Dataset Information

Application of CRS-PCR-RFLP to identify CYP1A1 gene polymorphism.

ABSTRACT: BACKGROUND:Cytochrome P4501A1 (CYP1A1) is a member of the cytochrome P450 gene family and plays an important role in the metabolism of exogenous and endogenous material. In recent research, it has been shown that its genetic polymorphisms are associated with many diseases. But the isoschizomers such as the BsrDI enzyme required for the detection of this polymorphism are expensive. METHODS:The study used an improved PCR-RFLP method with mismatched base for detection of the single-nucleotide polymorphism rs1048943. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and the restriction enzyme StyI for RFLP was cheaper than other enzymes. A total of 320 samples from Han Chinese were tested to evaluate this novel method. The PCR results were confirmed by DNA sequencing. RESULTS:After detecting 320 Chinese Han individuals, the genotype frequencies were 63.74% for AA, 31.54% for AG, and 4.72% for GG. The allelic frequencies were 75.48% for A and 24.52% for G. The x2 test showed the genotype and allele frequencies of CYP1A1 do not deviate from Hardy-Weinberg equilibrium, and the sequences of amplified products were consistent with the one published in GenBank with the exception of mismatched base. CONCLUSIONS:Based on PCR with mismatched primers we designed, the CYP1A1 polymorphism could be identified effectively with low cost.

SUBMITTER: Ding M

PROVIDER: S-EPMC6816910 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Application of CRS-PCR-RFLP to identify CYP1A1 gene polymorphism.

Ding Mingcui M Duan Xiaoran X Feng Xiaolei X Wang Pengpeng P Wang Wei W

Journal of clinical laboratory analysis 20170218 6

<h4>Background</h4>Cytochrome P4501A1 (CYP1A1) is a member of the cytochrome P450 gene family and plays an important role in the metabolism of exogenous and endogenous material. In recent research, it has been shown that its genetic polymorphisms are associated with many diseases. But the isoschizomers such as the BsrDI enzyme required for the detection of this polymorphism are expensive.<h4>Methods</h4>The study used an improved PCR-RFLP method with mismatched base for detection of the single-n ...[more]

PMID: 28213913

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Project description:BACKGROUND:Leishmania infantum is the causative agent of human visceral leishmaniasis (VL) and sporadic human cutaneous leishmaniasis (CL) in the Mediterranean region. The genetic variation of the Leishmania parasites may result in different phenotypes that can be associated with the geographical distribution and diversity of the clinical manifestations. The main objective of this study was to explore the genetic polymorphism in L. infantum isolates from human and animal hosts in different regions of Morocco. METHODS:The intraspecific genetic variability of 40 Moroccan L. infantum MON-1 strains isolated from patients with VL (n?=?31) and CL (n?=?2) and from dogs (n?=?7) was evaluated by PCR-RFLP of nagt, a single-copy gene encoding N-acetylglucosamine-1-phosphate transferase. For a more complete analysis of L. infantum polymorphism, we included the restriction patterns of nagt from 17 strains available in the literature and patterns determined by in-silico digestion of three sequences from the GenBank database. RESULTS:Moroccan L. infantum strains presented a certain level of genetic diversity and six distinct nagt-RFLP genotypes were identified. Three of the six genotypes were exclusively identified in the Moroccan population of L. infantum: variant M1 (15%), variant M2 (7.5%), and variant M3 (2.5%). The most common genotype (65%), variant 2 (2.5%), and variant 4 (7.5%), were previously described in several countries with endemic leishmaniasis. Phylogenetic analysis segregated our L. infantum population into two distinct clusters, whereas variant M2 was clearly distinguished from both cluster I and cluster II. This distribution highlights the degree of genetic variability among the Moroccan L. infantum population. CONCLUSION:The nagt PCR-RFLP method presented here showed an important genetic heterogeneity among Moroccan L. infantum strains isolated from human and canine reservoirs with 6 genotypes identified. Three of the six Moroccan nagt genotypes, have not been previously described and support the particular genetic diversity of the Moroccan L. infantum population reported in other studies.

Project description:BACKGROUND:Chagas disease, caused by the intracellular parasite Trypanosoma cruzi, is one of the most important parasitological infections in the Americas. It is estimated to infect approximately 6 million people from mostly low income countries in Latin America, although recent infections have been reported in southern US states. Several studies have described an extensive genetic diversity among T. cruzi isolates throughout its geographic distribution in the American continent. This diversity has been correlated with the pathology developed during an infection. However, due to a lack of a single reliable test, current diagnosis practices of the disease are not straightforward since several different tests are applied. The use of current genomic sequence data allows for the selection of molecular markers (MM) that have the ability to identify the Discrete Typing Unit (DTU) of T. cruzi in a given infection, without the need of any sequencing reaction. METHODOLOGY/PRINCIPAL FINDINGS:Applying three criteria on the genomic sequencing data of four different phylogenetic lineages of T. cruzi, we designed several molecular tests that can be used for the molecular typing of the parasite. The criteria used were: (1) single-copy orthologs of T. cruzi, (2) T. cruzi unique loci, and (3) T. cruzi polymorphic loci. All criteria combined allowed for the selection of 15 MM, 12 of which were confirmed to be functional and replicable in the laboratory with sylvatic samples. Furthermore, one MM produced distinct polymerase chain reaction (PCR) amplicon sizes among distinct T. cruzi DTUs, allowing the use of a AFLP-PCR test to distinguish DTUs I, II/IV, V and VI. Whereas two MM can differentiate DTUs I, II, IV and V/VI out of the six current DTUs with a PCR-RFLP test. CONCLUSIONS/SIGNIFICANCE:The designed molecular tests provide a practical and inexpensive molecular typing test for the majority of DTUs of T. cruzi, excluding the need to perform any sequencing reaction. This provides the scientific community with an additional specific, quick and inexpensive test that can enhance the understanding of the correlation between the DTU of T. cruzi and the pathology developed during the infection.

Dataset Information

Application of CRS-PCR-RFLP to identify CYP1A1 gene polymorphism.

Publications

Application of CRS-PCR-RFLP to identify CYP1A1 gene polymorphism.

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