Use of psoralen-coupled nucleotide primers for screening of COL4A5 mutations in Alport syndrome

Abstract

Alport syndrome [1] is a genetic renal disease characterized by hematuria and varying degrees of proteinuria, sensorineural hearing loss, and less frequently ocular lesions (lenticonus, macular lesions). Diverse mutations of the COL4A5 gene encoding the alpha 5 chain of basement membrane type IV collagen have been characterized in patients with X-linked Alport syndrome. Almost every affected family harbors a unique sequence change. Therefore, the mutation search in patients with Alport syndrome depends to a great extent on sensitivity and speed of the applied methods. In addition, future clinical diagnostic applications introduce economical aspects as to which method is chosen for mutation searches. Screening for mutations in Alport syndrome usually involves PCR amplification of single exons and subsequent analysis by single strand conformation polymorphism electrophoresis (SSCP) or, with even higher reported sensitivity, by denaturing gradient gel electrophoresis (DGGE) [2—4]. These techniques are based upon the detection of altered gel mobility of mutated DNA as compared to wild-type DNA. DGGE has been reported to be suitable for DNA fragments of 100 to 400 bp in length. One drawback is that mutations only in the domain with the lowest melting point are usually detectable. To circumvent this problem, long stretches (40 bp) of GC are added to one of the primers and then integrated into the PCR product to be analyzed. The so-called GC-clamps have very high melting temperatures and confine the adjacent DNA sequence to a single, flat melting domain that is amenable for DGGE detection of single base changes. GC-clamps have been reported to greatly increase the range of detectable mutations [2]. Besides the expense of synthesizing GC-primers, technical problems may occur with optimization of PCR conditions and yield because of the asymmetry of primer sets. Recently, psoralen phosphoramidites have become commercially available for use with oligonucleotide synthesizers. DNA amplified with a primer set containing one psoralen coupled primer can be covalently crossliriked by exposure to UV light. There is some evidence in the literature that psoralen photo crosslinked DNA behaves like GC-clamped DNA [5]. In this paper, we evaluated psoralen photo crosslinking as a less costly alternative to GC-clamping when applied to mutation screening