Long RT-PCR Amplification of the Entire Coding Sequence of the Polycystic Kidney Disease 1 ( PKD1 ) Gene

Abstract

Characterization of mutations of the PKD1 gene has been limited by the fact that three-fourths of this gene at its 5' end is homologous to sequences of at least three other genes on the same chromosome. We have therefore developed a method of long reverse transcription PCR for selective amplification of the entire coding sequence of the PKD1 gene from its mRNA. A PCR primer specific to the sequence in the 3' unique region of the PKD1 gene was synthesized for use coupled with a primer binding to sequence in the homologous region at a distance of about 13.6 kb apart. The commercial availability of RNase H-free reverse transcriptase for long cDNA synthesis and of an enzyme mixture containing Taq and Pfu DNA polymerases for long-range PCR have made this development possible. The long PCR product was proven to be derived from PKD1-mRNA. The results clearly indicated that the long PCR product contained the coding sequence derived from PKD1-mRNA. To our knowledge, this is the first report of a procedure that can reproducibly isolate full-length PKD1 coding sequence from its mRNA transcript, which will prove useful for screening and characterization of mutations in the PKD1 gene.

Novel and de novo PKD1 mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)

“…The full-length cDNA was then fractionated into nine overlapping fragments by nested-PCR methods as previously described [18]. The sizes of nested-PCR fragments were 1,473–1,678 bp (Table 1 and Figure 1A).…”

“…PKD1 mutations occur throughout the gene with apparently no mutational "hot-spots", rendering characterization of all disease causing PKD1 mutations in various ethnic groups essential for comprehension of molecular mechanism of the disease and for providing diagnostic genetic testing. Our group aim to fulfill PKD1- mutation database of Thai patients and have previously developed a long RT-PCR technique for isolation of the entire coding sequence of PKD1 (~13 kb) for the analysis of its mutations without interference from the homologous sequences [18]. The analysis of PKD1 transcript provides an opportunity for ex vivo study of splicing defects prior to identification of causative mutations.…”

CATSPER2, a human autosomal nonsyndromic male infertility gene

“…Full-length KIAA0377 transcripts were amplified, using long-range PCR of human cDNA libraries (Clontech) and of mRNAs from erythroid precursors grown in a two-phase liquid culture 17 as previously described. 18 The PCR products were cloned into a pCR2.1-TOPO vector to separate and sequence alternatively spliced variants expressed in the same tissue.…”

Full-Length Single-Gene cDNA Libraries: Applications in Splice Variant Analysis

“…Gene-specific second strand synthesis and PCR amplification of even large cDNAs with known 5Ј and 3Ј sequence thereafter was straightforward, as shown by others as well (11,12), but subject to controls described below. We found that addition of RNase H to remove RNA completely from heteroduplexes was critical for second-strand synthe- sis and subsequent PCR amplification of templates larger than 5 kb.…”