perform and less labor-intensive than previous methods for the detection of A2M polymorphisms. In addition, PCR-SSCP avoids misinterpretation that may occur because of partial digestion in the PCR-restriction fragment length polymorphism method.
Footnotes
fax 301-402-1885, e-mail gcsako{at}nih.gov
References
-2 Macroglobulin is genetically associated with Alzheimer disease [Letter]. Nat Genet 1998;19:357-360.
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2-macroglobulin-related genes (20M) on human chromosome 12p: cloning of the pregnancy-zone protein gene and an 2M pseudogene. Gene 1989;81:325-334.
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2-Macroglobulin, a multifunctional binding protein with targeting characteristics [Review]. FASEB J 1992;6:3345-3353.
[Abstract]
2-Macroglobulin attenuates ß-amyloid peptide 1–40 fibril formation and associated neurotoxicity of cultured fetal rat cortical neurons. J Neurochem 1998;70:1182-1188.
[Web of Science][Medline]
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2-Macroglobulin associates with ß-amyloid peptide and prevents fibril formation. Proc Natl Acad Sci U S A 1998;95:3275-3280.
2-macroglobulin complex. J Biol Chem 1996;271:8443-8451.
2-Macroglobulin complexes with and mediates the endocytosis of ß-amyloid peptide via cell surface low-density lipoprotein receptor-related protein. J Neurochem 1997;69:1904-1911.
[Web of Science][Medline]
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-2-macroglobulin (A2M) gene. Nucleic Acids Res 1991;19:5102.2-macroglobulin gene [Letter]. Clin Chem 1999;45:307-317.
The following articles in journals at HighWire Press have cited this article:
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  Y.-Y. Wu, R. M. Delgado, T. Sunderland, and G. Csako Semiautomated PCR-Single-Strand Conformation Polymorphism Method for Detection of a Novel Sequence Polymorphism (Ile1000Val) in Human {alpha}2-Macroglobulin Clin. Chem., May 1, 2000; 46(5): 715 - 718. [Full Text] [PDF]
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