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Objectives: The high-resolution melting (HRM) technique is fast, effective and successful method for mutation detection. The aim of this study was to determine the sensitivity and specificity of the HRM method for detection of a paternally inherited mutation in a fetus as a noninvasive prenatal diagnosis of β-thalassemia.Methods: Genomic DNAs were prepared from 50 β-thalassemia minor couples whose pregnancy was at risk for homozygous β-thalassemia. Ten milliliters of the maternal blood from each pregnant woman were collected and after separating plasma stored at -80 °C until analysis. The extracted DNAs were analyzed by HRM real-time PCR for detection of IVS-II-I (G-A) as a paternally inherited mutation. The gold standard was the result of a chorionic villus sampling by a standard reverse dot blotting test.Results: The sensitivity and specificity of HRM real-time PCR were 92.6% and 82.6%, respectively. Also, the positive and negative predictive values were 86.2% and 90.47%, respectively.Conclusions: HRM real-time PCR was a sensitive and specific method for determining the paternally inherited mutation in the fetus at risk with thalassemia major. [ABSTRACT FROM AUTHOR]

Nucleic acid amplification technologies are used in the field of molecular biology and recombinant DNA technologies. These techniques are used as leading methods in detecting and analyzing a small quantity of nucleic acids. The polymerase chain reaction (PCR) is the most widely used method for DNA amplification for detection and identification of infectious diseases, genetic disorders and other research purposes. However, it requires a thermocycling machine to separate two DNA strands and then amplify the required fragment. Novel developments in molecular biology of DNA synthesis in vivo demonstrate the possibility of amplifying DNA in isothermal conditions without the need of a thermocycling apparatus. DNA polymerase replicates DNA with the aid of various accessory proteins. Recent identification of these proteins has enabled development of new in vitro isothermal DNA amplification methods, mimicking these in vivo mechanisms. There are several types of isothermal nucleic acid amplification methods such as transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification. In this article, we review these isothermal nucleic acid amplification technologies and their applications in molecular biological studies. [ABSTRACT FROM AUTHOR]

There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA‐based detection system (PCR‐ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin‐11‐dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele‐specific capture probes bound to streptavidin coated plates. Use of the produced anti‐digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2′‐azino‐di‐3‐ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti‐digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti‐digoxigenin antibody HRP conjugate for use in the PCR‐ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost‐effective product. [ABSTRACT FROM AUTHOR]