Detection of a deletion polymorphism of the human [[alpha].sub.2]-macroglobulin gene (A2M-2) by a semi-automated PCR-single-stranded conformational polymorphism method.
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[degrees]C for 10 min; 95[degrees]C for 9 min; 30 cycles of 94[degrees]C for 1 min, 59[degrees]C for 40 s, and 72[degrees]C for 40 s. The PCR reaction mixture of 100 [micro]L contained 1 [micro]g of human genomic DNA, 20 pmol of each primer, 200 [micro]mol/L dNTPs, 2.5 mmol/L Mg[Cl.sub.2], 50 mmol/L KCI, 10 mmol/L Tris-HCI (pH 8.3), 0.1 g/L gelatin, and 3 U of Gold AmpliTaq polymerase (Perkin-Elmer). In addition, 5 [micro]mol/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 electro phoresis 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[degrees]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 devel opment were carried out in the semi-automated PhastSys tem~ (Pharmacia). The gels were prerun at 400 V,10 mA, 1.0 W for 100 V*h at 4[degrees]C. Sample (1[micro]L) was applied at 25 V, 10 mA, 1.0 W for 2 V*h at 4[degrees]C, and then electrophoresed at 200 V, 5 mA, 1.0 W for 800 V*h at 4[degrees]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).
[FIGURE 1 OMITTED]
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).
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.
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Yan-Yun Wu,  Rosario M. Delgado,  Trey Sunderland,  and Gyorgy Csako  * (Clinical Pathology Department, Warren G. Magnuson Clinical Center, and  Geriatric Psychiatry Branch, National Institute of Mental Health, NIH, Bethesda, MD 20892; * address correspondence to this author at: Clinical Pathology Department, NIH, Bldg. 10, Rm. 2C-407, Bethesda, MD 20892-1508; fax 301-402 1885, e-mail firstname.lastname@example.org)
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|Title Annotation:||Technical Briefs|
|Author:||Wu, Yan-Yun; Delgado, Rosario M.; Sunderland, Trey; Csako, Gyorgy|
|Date:||Sep 1, 1999|
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