scholarly journals “Mirror” Method to Estimate Mutagenic Activity of DNA Lesions

2018 ◽  
Vol 1 (3) ◽  
pp. 32 ◽  
Author(s):  
Leonid Gening ◽  
Oleg Shevchenko ◽  
Konstantin Kazachenko ◽  
Vyacheslav Tarantul
Keyword(s):  
Mutagenic Activity ◽  
Restriction Analysis ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Single Strand ◽  
Sscp Analysis ◽  
Improved Method ◽  
Single Strand Conformational Polymorphism ◽  
Conformational Polymorphism ◽  
Product Forms

We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains (“mirror” products) using a template containing 8-oxoG. The misincorporation of dAMP in the “mirror” product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of “mirror” product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded “mirror” products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered.

Genetic Characterization of Protein C Deficiency in Japanese Subjects Using a Rapid and Nonradioactive Method for Single-Strand Conformational Polymorphism Analysis and a Model Building

1996 ◽  
Vol 76 (03) ◽  
pp. 302-311 ◽  
Author(s):  
Toshiyuki Miyata ◽  
Toshiyuki Sakata ◽  
Yan-Zhen Zheng ◽  
Hiroaki Tsukamoto ◽  
Hideaki Umeyama ◽  
...  
Keyword(s):  
Protein C ◽  
Single Strand ◽  
Sscp Analysis ◽  
Polymorphism Analysis ◽  
Type I ◽  
Protein C Deficiency ◽  
Single Strand Conformational Polymorphism ◽  
Conformational Polymorphism ◽  
Molecular Defects ◽  
Japanese Subjects

SummaryWe studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phel39Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Argl69Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.

scholarly journals Comparison of Variant-Specific Hybridization and Single-Strand Conformational Polymorphism Methods for Detection of Mixed Human Papillomavirus Type 16 Variant Infections

1999 ◽  
Vol 37 (11) ◽  
pp. 3627-3633 ◽  
Author(s):  
Rebecca T. Emeny ◽  
John R. Herron ◽  
Long Fu Xi ◽  
Laura A. Koutsky ◽  
Nancy B. Kiviat ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Single Strand ◽  
Sscp Analysis ◽  
Clinical Samples ◽  
Long Control Region ◽  
Single Strand Conformational Polymorphism ◽  
Conformational Polymorphism ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
Specific Hybridization

PCR-based variant-specific hybridization (VSH) and single-strand conformational polymorphism (SSCP) analyses were compared for their capacities to detect mixed human papillomavirus type 16 (HPV-16) variant infections within clinical specimens. The SSCP assay used in this comparison targets a 682-bp fragment that spans nucleotides 7445 to 222 within the HPV-16 genome. This fragment includes portions of the HPV-16 long control region and the E6 open reading frame and identifies three categories of SSCP patterns: those identical to the patterns of prototype HPV-16 (P), those identical to the patterns of Caski-derived HPV-16 (C), or those that are different from the P and C HPV-16 patterns and that are therefore classified as belonging to novel (N) HPV-16 variants. VSH targets the entire HPV-16 E6-coding region (nucleotides 56 to 640) and distinguishes previously described variant nucleotides at positions 109, 131, 132, 143, 145, 178, 286, 289, 350, 403, and 532. Clinical samples used in VSH and SSCP analyses were subjected to multiple independent amplification reactions. The resultant amplicons were cloned, and 14 to 78 clones per clinical specimen were evaluated by VSH. VSH detected an HPV-16 variant that represented at least 20% of the amplified HPV-16 variant population. In contrast, SSCP analysis detected HPV-16 variants that represented 36% of the amplified HPV-16 population. Comparison studies were conducted with mixed HPV-16 variant laboratory constructs. Again, VSH had a higher sensitivity than SSCP analysis in detecting mixed HPV-16 variant infections in these constructed amplicon targets. Accurate detection of HPV-16 variants may enhance our understanding of the natural history of HPV-16 infections.

scholarly journals Differentiation of F18ab+ from F18ac+Escherichia coli by Single-Strand Conformational Polymorphism Analysis of the Major Fimbrial Subunit Gene (fedA)

1998 ◽  
Vol 5 (3) ◽  
pp. 299-302 ◽  
Author(s):  
Brad T. Bosworth ◽  
Evelyn A. Dean-Nystrom ◽  
Thomas A. Casey ◽  
Holly L. Neibergs
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Single Strand ◽  
Sscp Analysis ◽  
Polymorphism Analysis ◽  
Single Strand Conformational Polymorphism ◽  
Conformational Polymorphism ◽  
E Coli ◽  
Fimbrial Subunit ◽  
Enterotoxin Genes

ABSTRACT Toxin-producing Escherichia coli expressing F18 fimbriae colonizes the small intestines of weaned pigs and causes diarrhea, edema disease, or both. The F18 family is composed of two antigenic variants, F18ab and F18ac. Because many strains do not express F18 fimbriae in vitro, identification and differentiation of these two variants are difficult. Single-strand conformational polymorphism (SSCP) analysis is a rapid method for identifying genetic mutations and polymorphisms. The F18 major fimbrial subunit genes (fedA) of 138 strains were amplified by PCR, and genetic differences were detected by SSCP analysis. The SSCP analysis of thefedA gene differentiated F18ab+ strains from F18ac+ strains. Most strains classified as F18ab+ by SSCP analysis contained Shiga toxin 2e and enterotoxin genes. Most strains classified as F18ac+ by SSCP analysis contained only enterotoxin genes. The SSCP analysis was a useful method for predicting the antigenicity of F18+ E. coli and could also be used for analysis of other virulence genes in E. coli and other pathogenic bacteria.

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