PCR-SSCP: A Method for Reliably Detecting Single Nucleotide Polymorphisms in Multifactorial Diseases

Jäckel, Silke; Epplen, Jörg T.; Epplen, Cornelia

doi:10.1007/978-3-0348-7582-0_11

Cited by 3 publications

(2 citation statements)

References 23 publications

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“…SSCP is a powerful technique for scanning mutations in small DNA fragments, and has been successfully used to characterize the variation of nuclear genes and virus populations (Orita et al., 1989; Koenig et al., 1995). This technique is a simple method for reliably detecting single‐base polymorphisms (Prosser, 1993; Jäckel et al., 1999). SSCP was previously used to differentiate between CYSDV isolates as well as other viruses (Rubio et al., 1996; Offei et al., 2004; Turturo et al., 2005).…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of Jordanian Isolates of Cucurbit yellow stunting disorder virus

Sweiss¹,

Anfoka²,

Abou‐Jawdah³

2007

Journal of Phytopathology

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Cucurbit yellow stunting disorder virus (CYSDV) causes high yield losses to cucurbits in many parts of the world. In 1995, it was detected for the first time in Jordan but Jordanian isolates have never been characterized at the molecular level. In 2005 and 2006, leaf samples (2344) from symptomatic plants were collected from Jordan Valley, Ma'daba, Al-Mafraq, Al-Karak, Jarash, Al-Tafila, Al-BalqaÕ, Al-ZarqaÕ, Amman and Irbid. Detection by tissue blot immunoassay (TBIA) showed that the infection rate of CYSDV in collected samples was 58.5%. The coat protein (CP) gene of CYSDV was amplified from 36 selected samples by IC-RT-PCR. The amplicons (753 bp) from 16 isolates, representing all surveyed regions, were cloned and used for the single-strand conformation polymorphism (SSCP) analysis. Seven different SSCP patterns (P1-P7) were observed for the analysed Jordanian isolates. For further characterization, the CP genes from isolates with different SSCP patterns were sequenced, and the sequences were deposited in the GenBank under the accession numbers DQ903105-DQ903111. Sequence alignment and phylogenetic analysis revealed that all CYSDV isolates investigated in this study were most closely related to isolates previously reported to belong to the Western group of CYSDV.

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Section: Discussionmentioning

confidence: 99%

Molecular Characterization of Jordanian Isolates of Cucurbit yellow stunting disorder virus

Sweiss¹,

Anfoka²,

Abou‐Jawdah³

2007

Journal of Phytopathology

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“…Point mutations induced by lesions, also called single-nucleotide polymorphisms (SNPs), occur every day in living organisms. Traditional methods for detection of SNPs are diverse, including array-based comparative genomic hybridization (Mao, Young, & Lu, 2007), PCR-based methods (Jäckel, Epplen, & Epplen, 1999), and DNA sequencing (Kwok & Duan, 2003). Array-based hybridization is performed using oligonucleotide probes that hybridize fragmented DNA samples of complementary sequences.…”

Section: Commentary Background Informationmentioning

confidence: 99%

Mutation Analysis of L‐Thymidine‐Induced Replication Products Using a Restriction Enzyme–Mediated Assay

Kan

2020

CP Nucleic Acid Chemistry

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This article describes experimental and analytical procedures for evaluating the efficiency and fidelity of DNA replication containing mirror‐image thymidine (L‐T) in E. coli. The procedure involves construction of DNA recombinants containing a restriction enzyme (PstI) recognition site in which the L‐T lesion is site‐specifically located within the PstI recognition sequence (CTGCAG). The recombinants are transfected into DH5α cells. DNA is extracted, amplified, and cleaved into relatively short fragments using different combinations of restriction enzymes to facilitate electrophoretic analysis. Detailed explanations for the restriction enzyme–mediated assay for detection of mutagenic properties of mirror‐image thymidine at a predetermined site are also presented. Advantages and limitations of the assay are discussed by comparing it to other techniques used for detecting lesion‐induced mutation efficiency, and a troubleshooting guide is provided. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of oligonucleotides containing L‐T Basic Protocol 2: Construction of DNA recombinants Basic Protocol 3: Mutation analysis of L‐T‐induced replication products using a restriction enzyme–mediated assay

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High Genetic Stability of the Begomovirus Tomato yellow leaf curl Sardinia virus in Southern Spain Over an 8-Year Period

Sánchez-Campos¹,

Díaz²,

Monci³

et al. 2002

Phytopathology®

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The evolution of the plant single-stranded DNA virus Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae) has been monitored for 8 years after its appearance in southern Spain. Variation within three genomic regions of 166 TYLCSV isolates collected from three locations was assessed by single-strand conformation polymorphism (SSCP) analysis. According to SSCP, the intergenic region (IR) was the most variable. Low genetic diversity was found within the population and geographical or temporal differences were not evident. Nucleotide sequences of specific genomic regions of haplotypes identified by SSCP indicated close relationships among them. Therefore, the Spanish TYLCSV population appears to represent a single, undifferentiated population. The analysis of IR sequences for a subsample of 76 randomly chosen isolates confirmed the limited genetic diversity revealed by the SSCP analysis. A tendency to a lineal increase in diversity over time was observed in Málaga and Almería subpopulations; however, no accumulation of mutations in single isolates was evident. Negative selection to variation seems to operate to conserve certain regions of the genome. Thus, the low genetic diversity found in the studied TYLCSV population might be the result of a founder effect with subsequent selection against less fit variants arising by mutation.

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PCR-SSCP: A Method for Reliably Detecting Single Nucleotide Polymorphisms in Multifactorial Diseases

Cited by 3 publications

References 23 publications

Molecular Characterization of Jordanian Isolates of Cucurbit yellow stunting disorder virus

Molecular Characterization of Jordanian Isolates of Cucurbit yellow stunting disorder virus

Mutation Analysis of L‐Thymidine‐Induced Replication Products Using a Restriction Enzyme–Mediated Assay

High Genetic Stability of the Begomovirus Tomato yellow leaf curl Sardinia virus in Southern Spain Over an 8-Year Period

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