A reverse dot‐blot method for rapid detection of non‐deletion α thalassaemia

Abstract

Summary.A reverse dot blot method based on membranebound allele-specific oligonucleotides as hybridization targets for amplified a-gene fragments has been developed for the rapid detection of four non-deletion a thalassaemia defects found in the Chinese. Since these non-deletion defects account for 22·8% of haemoglobin H disease, a sensitive, specific and rapid screening method should be of value.Keywords: reverse dot blot, allele-specific hybridization, nondeletion a thalassaemia.Alpha thalassaemia (thal) is a group of hereditary anaemias which are commonly due to deletion of one or more of the aglobin genes on chromosome 16. In humans the a genes are duplicated and loss of one and two a genes on the same chromosome (a thal 2 and a thal 1 phenotype respectively) do not manifest any clinical symptoms. Loss of three genes (Hb H disease) results in a thal intermedia picture, and complete deletion of all four a-globin genes (Hb Barts hydrops foetalis) results in severe anaemia in utero. Although most cases of Hb H disease are due to gene deletion, it can also occur as a result of compound heterozygosity of a thal 1 and a non-deletion a thal (a T a). Such cases usually result in a more severe phenotype, some with severe iron overload, despite infrequent transfusion episodes (Todd, 1971;Chim et al, 1998). In a recent study of 114 Hb H patients the prevalence of such non-deletion cases accounted for up to 22·8% (unpublished observations). In South-East Asia four major types of non-deletion a thal are found, viz a2 codon 30 (DGAG), a2 codon 59 (G → A), a Quong Sze codon 125 (T → C) and a Constant Spring codon 142 (T → C). Since it is now possible to selectively amplify the two a genes (Dodé et al, 1990;Hall et al, 1993), we have devised a reverse dot blot allele-specific hybridization method for the rapid detection of the four types of non-deletion defects which will circumvent the need for direct genomic sequencing. MATERIALS AND METHODS150 ng of genomic DNA was used for selective PCR amplification of the a2 genes. A 5 0 primer C1 (nt 6464-6483) (nucleotide numbering according to Humhba4, GenBank/EMBL database J00153) and an a2-specific 3 0 primer C3 (nt 7548-7529) were used. Amplification was made using conditions as previously described (Chan et al, 1997) which generated an a2 gene fragment of 1085 basepairs (bp).For more specific amplification, a semi-nested second PCR (30 cycles) was made using an internal 5 0 primer 501 (nt 6692-6712) with C3, 2 ml of the first PCR product as template and the addition of 750 pmol biotin-dUTP (BRL, U.S.A.). The reactants and conditions were the same as before.Preparation of membrane strips. Biodyne C (Pall Biomedical, U.S.A.) membrane was activated briefly in 0·1 N HCl, rinsed with water and soaked in 16% 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) for 15 min; it was rinsed in water and air dried overnight. Oligonucleotide probes were diluted with 0·5 M NaHCO 3 /Na 2 CO 3 buffer, pH 8·4, to the desired concentration (ϳ5 pmol/ml) for application onto the membrane. Approxim...

Molecular diagnosis of inherited disorders: lessons from hemoglobinopathies

“…The latter can be considered the founding principle behind genotyping microarrays (see below). The reverse dot-blot is a widely used tool for routine screening of numerous mutant alleles in the HBA2/HBA1 [Chan et al, 1999, Foglietta et al, 2003] and HBB genes [Cai et al, 1994, Winichagoon et al, 1999. Automated platforms for preparing the reverse dot-blot membranes (strips) have been reported that allow printing of large numbers of strips with higher-density arraying [Lappin et al, 2001] and hence commercialization of the entire process.…”

An improved reverse dot hybridization for simple and rapid detection of adefovir dipivoxil-resistant hepatitis B virus

“…Second, PCR products for hybridization were digested into single strand by the exonuclease instead of traditional denaturation, the signal was stronger due to more single strand participating in hybridization. Since a critical requirement in the RDB mutation detection system is that the hybridization condition for all the mutations on the strip should be identical (Chan et al, 1999), different hybridization factors such as probe length, hybridization temperature and time were investigated to establish the optimization conditions.…”

Oligonucleotide array for detection of common severe determinants of alpha thalassemia

“…Currently, however, the diagnosis of thalassemia carrier states need several tests, which are not practical for population screening. Furthermore, separated tests are required for the deletion and non-deletion mutations (Chan et al, 1999). Both procedures are relatively complex, time-consuming, and cumbersome.…”