Rapid and optimized protocol for efficient PCR-SSCP genotyping for wide ranges of species

Badi, Milad A.; Al-Shuhaib, Mohammed Baqur S.; Aljubouri, Thamer R. S.; Al‐Shuhaib, Mohammed Baqur S.; Hassan, None Dawud, Halla; Hussein, Tamadhur Hani; Al-Nafii, Amir T.; Abdulmalek, Dhafer; Altamemi, Mohammed K. A.; Fadhil, Israa A.; Albakri, Ali H.; Hashim, Hayder O.; Mohammed, Amera Kamal

doi:10.1007/s11756-021-00776-z

Cited by 17 publications

(8 citation statements)

References 32 publications

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“…The observed association of the investigated EGFR fragments was based on the PCR-SSCP genotyping of four high-frequency SNPs located in four separate portions of the EGFR gene. Due to its low cost and high sensitivity for the genetic differentiation of PCR products ranging from 200 to 350 bp [30], the PCR-SSCP method was used instead of other commonly utilized genotyping methods in the current genotyping approach [37].…”

Section: Discussionmentioning

confidence: 99%

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Two missense variants of the epidermal growth factor receptor gene are associated with non small cell lung carcinoma in the Iraqi population

Lawi

Al-Shuhaib

Amara

et al. 2022

Preprint

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Background Lung carcinoma is a foremost cause of cancer-related mortality worldwide. Variable genetic factors are associated with the development of lung malignancy. This study was performed to evaluate the possible association between epidermal growth factor receptor (EGFR) gene polymorphisms and non small cell lung carcinoma (NSCLC) in Iraqi population. Methods DNA samples were extracted from 100 patients and 100 controls. Four PCR fragments were designed to amplify four high-frequency variants within EGFR, namely rs1050171, rs2072454, rs2227984, and rs2227983. The PCR fragments were genotyped by single-strand conformation polymorphism (SSCP) method, and each genotype was exposed to direct sequencing. Results Genotyping experiments confirmed the variability of three targeted variants, and logistic regression analysis showed that two of these variants (rs1050171 and rs2227983) exhibited a significant association with NSCLC. Individuals with rs1050171:GA genotype showed a significant association with the increased risk of NSCLC (P=0.0110; OD 5.2636; Cl95% 1.4630 to 18.9371). Individuals with rs2227983:GG genotype exhibited a highly significant association with NSCLC (P=0.0037; OD 5.2683; Cl95% 1.7141 to 16.1919). Linkage disequilibrium analysis showed that the effects of the investigated variants are independent of each other. Conclusions Our collective data indicated that EGFR-rs1050171G/A and EGFR-rs2227983G/G SNPs exerted significant and independent association with the increased risk of NSCLC. This study suggests employing both rs1050171 and rs2227983 SNPs as informative diagnostic markers for the early detection of NSCLC in the study population.

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Section: Discussionmentioning

confidence: 99%

“…1A). The PCR products were designed in these lengths to meet the recommended properties of PCR products that are necessary to give the best electrophoretic resolutions in PCR-SSCP protocols [30]. The nucleic acid sequences of PCR oligonucleotides and their corresponding details are shown in Table I.…”

Section: Pcrmentioning

confidence: 99%

Two missense variants of the epidermal growth factor receptor gene are associated with non small cell lung carcinoma in the Iraqi population

Lawi

Al-Shuhaib

Amara

et al. 2022

Preprint

View full text Add to dashboard Cite

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“…In PCR designing, four high-frequency SNPs (rs2735940, rs2736098, rs2736100, and rs10069690) were targeted within total lengths of 220 bp, 223 bp, 201 bp, and 206 bp, respectively ( Figure 1 a). The specified features of PCR products that are required to provide the best resolutions in PCR-SSCP methods were met by the PCR amplicons when they were prepared with these lengths [39] . The details of the PCR oligonucleotide sequences are displayed in Table 1 .…”

Section: Methodsmentioning

confidence: 99%

Two co-inherited SNPs of the telomerase reverse transcriptase (TERT) gene are associated with Iraqi patients with lung cancer

Lawi,

Alkhammas,

Elerouri

et al. 2023

J Med Biochemistry

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Background: Telomerase reverse transcriptase (TERT) gene is essential for the survival of the vast majority of malignant tumors. This study was conducted to assess the association between TERT gene and non-small cell lung carcinoma (NSCLC) in Iraq. Methods: Genomic DNA samples were extracted from a total of 200 samples of blood. Four specific PCR fragments were designed to amplify four high-frequency rs2735940, rs2736098, rs2736100, and rs10069690 SNPs within the TERT gene. Single-strand conformation polymorphism (SSCP) followed by sequencing reactions were used to genotype and validate the amplified fragments respectively. Results: Individuals with the genotype rs2735940:A/G had a significantly higher risk of developing NSCLC (P=0.0299; OD 2.3158; Cl95% 1.0853 to 4.9414). Individuals with the genotype rs2736098:C/T had also associated with the increased risk of NSCLC (P=0.0363; OD 2.1583; Cl95% 1.0503 to 4.4351). Linkage disequilibrium analysis showed that both SNPs showed a very high level of coinheritance in patients. The LD plot showed that allele T of rs2736098 had collaborated with allele G of rs2735940 to generate TG haplotype in patients. According to our findings, TERT- rs2735940:A/G and TERT- rs2736098:C/T SNPs exhibited significant associations with the increased risk of NSCLC. Both SNPs showed the highest values of co-inheritance in patients. This co-inheritance is mainly represented by allele rs2735940:A and allele rs2736098:C. Both pathogenic T and G alleles have generated TG haplotype that is only available in patients’ samples. Conclusion: This study suggests employing the haplotype TG as a promising biomarker for the early diagnosis of NSCLC. These findings need further validation by large-scale investigation with a larger size of samples in the study population.

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“…SSCP "experiments were performed using a rapid high voltage approach [14] [15] with several modifications [1. Briefly, each PCR amplicon was treated with an equal volume of SSCP denaturing-loading buffer (95% formamide", 0.05% xylene cyanol, 0.05% "bromophenol blue, and 20 mM EDTA, pH 8).…”

Section: Single-strand Conformation Polymorphism (Sscp)mentioning

confidence: 99%

Polymorphism of the GnRH1 gene and Its effect on some Physiological parameters of Local chickens

Badi¹,

Ayied²,

Al-Salhie³

2023

kujas

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The GnRH1 gene is among the genes that mainly affect reproduction and productive traits in local chickens. The GnRH hormone regulates reproductive activity in poultry by regulating the production of other reproductive hormones, such as LH and FSH, which significantly affect birds' productivity. This study was conducted to detect Genetic polymorphism of the GnRH1 gene in local chickens using PCR-SSCP sequencing. Samples were collected at ages 2, 4, 6, and 8 months of birds (n=50). The results found homogeneity in all encoded regions except for one. Polymorphism was detected in the Exon-4 1SNP site 147 C>G. The results indicated significant differences (P≤ 0.05) for the interaction of sex with the genotype of the LH, as the statistical results show that birds with the genotype CC for both, gender outperformed the birds carrying the genotype CG at the age of 2 and 4 months. The results showed significant differences (P≤ 0.05) in the mean genotype within each month in the mean testosterone concentration in males and estrogen in females. Age progression revealed that the homologous genotype CC-carrying birds had an arithmetic advantage over the CG-carrying birds in terms of the average sex hormone concentration. This gene may serve as a marker that is helpful in the marker-assisted selection of local chicken. This study is one of the first to study polymorphisms GnRH1 in local chickens.

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Rapid and optimized protocol for efficient PCR-SSCP genotyping for wide ranges of species

Cited by 17 publications

References 32 publications

Two missense variants of the epidermal growth factor receptor gene are associated with non small cell lung carcinoma in the Iraqi population

Two missense variants of the epidermal growth factor receptor gene are associated with non small cell lung carcinoma in the Iraqi population

Two co-inherited SNPs of the telomerase reverse transcriptase (TERT) gene are associated with Iraqi patients with lung cancer

Polymorphism of the GnRH1 gene and Its effect on some Physiological parameters of Local chickens

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