An Improved Method for Prenatal Diagnosis of Genetic Diseases by Analysis of Amplified DNA Sequences

Abstract

We report the development of a rapid nonradioactive technique for the genetic prediction of human disease and its diagnostic application to hemophilia A. This method is based on enzymatic amplification of short segments of human genes associated with inherited disorders. A novel feature of the procedure is the use of a heat-stable DNA polymerase, which allows the repeated rounds of DNA synthesis to proceed at 63 degrees C. The high sequence specificity of the amplification reaction at this elevated temperature permits restriction-site polymorphisms, contained in the amplified samples, to be analyzed by visual inspection of their digestion products on polyacrylamide gels. By means of this method, we have performed carrier detection and prenatal diagnosis of hemophilia in two families with use of the factor VIII intragenic polymorphisms identified by the restriction enzymes BclI and XbaI. Predictions can be made directly from chorionic villi, without previous DNA extraction, and fetal sex can be determined by amplification of sequences specific for the Y chromosome. Specific amplification of genomic sequences with heat-stable DNA polymerase is applicable to the diagnosis of a wide variety of inherited disorders. These include diseases diagnosed by restriction-site variation, such as Duchenne's muscular dystrophy and sickle cell anemia, those due to a collection of known mutations, such as beta-thalassemia, and those due to gene deletion, such as alpha-thalassemia.

Bioinformatic analysis of endogenous and exogenous small RNAs on lipoproteins

“…To isolate total DNA we used a standard protocol described by Kogan et al [15]. To generate bacterial source material, an overnight culture of DH5a E. coli was diluted 1:100 in PBS.…”

Allele frequencies of three factor VIII gene polymorphisms in Iranian populations and their application in hemophilia A carrier detection

“…The IVS7 nt 27 SNP, BclI, and HindIII markers were analyzed in several samples according to condition and primers described in [9,10]. This analysis was done to find positive control samples and to check the sensitivity of ARMS-PCRs.…”

“…Despite the great variety of mutations that have been described, FVIII gene markers can be used to trace any mutations in families afflicted with familial forms of hemophilia A [4][5][6][7][8][9][10][11]. Three potentially useful FVIII gene markers are BclI/intron 18, HindIII/intron 19, and IVS7 nt 27 G/A SNP.…”

“…Three potentially useful FVIII gene markers are BclI/intron 18, HindIII/intron 19, and IVS7 nt 27 G/A SNP. Until now these polymorphisms have been analyzed by PCR-based RFLP or multiplex heteroduplex analysis [7][8][9][10][11].…”

Sex identification by polymerase chain reaction using a Y-autosome homologous primer set

“…Recently, the polymerase chain reaction (PCR) has become popular since it requires minimal amount of samples, no isotope and a very short period of time (Witt and Erickson, 1989;Kogan et al, 1987): The PCR technique depends its judgments on the presence or absence of a Y-specific signal. Therefore, a failure of technique, or degraded samples could result in an erroneous conclusion.…”