1.
Polymorphism Analysis Of Exon 2,5 And 10 Of Bovine Lactoferrin Gene And Its Association Within Mastitis In Sahiwal Cows
by Sidra Mukhtar (2014-VA-223) | Dr. Sehrish Firyal | Dr. Sultan Ali | Dr. Muhammad Wasim | Dr. Muhammad Avais.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: A few factors militate against understanding the milk production capability of bovines. Mastitis is the most costly and the prevalent production-limiting disease of dairy animals in Pakistan and somewhere else on the world. Susceptibility and resistance to mastitis is a complex characteristic and impacted by hereditary variation of animals. Among these variations, the polymorphisms in LF assumes critical part in the immune responses to mastitis.
Susceptibility and resistant to mastitis is a complex trait and influenced by genetic variation of the immunity genes of the animals. Among these variations, polymorphism in Lactoferrin gene (LF) play important role in immune responses to mastitis. Polymorphism in exons 2, 5 and 10 of Lactoferrin gene are associated with mastitis susceptibility and resistance. It is a potential candidate gene for screening of the mastitis susceptible and resistant dairy cows.
The present study was designed for the identification of polymorphism in LF gene associated with mastitis. Blood samples from 20 Sahiwal cows having clinical and subclinical mastitis were sampled. Blood samples of 10 normal Sahiwal cows were also collected. DNA was extracted. Specific primers for amplification of LF gene were designed by using Primer 3 software.
LF gene was amplified and sequenced to get the full length sequence of the gene. Comparative analysis of resulted sequences was done with the help of NCBI BLAST. Multiple sequence alignment was done by using CLUSTAL W and BioEdit softwares. Protein analysis was done with ExPasy translate tool and the development of 3D structure were using PYMOL software.
Availability: Items available for loan: UVAS Library [Call number: 2582-T] (1).
2.
Snp Genotyping Of Cacna1a Gene Implicated In Childhood Absence Epilepsy (Cae)
by Wajeeha Tariq (2010-VA-487) | Dr. Muhammad Wasim | Dr. Ali Raza Awan | Dr. Muhammad tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Childhood absence epilepsy (CAE) is more pediatric epileptic syndrome. It is about 5 to 15% of all childhood epilepsies. CAE is polygenic and multifactorial syndrome. Many different genes other than CACNA1A gene are involved to cause the CAE collectively. Mutation in P/Q type alpha 1 A subunit channel (Cav2.1) gene CACNA1A, leading to the reduction of Cav2.1 activity in both neurons and in expression system. Reduction in Cav2.1 channel activity altered the neurotransmitter release at neocortical synapses. Molecular genetics techniques have identified various mutation in the genes of ion channels such (CACNA1A, CACNA1G, CACNA1H, CACNB4), sodium channel genes (SCN1A, SCN1B and SCN2A) and GABA receptor genes (GABRD and GABRG2). CACNA1A ion channels are the standard mediator of neurotransmission in Central nervous system (CNS) and mutations in this gene play significant role in the generation of absence seizures. Pore forming alpha 1 a (Cav2.1) channels encoded by CACNA1A gene and are usually located in presynaptic neuron.
Present study was aimed to examine coding regions of CACNA1A gene for analyzing the mutations involve in epilepsy.
Blood samples (n = 40) of true CAE representatives were collected from Children hospital Lahore. DNA was isolated from all blood samples through standard organic method. Amplification of CACNA1A gene exon 36 regions was done with specially designed primers.
Later on, results were analyzed through sequencing of target region. Sequenced samples were analyzed through BioEdit software and alignment was done through Clustal Omega software.
It has been identified that absence epileptic patients of Pakistan showed Mutation in exon 36 of CACNA1A gene at position 281258bp and 281285bp which alter the protein sequence. Due to frame shift mutation a stop codon was detected at position 1813 in protein sequence. So a truncated and loss of function Cav2.1 channel might be formed. In epileptic patients, mutation is responsible for the absence seizures.
In the conclusion, we can say that additional study with large number sample is required to amend the effects of these mutations and their associated factors are precisely and perfectly identified. Further, there is need to investigate the other gene variation causing epilepsy in the local population of Punjab Pakistan. This study will ultimately help to develop genetic counseling strategies, gene therapies and parental diagnostic procedures for the Pakistani population.
Availability: Items available for loan: UVAS Library [Call number: 2746-T] (1).
3.
Exploration Of Genetic Polymorphisms And Differential Expression Analysis Of Bovine Alpha-Lactalbumin And Osteopontin Genes Involved In Milk Composition
by Sidra Manzoor (2010-VA-92) | Dr. Asif Nadeem | Muhammad Imran | Dr. Abu Saeed Hashmi.
Material type: Publisher: 2017Dissertation note: Economically important traits of dairy animals are usually controlled by a large number of genes. The identification of the single nucleotide polymorphisms in potential genes has been associated with economically important traits. During lactation, mammary epithelial cells produced large amounts of specific milk proteins. Due to the expression sites, physiological properties and chromosomal localization, LALBA and SPP1 genes might be considered as candidate genes for milk composition in buffalo. Alpha-lactalbumin (LALBA) gene has been reported to be highly transcribed in transition and peak phase while late lactation exhibited its decline with progressive rise in SPP1 expression. This project was designed to investigate the effect of single nucleotide polymorphism that influencing the gene expression thus modulates the milk protein content in Nili Ravi. Samples of unrelated Nili-Ravi buffalo were collected from two Government, Buffalo Research Institute, Pattoki, and Livestock Production and Research Institute (LPRI) Bahadarnagar Okara, livestock farms. Milk samples were collected at 15, 90 and 250 days lactation for expression analysis. The genomic DNA was extracted by using the standard Phenol Chloroform Isoamyl alcohol (PCI) protocol. Specific set of primers was designed for the amplification of the LALBA and SPP1 genes. The amplified PCR products were sequenced for the identification of SNPs. To determine the differential expression of bovine LALBA and SPP1 genes, RNA was isolated from milk samples using the TRIzol reagent and converted it into cDNA. Taqman probes were used that are specifically designed to detect and target the DNA sequence. Five intronic polym orphic sites were identified in LALBA while exonic regions exhibited a complete homology with reference sequence. Additionally, eleven polymorphisms were identified in bovine SPP1 gene, six were in coding region and five were
Summary
122
found in intronic portion of the gene. The analysis and correlation of all identified polymorphism was done by using SNPs data analysis software “SNPator”. Results obtained from expression study was stored in in-build software of Real Time PCR and Cycle threshold (Ct) values of LALBA and SPP1 mRNA were compared in individuals of Nili-Ravi buffalo to determine the variation in expression levels. The LALBA gene expression was observed highest in transition phase with a gradual decrease of expression in mid and late lactation. The sample, NR-5, was observed highly expressed (79.30) while NR-2 with low expression (19.28) for alpha lactalbumin in early lactation. The change in LALBA regulation at same stage was considered due to genetic variation of the respective animal. While the SPP1 gene expression was observed with the highest values in peak lactation and remains elevated in late lactation. NR-4 has the highest (72.27) expression among all mastitis free healthy animals while NR-2 was observed with low expression. Thus, the identified SNPs might be used as genetic marker for milk production traits. Gene expression patterns may also help us to understand the molecular mechanisms of bovine LALBA and SPP1 genes influencing milk composition. However, the expression of both genes was considered in a correlation with other genes involved in milk production pathway. Also, the mutational effects of other milk proteins might be involved in determining the expression pattern of both genes in selected animals. Therefore, further studies are likely to explore the regulation of milk protein genes and their translational efficiency during the course of lactation in dairy animals. Availability: Items available for loan: UVAS Library [Call number: 2830-T] (1).
4.
Mutational Analysis Of Atp7b Gene Responsible For Wilson’s Disease And Its Homology Analysis In Primates And Mouse
by Amama Ghaffar (2011-VA-375) | Dr. M. Yasir Zahoor | Prof. Dr. Huma Arshad Cheema | Dr. M. Imran | Dr. Amjad Riaz.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Copper being an essential element to carry out different cellular processes normally is
maintained through proper regulation mechanisms to avoid its accumulation in the body. ATP7B
gene that codes for ATP dependent P type ATP7B protein controls the regulation of copper in
the body. It is required for the proper delivery of copper to apoceruloplasmin and its excretion
through bile in the form of feces. Therefore, mutation occurring in the ATP7B gene can cause
excessive cellular copper accumulation which results into Wilson’s disease. Variation in ATP7B
gene related to copper transportation leads to Wilson’s disease and transmitted in generation
through recessive pattern of inheritance.
For this study blood samples of fifteen Wilson’s disease affected patients along with
normal individuals of the same family were collected from Children's Hospital & Institute of
Child Health, Lahore. DNA was extracted from blood through organic extraction method
followed by DNA quantification. Amplification of exons 8, 13, 14 and 18 of ATP7B gene was
performed after designing specific primers for these specific regions. Sequencing of amplified
products was done through dideoxy chain termination method. A disease causing mutation of
ATP7B gene c.3155 C>T; p1052 Proline (CCC) to Leucine (CTC) has been mapped on exon 14
in family with Wilson’s disease. This mutation can be used for genetic testing, prenatal
diagnosis and genetic counseling. No mutation was found in exons 8, 13 and 18 which mean that
further study needs to be done to find more local mutation(s) that can be used for fast direct
genetic testing of Wilson’s disease patients or the carriers with heterozygotic conditions who can
develop this disease at any age of their life.
Results
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MUSCLE and Clustal Omega were used for homology analysis of ATP7B gene
nucleotide and protein sequences that revealed Gorilla to be closest to human regarding coding
sequences, while Clustal Omega output file showed all the species varied highly in their protein
structure homologies. Through the prediction of secondary structure homologies it was seen that
marmoset was closest to humans.
This study helped in providing prenatal diagnosis and genetic screening services in the
country. It has facilitated in selecting animal models for further study and research on ATP7B
gene and molecular pathogenesis of the Wilson’s disease leading to prevention and cure of
disease. Availability: Items available for loan: UVAS Library [Call number: 2892-T] (1).
5.
Identification Of Pkhd1 Gene Mutation In Polycystic Kidney Disease And In-Silico Molecular Characterization In Different Mammals
by Taslim Un Nisa (2015-VA-1105) | Dr. Wasim Shehzad | Dr Muhammad Yasir Zahoor | Prof.Dr. Aftab Ahmad Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Fibrocystin is a large, receptor-like protein that is involved in the tubulogenisis and maintenance of duct-lumen architecture of epithelium. Fibrocystin has a combination with the primary cilia of epithelial cell. Renal tubules (small tube) of kidney where urine is formed lined by tiny hair like projection.
Twenty five suspected patient was selected and DNA extracted through organic extraction method from the suspected patient blood. Primers were designed PKHD1gene’s coding sequence located at 6p12.2 in human. The coding region sequenced using the ready mate terminator Sequencing Reaction Kit by Perkin Elmer/ABI and read in an automated sequencer. The allele’s variants have been only reported for Fibrocystin protein in human.
All of the sequences are evaluated by using Chromas and Bioedit software for sequence analysis. The in-silico protein analysis is done for normal and mutated alleles through UCSC and RAPTROX. Homology analysis was also done between human and mammals DNA sequence.
We found mutations which are associated with ARPKD disease and these variants are most common in other population whereas we also found some new variants. There are some reported mutations which we found in our study such as (c3790C>T),(c3891G>T),(c3790C>T). We found three new mutations in PKHD1 gene. The new mutations which we found are (c3681G>A),(c3804C>T),(c3931A>C).These mutations (c3790C>T) and (c3931A>C) in the exon 32 show significant effect on the gene and protein function. Geneticanalysis of PKHD1gene show thatPakistanifamilies have mutations as compared to other population along with some common exonic regions such as exon 32 whichisalsodescribed by others in two different studies.We also analyze the pedigrees of these patients which are consanguineous families and autosomal recessive polycystic disease.
We found total six mutations in this gene including missense/ synonymous mutations. In which, three novel mutations and others are reported mutations. These variations from the results are due to the population and consanguineous families’ pattern.In our study, we also found that Mouse and Chickencan preferably be used as a modal organisms in pathology.This study will also help us in the development of molecular genetic testing for their detection in Pakistani families and population.
Availability: Items available for loan: UVAS Library [Call number: 2941-T] (1).
6.
Bacterial Profiling And Development Of Molecular Diagnostic Assays For Detection Of Bacterial Pathogens Associated With Bovine Mastitis
by Aqeela Ashraf (2012-VA-388) | Dr. Muhammad Imran | Prof. Dr. Tahir Yaqub | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: The livestock sector plays a critical role in strengthening the economy of Pakistan. Control of livestock diseases is the primary objective of government livestock departments. Bovine mastitis is among the most significant diseases of livestock as reported by various field surveys in Pakistan. Despite considerable knowledge about mastitis and its etiology, this disease is still prevalent in many dairy herds; it remains most difficult to eradicate or control. It is an inflammation of mammary gland resulting in decreased milk production, veterinary care costs and culling losses.
In animal health improvement, there is a paradigm shift from treatment of clinical illness to disease prevention. Recognition of disease is the foundation of disease control and prevention. California mastitis test and somatic cell counting are the most commonly used methods for diagnosis of bovine mastitis. These methods are unable to identify the causative agent. Detection of pathogen is critically important for better control of mastitis. Microbial culturing and biochemical tests are traditionally used methods for pathogen identification. But, these methods are very time consuming and can only detect viable bacteria from the sample and can lead to false negative results. The progress in molecular methods based on PCR has improved the veterinary diagnostics.
For the identification of bovine mastitis pathogens, an economical, rapid and sensitive molecular diagnostic assay was developed using multiplex PCR, detecting the pathogenic species-specific DNA. The target species areS. aureus,E. coli, S. uberis, S. agalactiae, S. dysagalactia, S. haemolyticus, S. epidermidis, S. chromogenes andM. bovis. Multiplex PCR assay was developed for the detection of these significantly important bacterial pathogen
causing bovine mastitis. Species specific primers were designed which have the ability to specifically amplify the particular gene in the target species. For this purpose various gene regions were selected for different bacterial species which included 16S rRNA, cpn60, phoA and rdr. Initially monoplex PCRs were optimized individually for each target species. For optimizing multiplex PCR assay, various combinations of individual PCRs with varying concentrations of primers and template DNA were used. The final protocol included all the nine sets of primer pairs, every set targeting a unique mastitic pathogen. Multiplex PCR assay was checked for its specificity and analytic sensitivity was calculated. Mastitic milk samples were collected aseptically from various farms. Initial screening was based on Surf field mastitis test and California mastitis test. Milk samples were cultured on nutrient agar, blood sheep agar and MacConkey’s agar. The bacterial isolates were identified and further sub-cultured in nutrient broth. All the isolates were identified on the basis of 16S rRNA sequencing analysis.
The developed multiplex PCR assay was used to detect the target bacterial pathogens from the collected milk samples. Limit of detection of developed assay was up to 50 pg for DNA isolated from pure cultures and 104 CFU/ml for spiked milk samples. The results obtained by 16S rRNA sequencing, bacterial culture based identification and multiplex PCR assay were compared. Sensitivity and specificity were calculated using latent class analysis, specificity was up to 88% and sensitivity was up to 98% for targeted mastitic pathogens. The developed multiplex PCR detected nine bacterial species in a single reaction. Multiplex PCR assay has also detected the bacterial pathogens in a few culture-negative mastitis milk samples. This is the first multiplex PCR assay which can efficiently detect nine important mastitic bacterial pathogens in a single reaction. The development of multiplex PCR assay is useful in early diagnosis and prevention & control of bovine mastitis.
Mycoplasma is often ignored as a major mastitis-causing pathogen due to lack of rapid and accurate diagnostic tools. In this study a LAMP assay was developed for the identification of M. bovis from clinical mastitic milk samples. LAMP primers were designed from three gene regions including uvrC, 16S rRNA and gyrB. Bacterial reference strains and mastitic milk samples positive for M. bovis were collected from Quality Milk Production Services, Cornell University, Ithaca, NY. Bacterial strains were further cultured on Hayflick medium containing 15% horse serum and incubated for up to 7 d at 37°C with CO2 enrichment. DNA was isolated from mastitic milk samples and bacterial culture using Qiagen DNeasy Blood and Tissue Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions with few modifications. PCR and LAMP assay was performed for all the samples obtained. Analytic sensitivity was calculated and the limit of detection was up to 50pg/reaction for LAMP assay which is higher as compared to PCR.
Sensitivity and specificity was calculated for each of the three tests. Cohen’s kappa values obtained were 0.940 for uvrC, 0.970 for gyrB and 0.807 for 16S rRNA. All three tests showed a high level of agreement between test results and the true mastitis status, indicated by the receiver operating characteristic (ROC) curve. A robust, sensitive and specific LAMP assay has been developed for the detection of M. bovis from mastitic milk. These novel molecular assays could be helpful for correct and timely identification of bovine mastitic pathogens, which is crucial for the control and treatment of the disease.Molecular diagnostic assayshave been developed in the current study based on multiplex PCR assay and loop-mediated isothermal amplification assay.
Availability: Items available for loan: UVAS Library [Call number: 2930-T] (1).
