Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

Point mutations and small insertions or deletions in the human αglobin genes may produce α-chain structural variants and α-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the α-globin genes are duplicate (α2 and α1) and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phastSSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per αgene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystemTM and GenePhorTM, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystemTM and 20% gels at 15oC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer. Correspondence

Mutations in the human α-globin genes have not been as extensively investigated as ß-globin gene alterations in the Brazilian population, although it has been demonstrated that they are prevalent, frequently in association with other hemoglobinopathies and sometimes resulting in important clinical and hematological manifestations (1)(2)(3)(4).
Because the α-globin genes are G-C rich, artifacts in the sequencing reaction are common.Thus, the use of mutation screening methods is interesting, especially for laboratories which do not have an automated sequencer available.
Selective amplification of the α 1 -and α 2globin genes was carried out by PCR as described by Dodé et al. (11), and the product was used as template for a second PCR round, resulting in 24 subfragments (see Figure 1A).The general SSCP conditions were as follows: the second round of PCR products was mixed with a formamide solution (95% formamide, 20 mM Na 2 EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol) in equal volumes (1:1), heated at 95ºC for 5 min and then cooled on ice.The mixture was then loaded onto a homogeneous polyacrylamide gel.Electrophoresis was performed in both PhastSys-tem™ and GenePhor™ apparatus (Amersham Biosciences, Uppsala, Sweden).After a pre-run at 400 V, 100 Vh, 15ºC, different conditions were tested: for Phast-System™, 12.5 and 20% polyacrylamide gels (PhastGels™) and run temperatures of 4º and 15ºC, at 400 V; for GenePhor™, only 12.5% gels (GeneGels™) and run temperatures of 5º and 15ºC, at 600 V. Run time was about 3 h for both systems.Gels were stained with silver (10).
A second denaturing solution, 10% low ionic strength (LIS) (12), was also tested, as well as complete and shortened fragments (cut with restriction enzymes, see Figure 1A).
The fragments and conditions that showed the best quality of electrophoretic separation and allowed 100% mutation detection are presented in Table 1.No difference was observed between LIS and formamide denaturing solutions.The PhastSystem™ apparatus was the best system, probably because 20% gels could be used, which resulted in better electrophoretic performance.Reducing fragment length, in this case, did not improve the sensitivity of mutation detection.
The basic composition of the fragments rather than their length was the most important parameter examined and was dependent on the primer pair choice, since each fragment produces one main stable conformation which is determined by the intramolecular interactions of the primary sequence (13).Therefore, after optimization, nonradioactive SSCP proved to be a sensitive and efficient method for the screening of mutations in the α-globin genes.This strategy may be important for laboratories that wish to work on these genes and do not have an automated DNA sequencer.It will speed up the procedures, will reduce the costs involved in frequent sequencing repetitions, and will increase the reliability of the results.

Figure 1 .
Figure 1.A, Schematic representation of fragment and primer origin.B, Schematic representation of the α-globin gene mutations screened by nonradioactive single-strand conformation polymorphism in the present study.

Table 1 .
Recommended single-strand conformation polymorphism primer combinations and conditions.
*Primer sequences are described in Figure1A.