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Methylated DNA and Unmethylated DNA

Methylated DNA and Unmethylated DNA. After the Bisulfite treatment 5’-ACGTTAGTCG-3’ 5’-ATGTTAGTTG-3’ _ Figure … Bisulfite conversion of unmethylated cytosine followed by PCR. (methyaltion ffpe—11.8.9 –bs con) Bisulfite-conversion of unmethylated cytocine to uracil is a three step process. First step is the reversible addition of HSO3– to cytosine. Second step is the liberation of NH3 by hydrolysis, and third step is the release of HSO3– to form uracil. The [C-SO3–] formed in first step rapidly regenerates in to [C] because it is unstable in neutral solutions. On the other hand, [C-SO3–] is stable in acid. The equilibrium between [C] and [C-SO3–] is reached rapidly at pH 7 when bisulfite concentration is 0.5M or higher. Second step is the key process of the overall procedure. It is the rate-limiting step for the conversion of [C] to [U-SO3–]. [U-SO3–] is stable in neutral conditions, but can be easily converted to [U] by alkali (Step 3). With respect to pH, the [C] to [U-SO3–] conversion is optimal at pH 5–6. (Hayathsu et al 2008) (Coffee et al 2009) _ Figure …. Bisulfite conversion of cytosine to uralic 5- methyl cytosine can be deaminated by bisulfite, but the rate is much more slower than cytosine. The rate of deamination is approximately two orders of magnitude lower than that for cytosine. T he deaminated products, thymine-bisulfite adducts are a mixture of two diastereomers. The structures of these isomers are shown in Fig. ….. Figure … Two diastereomeric thymine -bisulfite adducts formed in the deamination of 5- methylcytosine. They are produced in approximately 1 to 1 ratio. The treatment of trans isomer with alkali rapidly generates thymine. In contras the cis adduct, is quite stable. It can be converted to thymine only with a harsh alkaline treatment because cis elimination is difficult. This situation creates an issue in bisulfite modification of methylated cytosine. (Hayathsu et al 2008) DNA degradation is the major side reaction occurs during this conversion. If the incubation temperature and time of the deamination step is increased DNA degradation will be increased. Therefore bisulfite modification is carried out in 55 C temperature for 3 hours. A total of 500 ng genomic DNA is enough for this modification. (BS 1— BS CONVERSION) _ 1.12.6. Melting curve analysis of combined direct 5’ and 3’ triplet primed PCRs PCR is performed in the presence of intercalating dyes such as SYBR green 1, eva green and BRYT green. The intercalating dye binds to dsDNA producing fluorescence. When the DNA is heated, the strands begin to separate and the dye is released and fluorescence is reduced. The temperature at which of the 50% DNA is in single stranded is the melting temperature (Tm). Tm is influenced by GC content and length, therefore Tm is an indicative of the identity of a PCR product. _ Individuals with expanded alleles (PM and FM) from both males and females are produced melt peak profiles that are distinct from those individuals with the NL alleles. Individuals with high GC content have higher Tm than individuals with low GC content. This approach consists of 2 complementary FMR1 triplet-primed PCR (TP-PCR) assays that use unmodified DNA as template. Both assays are based on the same principal feature use of a primer that anneals within the triplet repeats. The combined analysis of both 5’ and 3’ TP-PCRs avoid false negatives caused by flanking-sequence deletion. This assay works as a rapid first-line screen for FMR1 expansions, thereby reducing the number of samples that require more labor-intensive approaches such as Southern analysis for exact repeat sizing and methylation analysis. Previously described methods such as conventional PCR and MS PCR are two step procedures, initially PCR and then product analysis. dTP-PCR followed by MCA is a single step assay with high sensitivity and specificity for rapid screening of FXS. The closed-tube format of this screening strategy eliminates post-PCR transfers, thereby reducing the possibility of human error and sample mix-ups or contamination, an important consideration in the multi-sample screening setting, which is currently minimized via automated sample handling. By eliminating the electrophoresis step, we also reduced the total hands-on and turnaround time. 1.12. 7. Triplet primed PCR and capillary electrophoresis The CE is the most successful analytical tool for high-throughput, cost-effective, and reliable DNA analysis. CE separates ions based on their electrophoretic mobility with the use of an applied voltage. CE is predominantly used in nucleic acid fragment analysis and DNA sequencing. Triplet-primed repeat PCR combined with CE simplifies detection of FX expanded alleles. In triplet primed PCR, forward and reverse primer flanks the CGG repeat region. (CGG)5 primer anneal at multiple targets within the repeat region generating multiple amplicons with a length difference of 3 bases. When electrophoresed in agarose, the multiple products appear as a smear. If PCR products subjected to the CE, the smear becomes a characteristic stutter pattern. The number of peaks corresponded to the number of repeat units (tp pcr 2—tppcr 2010). PMs and FMs can be discriminated as expanded from NLs in males and females by evaluating the peak pattern. Then individuals with expanded alleles can be subjected to Southern blot analysis to determine precise allele sizing and methylation pattern. ( Nahhas et al 2012 ). This largely overcome the need for Southern analysis, (Lyon, et al 2010). However CE provides resolution up to approximately 200 repeats. Above 200 all FM alleles migrated late as an aggregate peak independent of length, located at approximately 850 to 1100 bp range () (TPPCR 1—TP PCR 2010) _ Figure …. Schematic representation of triplet primed PCR 1.12.8. Methyaltion specific triplet primed PCR and capillary elecrophoresis Another simplified strategy based on fluorescent methylation-specific PCR (ms-PCR) and CE analysis for molecular diagnosis of FXS is available. Sodium bisulfite modification is performed to selectively modify genomic DNA. Then msPCR is perforemed using fluorescently labeled three primer sets with different fluoropores to amplify modified methylated or unmethylated DNA and amplicons are resolved by CE. This assay accurately size the NL and PM in both male and females according to the fluorescent peak sizes and patterns on the electropherogram. (Zhou et al 2006) 1.12. 9. Methyaltion specific triplet primed PCR and Melting curve analysis (msTP-PCR MCA) In this assay MCA is performed for the amplicons generated from MS PCR. The melting temperature of an amplicon is dependent on its length and base composition. Amplicons generated from methylated templates have 5-methylcytosine in the positions corresponding to cytosine and they have higher melting temperatures than amplicons from unmethylated templates, which contain thymines. When methylation-independent primers are used, this allows the amplification of both methylated and unmethylated sequences which are seen as distinct melting peaks. (Candiloro et al 2011) Previously described dTP-MCA assay does not discriminate between PMs and FMs. However the msTP-PCR MCA can differentiate PMs from FMs. Therefore only FMs cab be subjected to sizing by Southern analysis. The msTP-PCR MCA assay is rapid and less labor-intensive than Southern blotting or even the newer PCR and capillary electrophoresis– based tests. This assay also ideal for population-based newborn or early childhood screening of FXS. (bs-mca——–ref nus 2013) 1.12.10. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) Southern blot analysis provides the number of the CGG repeats and methylation status for all types of FMR1 alleles. But one of its limitations is that it targets methylation of only a few CpG sites located on the CpG island in 5′ of the repeat region. Recently developed test for FXS targets biomarkers located in the CpG sites. Two novel epigenetic markers called fragile X–related epigenetic elements 1 and 2 (FREE1 and FREE2) were identified and they are related to FMRP expression (inversely correlated with FMRP production) and cognitive impairment in FM individuals. These elements are located on either side of the repeat region and are different from the sites routinely analyzed using methylation-sensitive SB. FREE1 is located on the 5‘ region of CGG repeats. FREE2 is located on the exon 1/intron 1 boundary of the FMR1 gene. FREE2 allows identifying FM males and females with 100% specificity and sensitivity but could not distinguish between PM carriers and healthy controls or between PM carriers and high functioning males with un methylated FM alleles (Godler et al 2010, Godler et al 2011, Godler et al 2012, Godler et al 2013, ) . 1.13 Ethical aspects Genetic testing is a complex ethical and social concern. The use of patient sample requires informed consent from individual. If the study population is children or mentally impaired consent should be obtained from parent or guardian. Explanation of the research and its potential outcome is must. Each individual is entitled to privacy and confidentiality in terms of the protection of their personal data and research findings. Current guidelines state that genetic testing of children is recommended only if a clear benefit can be offered. Fragile X testing for children less than 18 years of age must be approached carefully, with medical and emotional benefits to the child weighed against potential harms. Methylated DNA and Unmethylated DNA
Low-income African-American Caregivers Critical Essay. The article in question dwells upon attitudes of African-American caregivers whose children and grandchildren were diagnosed with emotional and behavioral disorders towards school counselors. It is necessary to note that this matter has not received sufficient attention from researchers. The aim of the research is to reveal attitudes of African-American caregivers with low income whose children/grandchildren have to get mental health counseling. The hypothesis was not explicitly stated but it is clear that the researcher hypnotized that these people have negative experience due to the lack of understanding form the side of school staff that is primarily focused on white middle-class values (even in school where minorities are prevailing). Six caregivers took part in the study. Tucker (2008) notes that three interviews during several months were held with the participants. It is also added that not all caregivers could attend the final interview. The participants were selected by school counselors of local schools. Moustakas’ transcendental phenomenological method was utilized. Tucker (2008) analyzed transcripts of the interviews identifying themes. The findings of the study are as follows. The participants were frustrated as they felt they were let alone with their problems. They also felt that school staff tended to have the so-called ‘my way or highway’. Caregivers also felt that the blame of the behavior was put on them. Only one participant reported that a school counsellor was helpful and gave her all the necessary information concerning mental health services available. Tucker (2008) argues that this is mainly the result of the white middle-class perspective shared by the vast majority of school staff. The author also notes that counsellor have to be more attentive and find the most appropriate approaches to reach caregivers. The author also notes that the number of participants is one of major limitations of the study. It is possible to note that the article in question is rather relevant as it can be regarded as a starting point of further research of the matter. The author used relevant research method, as it is necessary to use a qualitative technique to identify people’s attitudes. Nonetheless, the number of participants as well as their geographic location makes the study quite irrelevant. Though the researcher notes that the number of participants may be between five and eight, it is inappropriate to make conclusion based on only three interviews held with six people living in a local neighborhood. The results may also be inaccurate as the interviewer was white and African-American participants could be reluctant to reveal their true emotions and concerns, as they could not trust a white person. Therefore, there is a chance that the conclusions may be quite inaccurate. However, it is possible to agree with the author’s conclusions even though they need further justification. In conclusion, it is necessary to note that the article contributes greatly to the field of children development. Of course, further research is needed as there are numerous gaps. It is essential to reach low-income African-American caregivers in different states and different schools. One of the major advantages of the study is that the author provides certain recommendations on the way trust among low-income African-American caregivers and schools staff (especially school counsellors). The article can be very helpful for school counselors and principals as it provides an important perspective on possible improvements of the situation. Reference List Tucker, C. (2008). Low-income African-American caregivers’ experiences of being referred to mental health services by the school counselor: Implications for best practices. Professional School Counseling, 12(3), 240-252. Low-income African-American Caregivers Critical Essay
Grand Strategies Differentiation between Horizontal and Vertical integration Horizontal Integration: When additional business is acquired at the similar stage of the value chain, this type of integration is called horizontal integration, which is quite opposite to the vertical integration. By expanding externally and internally, horizontal growth can be attained easily by the company. External expansion includes the merger and acquisition. Examples of horizontal integration are: Manufacturer of automobile acquiring a manufacturer of SUV (sport utility vehicle) and an oil company acquiring forty refineries (Ramaswamy

1st ASSIGNMENT AS1: Cells have a specific structure which dictates their proper function. Sometimes, however, cells malfunction. Read the Case

1st ASSIGNMENT AS1: Cells have a specific structure which dictates their proper function. Sometimes, however, cells malfunction. Read the Case Study “When Cells Malfunction” (Chapter 3, page 43) Analyze the study and answer the three questions. Detail your answers. What recent information, other than that in the text, should should be considered in this case study. Also, consider the mitochondria. What would occur if the mitochondria were to malfunction? How would homeostasis be affected? Consider possible repercussions if there were an overabundance of lysosomes, instead of malfunctioning lysosomes. Cite specific examples and scientific principles to substantiate your points. Remember to incorporate scientific principles and recent, outside resources/studies/examples (other than the text) in your discussion. Cite the resources. The report should be between 500-600 words. APA or MLA format is acceptable. (25 points) (4 CU) Deduction of 4 points is submission is late. Refer to syllabus. 2ND ASSIGNMENT Although organ transplantation is considered an acceptable surgical treatment, face transplantation is considered more of a quality of life rather than a life or death treatment option. Read Biology Matters-Face Transplantation (Chapter 4 page 81) Answer the two questions at the bottom of the reading. Detail your answer. What other biological considerations regarding face transplantation should be analyzed. Citing specific examples and scientific principles, describe your feelings regarding ‘face transplantation.” Remember to incorporate scientific principles and recent, outside resources/studies/examples (other than the text) in your discussion. Cite the resources. The report should be between 500-600 words. (25 points) (4 CU) Deduction of 4 points if submission is late. Refer to the syllabus.

RELG300 Norwich University Islamic Religion and Conflict Research Paper

term paper help RELG300 Norwich University Islamic Religion and Conflict Research Paper.

You would just need to use the Annotated Bibliography and outline from Week 6 you just helped me with!WEEK 8 RESEARCH PAPERStudents will submit a research paper of 2500 to 3500 words (approximately 10-14 pages) of text (exclusive of title page, notes, and bibliography) describing and analyzing the key tenets of one (or at MOST two) of the religions covered in the course (Hinduism, Buddhism, Judaism, Christianity, or Islam) as they relate to justice, the resolution of conflict, and forgiveness. Choose an area of conflict in which your chosen religion is implicated. Examine findings and distill into useful strategies for 21st-century nonlethal warfare intervention.The Week 08 Research Paper will assess the students’ mastery of course outcomes 1,3 & 4 (CO1, CO3, CO4)CO1 – Analyze key tenets of selected world religions.CO3 – Evaluate the impact of belief systems, religious themes, myths and rituals on culture, history and current affairs.CO4 – Analyze the role of religious thinking in extremism, conflict, and peace building.
This assignment is graded: 350 points.All assignments should be submitted in Times New Roman 12 font with standard one-inch margins.
RELG300 Norwich University Islamic Religion and Conflict Research Paper

Achieving Universal Primary Education

In 1998, the then government of Pakistan, accorded full recognition to the fundamental responsibility of restructuring of education system of Pakistan, by announcing the National education policy on 27th March 1998. The National Education Policy 1998 was devised with a sight to transform the Pakistani nation into an integrated, cohesive entity that can stand up and compete against future challenges by setting one of the main objectives of achieving universal primary education by providing the maximum opportunities for free access to every child. In 2000, world leaders from 189 nations voted to implement the MDGs.Pakistan was also one of them. This declaration set eight goals to achieve the unanimous vote to instill the Millennium Declaration may suggest that the political will to accomplish these targets is promising. The achievement of universal primary education was second goal set after eradicating poverty.The target set under this goal was that by 2015 universally children will be able to complete full course of primary schooling.The indicators to achieve this target were completion/survival rate , net enrollment ratio and literacy rate of 15-24 years old men and women. However, critics of the MDGs are not sure about the likelihood of every nation attaining universal education by 2015. The critics of the MDGs suggest that greater focus should be placed on the overall progress rather than the final outcome as the evidence implies that the educational MDGs are not likely to be achieved in most countries. By concentrating primarily on the rates of progress over time, (Sahn, 2003) claim the results will be more beneficial in providing encouragement to countries, as “failure to achieve the goals is not synonymous with failure to achieve social progress”. So, greater attention should be placed on rates of progress towards completing these goals rather than the final outcome. As failing to achieve these goals by 2015 does not indicate a lack of improvement in Pakistan, it is more appropriate for policy analysts to focus on rates of progress. Since Pakistan has signed Millennium development declaration, it is facing major problems like war on terror, political instability, and earthquakes 2004 and floods 2009-10 which hindered its progress towards these goals. There are so many other factors that are obstructing Pakistan in achieving its millennium development educational goals. 2.2 Factors that affect the achievement of millennium development goals of primary education in Pakistan in light of previous researchers and critics. As this paper intends to provide the reasons for critical lag and gap in achieving primary educational millennium developments goals in Pakistan and how these goals may be achieved, I chose to focus on factors which are common and have greater influence on educational progress and aspects that government and other educational authorities in Pakistan can have a direct influence over through policy change. For the purposes of this research, the model that signifies the determinants of educational progress are completion survival rate, female teachers as percentage of total primary teachers, population aged between 0-14 as percentage of total population , poverty rate and literacy rate gap.But other variables that are considered and have an impact are net enrolment ratio, rural population, pupil-teacher ratio, public expenditure, gender parity index, and non-formal education (deeni madrassas), low birth weight babies. The debate that whether educational progress is more affected by personal back ground of students ( that includes rural population, poverty rate, public expenditure , gender parity index) or school quality factors ( that are pupil teacher ratio, trained teachers ,female teachers and non formal education) has been ongoing since the Coleman Report (Coleman et al. 1966). Coleman et al. (1966) found that family background of students, that is their parent’s socioeconomic status, parent’s education and occupations are more indicative of student’s educational progress than school-level factors. Since the publication of the Coleman Report, researchers have continued to examine and debate whether quality of education or home environment determines student’s academic achievements. Especially in regard to how national economic development affects which factors are more important on a global scale, policy makers continue to look for which factors may best explain educational progress. In this research, I intend to observe whether economic or educational investment factors best explain progress towards the educational MDGs in Pakistan. In order to determine how Pakistan can use its resources most efficiently, I have used statistical data for different variables covered under socio economic status of students and educational quality and their effect to reduce literacy gap. Though 70% of population in Pakistan is still living in rural areas. But growth in rural areas is moving towards down and literacy rate is improving so we can see that social background of people has high impact on educational progress. (DR .P.A.Shami, 2005) in their study on basic education in Pakistan raised issues like lack of access to quality education in rural areas and unequal distribution of educational resources in rural and urban areas. These issues still prevail in society as a hindrance to achievement of MDGs for education. The Pakistan has The average literacy rate of Pakistan is 57 per cent with combination of 69 per cent for males and 45 per cent for females.The literacy rate in urban areas remains much higher than in rural parts of the country , 74 per cent and 48 per cent respectively The average provinicial literacy rate follows as Punjab and Sindh 59 per cent, Khyber Pakhtunkhwa (50 per cent) and Balochistan (45 per cent) (Pakistan Economic Survey, 2009-2010). As I have taken population aged between (0-14) as percentage of total population as one of my variables and more than half of population in Pakistan lives in rural areas so it ultimately effects my determinant. As past research indicates that the effects of educational investment factors or school quality factors which include total educational expenditures and number of trained teachers for the universal enrolment MDG, pupil-teacher ratio and repetition rate for the universal completion MDG, and pupil-teacher ratios and number of female instructors for the gender parity goal may vary depending on the economic status of a nation (Heyneman

HRMN 400 UMUC Hubbs The Onboarding Process & Candidates Lacking Skills Case Study

HRMN 400 UMUC Hubbs The Onboarding Process & Candidates Lacking Skills Case Study.

Assignment 2: Case Study 1 Part B: Onboarding and Performance
Worth up to 25 points and 25% of course grade
The purpose of the activity is for you to explain and evaluate different recruiting, selection,
onboarding, and performance management methods.
Assignment alignment with Course Competencies:
• Recommend recruiting, selection and performance management solutions or initiatives to
address dynamic customer and stakeholder needs.
• Interpret HR recruiting, selection and performance management issues and challenges to
develop strategic solutions and interventions.
• Critique recruiting, selection and performance management initiatives to ensure
alignment with HR and organizational strategies.
1. Read the entire case study carefully and then respond to the seven Discussion Questions
on page 5. Answer all questions and all parts of each question.
2. Develop each answer to the fullest extent possible, including citations from course
resources, where applicable, to support your arguments.
3. Submit your assignment as a separate MS Word document in your assignments folder.
Do not type your answers into the case study document.
4. Include a Cover Page with Name, Date, and Title of Assignment.
5. Do not include the original question. Use the following format: Question 1, Question 2,
6. Each response should be written in complete sentences, double-spaced and spell-checked.
Use 12-point Times New Roman font with 1-inch margins on all sides
7. Include page numbers according to APA formatting guidelines.
8. Include citations in APA format at the end of each answer.
9. You must submit to the assignment link by the due date. A missing assignment will be
assigned a grade of 0.
HRMN 400 UMUC Hubbs The Onboarding Process & Candidates Lacking Skills Case Study

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