Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia

Abstract

Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.

Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis

“…The other controls incorporated with each real-time PCR 16S rDNA assay included a no template control (NTC), which was used to monitor potential contamination in the master mix, DNA adequacy of the sample was assessed using ␤-globin PCR as described previously, 25 and specimen inhibition was assessed by spiking 1000 CFU of GBS into each of two separate 25-l 16S rDNA PCR reactions that contained master mix either with or without the prepared blood specimen. The cycle threshold (CT) value of the spiked master mix, containing specimen, was compared with the CT value of the spiked master mix alone lacking specimen.…”

“…All of the whole-blood specimens were analyzed for DNA adequacy using ␤-globin PCR 22,25 and found to be positive. The whole-blood specimens were also tested for inhibitory substances by performing "spiking" experiments.…”

Molecular phylogenetics and biogeography of transisthmian and amphi-Atlantic needlefishes (Belonidae: Strongylura and Tylosurus): perspectives on New World marine speciation

“…Amplifications using the polymerase chain reaction (PCR) (Saiki et al, 1985) were carried out in 50 ll reactions: 5 ll of 10Â buffer (100 mM Tris, pH 8.3, 20 mM MgCl 2 , 500 mM KCl, and 0.1% Gelatin), 4 ll MgCl 2 , 5 ll of 2 mM dNTP, 0.25 ll Taq polymerase, 29.75 ll ddH 2 O, 1-2 ll genomic DNA as template, and 2.5 ll of each 10 mM primer. For ATPase8,6 we used primers ATPase8.2, ATPase8.3, COIII.2, and COIII.3 (http:// nmg.si.edu/bermlab.htm).…”

Polymerase chain reaction cloning of L‐type calcium channel sequences from the heart and the brain

“…The polymerase chain reaction is a powerful method for the enrichment of specific target sequences [16]. It has been used for the cross-species isolation of homologous genes [17,18], and for isolation of members of gene families [19].…”