Identification of bacteria in the Rocky Mountain wood tick, Dermacentor andersoni, using single-strand conformation polymorphism (SSCP) and DNA sequencing

Abstract We designed oligonucleotide primer pairs to amplify the promoter region, the translated exon sequences, and the flanking intron sequences of all 18 exons of the LDL receptor gene to compare the ability of the PCR single-strand conformation polymorphism (PCR-SSCP) method with semiautomated solid-phase genomic DNA sequencing to detect sequence variations. In 20 apparently unrelated Danish patients with a clinical diagnosis of heterozygous familial hypercholesterolemia (FH), we identified 13 different mutations in the LDL receptor gene: two silent (C331C, N494 N); five missense (W66G, E119K, T383P, W556S, T7051); one nonsense (W23X); three splice-site (313 + 1G-->A, 1061-8T-->C, 1846-1G-->A); and two frameshift (335del10, 1650delG) mutations. Four of these mutations, N494 N, T383P, 1061-8T-->C, and W556S, have not been reported earlier. The pathogenicity of the T383P, 1061-8T-->C, and W556S mutations remains to be established by in vitro mutagenesis and transfection studies. One patient had three mutations (335del10, 1061-8T-->C, and T705I) on the same allele. Further, nine well-known polymorphisms were detectable with this methodological setup. Direct DNA sequencing of the PCR products used for the SSCP analysis did not reveal any sequence variations not detected by the PCR-SSCP method. In two patients we did not detect any mutation by either method. We conclude that the PCR-SSCP analysis, performed as described here, is as sensitive and efficient as DNA sequencing in the ability to identify the sequence variations in the LDL receptor gene of the patients with heterozygous FH of this study.

Download Full-text

Low-stringency single specific primer PCR, DNA sequencing and single-strand conformation polymorphism of PCR products for identification of genetic variants of human papillomavirus type 16

Journal of Virological Methods ◽

10.1016/0166-0934(95)00096-6 ◽

1995 ◽

Vol 55 (3) ◽

pp. 435-443 ◽

Cited By ~ 4

Author(s):

Alex van Belkum

Keyword(s):

Human Papillomavirus ◽

Dna Sequencing ◽

Genetic Variants ◽

Single Strand Conformation Polymorphism ◽

Single Strand ◽

Specific Primer ◽

Human Papillomavirus Type 16 ◽

Pcr Products ◽

Conformation Polymorphism

Download Full-text

Molecular Differentiation of Four Species of Oropsylla (Siphonaptera: Ceratophyllidae) Using PCR-Based Single Strand Conformation Polymorphism Analyses and DNA Sequencing

Journal of Medical Entomology ◽

10.1093/jme/tjaa161 ◽

2020 ◽

Author(s):

Jessica T Thoroughgood ◽

James S Armstrong ◽

Brandon White ◽

Clare A Anstead ◽

Terry D Galloway ◽

...

Keyword(s):

Dna Sequencing ◽

Dna Sequences ◽

Related Species ◽

Single Strand Conformation Polymorphism ◽

Single Strand ◽

Morphological Identification ◽

Rrna Gene ◽

28S Rrna ◽

Closely Related Species ◽

Conformation Polymorphism

Abstract It is often difficult to distinguish morphologically between closely related species of fleas (Siphonaptera). Morphological identification of fleas often requires microscopic examination of internal structures in specimens cleared using caustic solutions. This process degrades DNA and/or inhibits DNA extraction from specimens, which limits molecular-based studies on individual fleas and their microbiomes. Our objective was to distinguish between Oropsylla rupestris (Jordan), Oropsylla tuberculata (Baker), Oropsylla bruneri (Baker), and Oropsylla labis (Jordan & Rothschild) (Ceratophyllidae) using PCR-based single strand conformation polymorphism (SSCP) analyses and DNA sequencing. A 446 bp region of the nuclear 28S ribosomal RNA (rRNA) gene was used as the genetic marker. The results obtained for 36 reference specimens (i.e., fleas that were morphologically identified to species) revealed no intraspecific variation in DNA sequence, whereas the DNA sequences of the four species of Oropsylla differed from one another at two to six nucleotide positions. Each flea species also had a unique SSCP banding pattern. SSCP analyses were then used to identify another 84 fleas that had not been identified morphologically. DNA sequencing data confirmed the species identity of fleas subjected to SSCP. This demonstrates that PCR-SSCP combined with DNA sequencing of the 28S rRNA gene is a very effective approach for the delineation of four closely related species of flea.

Download Full-text