Rapid detection of point mutations and polymorphisms of the α‐globin genes by DGGE and SSCA

Abstract

We report the application of DGGE and SSCA for the identification of point mutations causing alpha-thalassemia. The alpha-globin genes were amplified in three overlapping fragments of 250 bp (I), 540 bp (II), and 600 bp (III), respectively. Fragments II and III were analysed by DGGE, while fragments I and II were analysed by SSCA. A panel of seven previously identified mutations was employed to test the combined DGGE/SSCA strategy: 5/5 and 6/7 mutations were detected by SSCA and DGGE, respectively. The same approach has also led to the identification of eight disease-causing mutations in a sample of 18 presumed non-deletional alpha-thalassemia carriers. During this pilot study, two novel mutations as well as three new polymorphisms were found. The combined application of SSCA and DGGE allows the rapid identification of mutations responsible for alpha-thalassemia and abnormal globin chain variants. Moreover, it will prove extremely useful for pre- and postnatal diagnosis and in screening programs for non-deletional alpha-thalassemias.

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.…”

Prenatal Diagnosis of Hemoglobinopathies: From Fetoscopy to Coelocentesis

“…This tail has a high Tm and the DNA fragment has a lower Tm. 81,82 Denaturing high-pressure liquid chromatography Denaturing high-pressure liquid chromatography (DHPLC) is a relatively new technique analogous to DGGE. Heteroduplex molecules are separated from homoduplex molecules by ion-pair, reverse-phase liquid chromatography on a special column matrix with partial heat denaturation of the DNA strands.…”

Incidence of Variant Hemoglobin (Hb) and Increased Fetal Hb Concentrations and Their Effect on Hb A1c Measurement in a Korean Population

“…Ion-exchange HPLC is commonly used for measuring hemoglobin A 1c ( (4,5 ). In the group with increased Hb F, we performed a gene dosage analysis on exon 1 of the ␤-globin gene with the LightCycler (Roche Diagnostics), with albumin coamplified as the reference gene.…”

“…In detection of RNA viruses, which are often present at low concentrations and are prone to degradation, stringent monitoring is needed for all aspects of assay performance, including virus lysis, RNA isolation, reverse transcription, amplification, and detection steps. Among many proposed RNA control preparations (4,5 ), armored RNA is currently the most suitable for clinical applications as it carries the viral RNA target of interest in a form that is ribonuclease-resistant, noninfectious, and stable after prolonged incubation in clinical matrices, and the preparations are substantially less expensive to manufacture than virusinfected plasma (6)(7)(8). Thus, armored RNA has been applied as a positive control for a variety of RNA viruses (9 ).…”