Rapid identification of beta-globin structural mutations by sequencing the mRNA from peripheral blood reticulocytes.
A simple and rapid method for the molecular detection of beta-globin structural mutations is described using a reverse transcription-polymerase chain reaction of reticulocyte mRNA and direct sequencing of the product. The amplified segment (employing a sense primer 5'-ATTTGCTTCTGACACAACTGT-3', located at position + 1 with respect to the Cap site and an antisense primer 5'-TCCAGATGCTCAAGGCCCTTC-3', located at position + 1772 with respect to the Cap site) encompasses the cDNA sequence including the three globin exons. Employing this method we were able to characterize two hemoglobin structural variants: Hb S (beta 6 (A3) Glu-Val: GAG-GTG) and Hb Porto Alegre (beta 9 (A6) Ser-Cys: TCT-TGT). The approach described in this paper should be very useful to detect hemoglobin structural variants because the RNA extraction is simple, rapid and does not require cesium chloride, guanidinium and proteinase K. In addition, the direct sequencing of the RT-PCR product permits the screening of the entire globin genes with only two reactions.
Miranda
S R
SR
Departamento de Clínica Médica e Hemocentro, UNICAMP, Campinas, SP, Brasil.
Gonçalves
M S
MS
Sonati
M F
MF
Saad
S T
ST
Costa
F F
FF
D001483:Base Sequence; D004252:DNA Mutational Analysis; D005914:Globins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*; D012156:Reticulocytes / Q000737:chemistry; D014158:Transcription, Genetic
D012333:RNA, Messenger; D005914:Globins