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

Abstract

Point mutations and small insertions or deletions in the human α-globin 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 (α 2 and α 1 ) 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 phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per α-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem™ and GenePhor™, 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 PhastSystem™ and 20% gels at 15 o C, 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.

Microsatellite Markers within —SEA Breakpoints for Prenatal Diagnosis of HbBarts Hydrops Fetalis

“…Several techniques based on PCR amplification of normal and affected chromosomes (26 -28 ) have been developed to more rapidly identify globin gene mutations. These techniques include single-strand conformation polymorphism analysis, denaturing gradient gel electrophoresis (29,30 ), direct sequencing, amplification refractory mutation system PCR (31 ), reverse dot-blot analysis (32 ), and Gap-PCR, which is based on the multiplex amplification of junctional segments of several different breakpoints (33)(34)(35)(36). The latter technique enables screening and diagnosis of several common deletions in a single test.…”

Coinheritance of a Novel Mutation on theHBA1Gene: c.187delG (p.W62fsX66) [codon 62 (–G) (α1)] with theα212 Patchwork Allele and Hb S [β6(A3)Glu→Val, GAG>GTG;HBB: c.20A>T]

“…The HBA1: c.187delG was validated by single strand conformation polymorphism (SSCP) using the exon 2 product obtained by nested PCR (12). The gel matrix for SSCP analysis contained 10.0% polyacrylamide (29:1), with or without 10.0% glycerol.…”

Molecular diagnosis of inherited disorders: lessons from hemoglobinopathies

“…Although SSCP does not enjoy wide applicability in globin gene genetic screening, as in a variety of other genomic loci [Garinis et al, 2005], a number of SSCP-based mutation-screening strategies have been described for the HBA2/HBA1 [Harteveld et al, 1996, Jorge et al, 2003, HBB [Takahashi-Fujii et al, 1994, Gupta andAgarwal, 2003], and HBE1 genes (Papachatzopoulou et al, in press). However, careful adjustment of the experimental conditions is required in order to obtain reproducible results between different runs, particularly for the purposes of prenatal diagnosis.…”