Genetic Study for G-Protein Coupled Receptor from Saccharomyces Cerervisiae and From Sera of Patients with Heart Thrombosis
Abstract
Aim of the present study is Identification of specific gene for GPCR using specific primers .and identification of difference in PCR analysis in patients with heart thrombosis and compared with healthy, Sequencing of PCR product regarding GPCR compared for all three subject, Identification the similarity of human GPCR with local strain of yeast fifty healthy control and fifty patients with thrombosis which diagnosed medically with cardiac specific troponin t, troponin 1 levels and electro myocardiogram ECG. The aged for all subjects ranged (39-75) years patients were lying in cardiac care unit at Ibn- al- Nafees teaching hospital and Sheikh Zayed teaching hospital. Genomic DNA of whole blood was extracted from buffy coat and cell cultured handbook protocol using Bioneer- kit and Genomic DNA fungus/yeast kit was used in isolation and purification of DNA. patients divided into three groups according to their age: group A (60-75) years , group B (50-59) years , group C (39-49) years the results of genomic DNA isolation from blood cells extracted in pure form which ensured by the absorbance ratio (260/280 ) was (1.6 – 1.9 ) with a concentration of 50µg/ml and one DNA band with high resolution in gel electrophoresis. The result of genomic DNA extracted from the local strain of S. cerevisiae showed that DNA extracted with high purity because the absorbance ratio (260 /280 )was (1.7 to 2.0) with a concentration of 60 µg/ml and presence one DNA band with high resolution in gel electrophoresis. primers were designed depending on the sequence of the gene responsible for the production of GPCR on the chromosome 11 , GPCR contain three exons which covered with six primers to detect a defect in gene sequence among. Results of gel electrophoresis are showed that primer GPRX1 gave one band for (Control , A,B ) groups but absent amplified band in the patient eight and nine from group C. with molecular weight of this band is 1000 bp. The GPRX2 primer used to amplify second exon in the GPCR gene ,the molecular weight of amplified bands are 400 bp were present in all control samples and three groups of thrombosis patientsand yeast. GPRX2A primer that designed to amplify part two from second exon of GPCR gene by PCR gave one band for all samples which include control and patient, the molecular weight of this band is 500 bp. PCR analysis showed one amplify band for all control and patients group with molecular weight 500 bp for GPRX3 primer and 400 bp for GPRX3A ,300 bp for GPRX3B. The specific primers which designed to covering GPCR gene used to amplification genomic DNA of the local strain S.cerevisiae by PCR technique. Results showed all six primers which gave one band with difference molecular weight for each primer. All samples demonstrate identity planned sizes to control and local strain of S. cerevisiae samples except patient number 8 and 9 in the group(C) that showed non specialist bands in specific primer with first exon (GPRX1) .So the genetic sequence analysis of these two case based on the sequence of the remainder exons to detect the genetic defect in these case. The sequence of the first part for the second exon (X2) was identity standard sequence found on the NCBI web site for case( 8). The case (9) showed identity with the sequence present in the human gene bank but some difference in the first of sequence which neglected because it is in the place link of primer. The results for case 8 showed some mutation for Exon X2(part2). but case (9) demonstrate one deletion and one substitution. The results, also, illustrated that the ether48 thrombosis patients didn't appeared any mutation despite the positive results for ( Troponin) that gives strong indication of thrombosis. The conclusion Primer GPRX1 gave one band for (Control , A,B ) groups but absent amplified band in the patient eight and nine from group C. The molecular weight of this band is 1000 bp. The amplified band with molecular weight 400 bp were present in all control samples and three groups of thrombosis patients with primer GPRX2 and 500 bp with primer GPRX2A.PCR analysis showed one amplify band for all control and patients group with molecular weight 500 bp for GPRX3 primer.,400 bp with GPRX3A and 300 bp for primer GPRX3B.Similarity between results given by healthy group and local strain of yeast. Genetic study showed that there are only two case of patients eight and nine demonstrated mutation in nucleic location on exon two and three from GPCR gene
Keyword: GPCR, Genetic Study, Saccharomysis cerevisiae, Patient with Thrombosis.
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ISSN (Paper)2224-3186 ISSN (Online)2225-0921
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