Allele-specific PCR Primers Research Articles

Use of site-directed mutagenesis of allele-specific PCR primers to identify the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms at the glutathione S-transferase, GSTM1 locus.

We describe the identification of the GSTM1 null, GSTM1 A, GSTM1 B and GSTM1 A,B polymorphisms at the glutathione S-transferase GSTM1 locus using a single-step PCR method. Target DNA was amplified using primers to intron 6 and exon 7 with site-directed mutagenesis being used to introduce a restriction site in DNA amplified from GSTM1 *A, thereby allowing differentiation of this allele and GSTM1 *B. The accuracy of this approach in identifying the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms was confirmed by comparison with, firstly, an established PCR method that distinguishes GSTM1 *0 homozygotes from individuals with the other GSTM1 genotypes and, secondly, GSTM1 phenotypes determined using chromatofocusing.

29] Polymerase chain reaction amplification of specific alleles: A general method of detection of mutations, polymorphisms, and haplotypes

Publisher Summary The polymerase chain reaction (PCR) utilizes two oligonucleotide primers to amplify a segment of DNA more than 1 million-fold. The PCR can be adapted for the rapid detection of known single-base changes in DNA by using specially designed oligonucleotides in PCR amplification of specific alleles (PASA). The principle of this method is to design an oligonucleotide primer that will preferentially amplify one allele over another. PASA is a generally applicable technique for detection of point mutations or polymorphisms and the presence of small deletions or insertion. But PASA has certain disadvantages that are eliminated by another technique, PCR amplification of multiple specific alleles (PAMSA). This technique allows the rapid detection of more than one allele in a single PCR reaction. PAMSA is useful for detecting mutations and polymorphisms. This chapter discusses general method of detecting haplotypes. PASA has been used to perform population screening, haplotype analysis, patient screening, and carrier testing. Haplotypes are useful in population genetics and medicine. However, determining linkage of haplotypes in the absence of DNA samples from appropriate family members can be difficult and laborious. PASA can be adapted to provide a rapid and reproducible method for haplotyping an individual in the absence of relatives. This method, termed “double PASA,” uses four pairs of allele-specific PCR primers to differentially amplify each of the four possible haplotypes from two biallelic polymorphisms. Double PASA is an important tool for haplotyping doubly heterozygous individuals because the physical linkage of alleles on a strand of DNA is necessary to determine the haplotype.

Allele-specific MVR-PCR analysis at minisatellite D1S8.

Minisatellite variant repeat mapping by the polymerase chain reaction (MVR-PCR) provides a digital approach to DNA typing of great potential use both in forensic medicine and, by mapping single alleles, for exploring allelic variability and mutation processes at minisatellites. The MVR haplotypes of single alleles can be determined either from physically separated alleles or by pedigree analysis of digital diploid codes generated from both alleles simultaneously. We now show that single alleles can be rapidly mapped from total genomic DNA using allele-specific PCR primers directed to polymorphic sites in the DNA flanking the minisatellite. This approach can also be used to dissect mixed DNA samples such as those often encountered in forensic DNA analysis.

Detection of mutation delta F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry.

A method to detect the main cystic fibrosis (CF) mutation delta F508 from dried blood spots, whole blood, or saliva using the polymerase chain reaction (PCR) and time-resolved fluorometry (TRF) is described. Samples are treated by boiling in mild alkaline solution, after which two allele-specific PCR reactions are performed. Allele-specific primers and a common biotinylated primer are used in the amplification reactions. To detect the PCR product, an europium-labeled oligonucleotide, complementary to the biotinylated strand of the PCR product, is used in a solution hybridization. Hybridization is done in streptavidin-coated microtitration wells, making the detection easy to perform. After a washing step, the bound label is detected using a time-resolved fluorometer. To analyze function of the assay, 20 dried blood spot samples were tested. PCR amplification of the deletion region combined with gel retardation assay was used as a control method. In the initial testing, 2 samples giving discrepant results in the two assays were found. In addition, 17 samples from known CF patients together with 6 normal control samples were analyzed. Among these patient samples, 10 homozygotes and 6 carriers for mutation delta F508 were found.

Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction.

We have developed a procedure that allows the detection of polymerase chain reaction (PCR) products derived from a single target DNA molecule in a human sperm without using radioactive probes. With this method, three genetic loci present in a single sperm can be amplified simultaneously. The amplification procedure is specific as well as efficient and permits detection of the PCR product by ethidium bromide staining after polyacrylamide gel electrophoresis. When allele-specific PCR primers that differ in length are used, the size of the PCR products of different alleles also vary in length, allowing the allelic state at each locus to be determined electrophoretically. Studies on individual sperm by using this procedure should facilitate the measurement of genetic recombination in humans over small physical distances. The ability to directly analyze the allelic state of PCR products from one cell rapidly and simply will also be useful for the prenatal diagnosis of genetic disease, especially in the analysis of single blastomeres taken from in vitro fertilized eggs prior to implantation.