Primed in situ labeling for detecting single-copy genes

Abstract

ABSTRACT. In order to analyze male sterility caused by deletion of SRY and DAZ, we examined the accuracy and cost-effectiveness of a modified primed in situ labeling (PRINS) technique for detection of single-copy genes. Peripheral blood samples were collected from 50 healthy men; medium-term cultured lymphocytes from these samples were suspended in fixative solution and then spread on clean slides. We used four primers homologous to unique regions of the SRY and DAZ regions of the human Y-chromosome and incorporated reagents to increase polymerase specificity and to enhance the hybridization signal. PRINS of SRY and DAZ gave bands at Yp11.3 and Yq11.2, respectively, in all 50 metaphase spreads. The PRINS SRY signals were as distinct as those obtained using traditional fluorescence in situ hybridization (FISH). This new method is ideal for rapid localization of singlecopy genes or small DNA segments, making PRINS a cost-effective alternative to FISH. Further enhancement of PRINS to increase its speed of implementation may lead to its wide use in the field of medical genetics.

Efficient combined FISH and PRINS technique for detection of DAZ microdeletion in human sperm

“…The traditional PRINS protocol utilized primers specific for repetitive alpha satellite sequences thus because of the large number of copies available for hybridization, interference with hybridization by chromatin or other proteins would still allow a strong enough signal to be visualized. PRINS was also used to localize single copy genes including DAZ and SRY on metaphase chromosome [33][34][35]. However, utilizing PRINS technique for the detection of single-copy genes, modifications must be made to the protocol to allow for efficient and accurate labeling [33,35,43].…”

“…PRINS was also used to localize single copy genes including DAZ and SRY on metaphase chromosome [33][34][35]. However, utilizing PRINS technique for the detection of single-copy genes, modifications must be made to the protocol to allow for efficient and accurate labeling [33,35,43]. Similar sets of primers were used to detect DAZ gene on Y chromosome at metaphase stage.…”

“…For each slide, the reaction mixture was prepared in a final volume of 50 μL containing: 10x PCR buffer containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , 0.2 mM dATP, dCTP, and dGTP, 0.02 mM dTTP, 0.02 mM fluorescent dUTP, 0.01 % BSA, 2.5U of Taq DNA polymerase (All reagents from Roche Diagnostics, USA) and 200 pmol of each oligonucleotide primer for DAZ. The four primers specific for DAZ were as follows: (5′-CTCTGCCTCTGGCTTTACCA-3 ′ , 5'-G A GG A GG C ATC TG GA A AT C AT T-3 ' , 5' -GGAAGCTGCTTTGGTAGATAC-3', 5′-TAGGTTT-CAGTGTTTGGATTCCG-3′) [33,34]. A blast search of the primer sequences showed that they were specific for their intended targets.…”

“…PCR based DAZ analysis does not show situation of DAZ gene in individual cells. There has been attempt to show DAZ gene by the use of FISH on sperms or metaphase lymphocytes [31][32][33] or PRINS [33][34][35] in lymphocytes either as interphase or metaphase cells. However, the PRINS method was not used on sperms for DAZ localization.…”

“…In this report we describe a combined PRINS and FISH technique to show the DAZ gene on sperm nuclei. PRINS, Primed in situ labeling, [33,35,36] is a method of target DNA sequence detection and localization which combines features of fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). In this procedure, introduced by Koch et al (1989) [37] the chromosomal identification is performed by in situ annealing of specific and unlabeled oligonucleotide primers to complementary sites on denatured chromosome spreads or nuclei.…”