Diagnosis of Sickle Cell Anemia and β-Thalassemia with Enzymatically Amplified DNA and Nonradioactive Allele-Specific Oligonucleotide Probes

Abstract

We have developed a simple and rapid nonradioactive method for detecting genetic variation and have applied it to the diagnosis of sickle cell anemia and beta-thalassemia. The procedure involves the selective amplification of a segment of the human beta-globin gene with oligonucleotide primers and a thermostable DNA polymerase, followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes covalently labeled with horseradish peroxidase. The hybridized probes were detected with a simple colorimetric assay. We demonstrated the usefulness of this method in a retrospective analysis of two pregnancies at risk for beta-thalassemia and one at risk for sickle cell anemia, as well as in an analysis of nine DNA samples simulating three family sets.

Overexpression of the phytochrome B gene from Arabidopsis thaliana increases plant growth and yield of cotton (Gossypium hirsutum)

“…Genomic DNA isolated from apical leaves of transgenic and control cotton plants (growing in the greenhouse as well as in the field) was analyzed by PCR for detection of PHYB by amplifying internal fragments of PHYB genes with the method of Saiki et al (1988) with a little modification. The sequence of PHYB forward primer was 5′-TAGGGCTCCTC ATGGTTGTC-3′ and the sequence of reverse primer was 5′-TCGCAGTGTGAGATCGAAAC-3′.…”

Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spectrum of human papillomavirus types

“…PCR amplification was performed according to the method of Saiki et al (1988) in 0.1 ml of reaction mixture containing 50 mM-KCI, 10 mM-Tris-HC1 pH 8-8, 3.6 mM-MgCI2, 0-01 mg/ml BSA, 0.2 mM of each dNTP, 2 units of Taq DNA polymerase (Perkin-Elmer Cetus) and 300 ng of each primer. Forty cycles of amplification (1 min at 95 °C, 1 min at 55 °C and 2 min at 72 °C) were performed in a Perkin-Elmer Cetus thermal cycler.…”

“…For epidemiological purposes, methods based on the polymerase chain reaction (PCR) (Saiki et al, 1988) have been designed for the detection and typing of HPV DNA in fresh or frozen cervical biopsies (Li et al, 1988 ;Manos et al, 1989), unprocessed cervical smears (van den Brule et al, 1990), paraffin-embedded tissue specimens (Shibata et al, 1988;Cornelissen et al, 1989) and archival cervical smears (Rakoczy et al,, 1990). The prevalence of HPV in cervical smears from women participating in a screening programme for cervical cancer (van den Brule et al, 1991) and specific groups of patients (Young et al, 1989;Manos et al,, 1990;van den Brule et al, 1990van den Brule et al, , 1991 has been reported.…”

Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.

“…The PCR can selectively increase the number of copies of a particular DNA segment in a sample by many orders of magnitude. As a result ofthis 106-to 108-fold amplification, more convenient assays and nonradioactive detection methods have become possible (10)(11)(12). These PCR-based assays are usually done by amplifying the target segment in the sample to be tested, fixing the amplified DNA onto a series of nylon membranes, and hybridizing each membrane with one of the labeled oligonucleotide probes under stringent hybridization conditions.…”