Basics of Genomic Technology

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Genomics is the study of all the genes present in an organism, including their structure, function, and interactions. It involves the use of advanced technologies such as DNA sequencing, PCR, and biochip technology to analyze the entire genetic complement of an organism. Genomics plays a crucial role in understanding genetic variations, disease mechanisms, and personalized medicine.
Some key technologies used in the field of genomics to analyze genetic information include:

1. DNA sequencing: This technique allows the determination of the precise order of nucleotides within a DNA molecule.
2. Polymerase chain reaction (PCR): PCR is a method widely used to amplify a single copy or a few copies of a segment of DNA to generate thousands to millions of copies of a particular DNA sequence.
3. Electrophoretic methods: Techniques such as SSCP (single-strand conformation polymorphism) and DGGE (denaturing gradient gel electrophoresis) are used to separate and analyze DNA fragments based on their size and electrical charge.
4. Biochip technology: DNA microarray and protein microarray are used for large-scale analysis of gene expression, genotyping, and protein-protein interactions.
5. Hybridization techniques: Methods such as Southern blot and Northern blot involve the use of labeled DNA or RNA probes to detect specific sequences in DNA or RNA samples.
6. Recombinant DNA technology: This technology is used to manipulate DNA sequences, such as cloning genes, constructing genetically modified organisms, and producing recombinant proteins.

Overall, these technologies play a crucial role in the field of genomics by allowing researchers to analyze and understand genetic information at a molecular level.

The DNA sequencing process involves several key steps to accurately determine the order of nucleotides within a DNA molecule. Firstly, in the library preparation stage, DNA is extracted and fragmented into smaller pieces. Subsequently, during sequencing, fragmented DNA undergoes third-generation sequencing techniques, such as PacBio Single Molecule, Real-Time (SMRT) sequencing, or Oxford Nanopore sequencing, enabling single molecule sequencing without amplification. Finally, in the data analysis phase, bioinformatics tools come into play. These tools assemble the sequence reads, align them to a reference genome, and decipher the exact order of nucleotides. This comprehensive process yields precise DNA sequences, providing invaluable insights for diverse applications in genomics.

Bioinformatics tools are instrumental in analyzing the obtained DNA sequences. They facilitate the assembly and alignment of reads to a reference genome, enabling the identification of variations, mutations, and specific genetic markers. Moreover, bioinformatics software predicts the functions and structural features of genes and proteins encoded by the DNA sequences. These tools also contribute to the identification of regulatory elements, non-coding regions, and genetic motifs within the DNA sequences. In essence, bioinformatics plays a pivotal role in interpreting and extracting valuable information from DNA sequences, enriching our understanding of genomics and supporting advancements in sequencing technologies.

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Proteins encoded by corona virus (SARS-CoV2)

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by Swikriti Adhikari, Pratika Regmi, and Sangharshika Chaudhary

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the betacoronavirus of the family Coronaviridae that causes coronavirus disease 2019 (COVID-19). It is an enveloped and bear a non-segmented, positive sense, single-stranded RNA. This 30 kb genome (RNA)  encodes 14 open reading frames, Orfs (Orf is the part of the genetic material that has the ability to be translated). In particular, the 5’ Orf1a / Orf1ab of SARS-CoV-2 encodes for a single polyproteins, which is cleaved by proteolysis of virus’s own protease enzyme and fragmented into 16 non-structural proteins (Nsp1–16) creating the replicase / transcriptase complex (RTC). The proteins encodes by SARS-CoV-2 that are structural proteins, non-structural proteins and accessory proteins.

1. Structural Proteins
Structural viral proteins usually make up the outer coat of the virus and help in packaging of genome. SARS-Cov-2 encodes for four structural proteins and are encoded by the 3’-terminus of the viral genome. These are: 

Spike (S) glycoproteins: It is glycoprotein that gives crown like appearance thus named corona. It represent the largest structures of the virus and are essential for the entry into host cells. S protein attaches to the receptor protein ACE 2 (Angiotensin converting enzyme 2) of host alveoli and facilitate entry. Spike protein together with Hemaglutin esterase (HE) protein assist in entry to the human cell.

Envelope (E) proteins: E protein interacts with membrane protein to form the viral envelope. There is 2 types of  Envelope protein E1 & E2.  E1 is matrix glycoprotein transmembrane protein where as E2 is peplogenic glycoprotein. They are only present in small quantities and most likely function as ion channels, not necessarily needed for viral replication but essential for pathogenesis. 

Membrane/Matrix (M) proteins: They are the most abundant proteins in the virus structure and are responsible for viral membrane curvature and binding to the nucleocapsid.

Nucleocapsid (N) proteins: They bind to the viral RNA genome and ensure the maintenance of the RNA in a ‘beads-on-a-string’ conformation.

   Figure 1: Structural Proteins of SARS-CoV-2 (Picture source: biovendor.com)
  

2. Non-structural Proteins
The non-structural proteins are produced by the 5’-terminal end of the genome. Majority (~66%) segment of viral RNA located in the first ORF translates two polyproteins, pp1a and pp1ab, and encodes 16 non-structural proteins (NSP1-16), while the remaining ORFs encode accessory and structural proteins. The 16 non-structural protein includes;

1. NSP3: Papain-like protease-Plpro, supplement polyproteins
2. NSP5: Main protease-Mpro, also called Chymotrypsin-like protease (3CLpro): cuts the polyproteins translated from viral RNA to yield functional viral proteins.
3. NSP7-8: Primase complex,
4. NSP12: RNA-dependent RNA polymerase-RdRp
5. NSP13: Helicase-Hel
6. NSP14: 3′–5′ Exoribonuclease-ExoN, initiates cap formation
7. NSP15: Endoribonuclease-NendoU
8. NSP16: N7- and 2’O-methyltransferases: modify the cap of viral RNAs
9. other NSPs (1,2,4,6,9,10,11)

Figure 2: Non-structural proteins (upper) and spike protein (lower) encoded by SARS-CoV-2
Picture Source: Viruses 2019, 11(1), 59; https://doi.org/10.3390/v11010059

Nonstructural proteins of corona-viruses and their function

Nonstructural protein (nsp)	functions
nsp1 & 3	Inhibit interferon signaling and block host innate immune response by promotion of cellular degradation and also blocks translation of host’s RNA
nsp2	Binding to proinhibition protein
nsp3 & 5	Promoting cytokine expression and cleavage of viral polyprotein
nsp4 & 6	Contribute to structure of DMVs as transmembrane scaffold protein (DMVs formation)
nsp7/8 complex	Processivity clamp for RNA polymerase by orms hexadecameric complex
nsp9	RNA binding protein phosphatase
nsp10, 16 & 14	Stimulation of ExoN and 2-O-MT activity
nsp12	Replication enzyme (RNA-dependent RNA polymerase)
nsp13	RNA helicase, 5′ triphosphatase
nsp14	Proofreading of viral genome
nsp15	Viral endoribonuclease and chymotrypsin‐like protease
nsp16	Avoiding MDA5 recognition and inhibit innate immunity regulation

3. Accessory Proteins
Apart from structural and non-structural proteins, SARS-CoV-2 encodes a group of unique proteins called accessory proteins. They help to change the environment inside the infected cell to make it easier for the virus to replicate. Many of these accessory proteins have no known homologues. The SARS-CoV-2 genome has an unusually high number of accessory genes in the 3′-end of the genome. The accessory proteins present in SARS-CoV-2 are named under the specific position of the ORFs and they are:

3a protein
3b protein
6 protein
7a protein
7b protein
8a protein
8b protein
9b protein
   

Applied Biotechnology

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Modern Technique in Biochemistry

1. Describe how affinity chromatography is useful for protein purification. 7
2. Describe the size exclusion gel chromatography in detail.  7
3. Describe the principle, procedure and uses of  paper chromatography.  8
4. What is immunodetection? Describe the fluorescence immunoassay.  2+6
5. Tabulate the difference between RIA and ELISA.    7
6. Describe the principle and instrumentation of PCR.    3+5
7. Explain about the vectors in gene cloning.   7
8. Describe the principle and uses of western blotting.  7
9. Write the Nernst equation.  Write the application of biosensor.  2+5
10. Describe the mechanism of pH electrode.    8
11. What is electrophoretic mobility (μ)? Describe the agarose gel electrophoresis.    2+7
12. How PAGE is prepared, explain.        6
13. What is redox reaction? Write the application of redox couple.    2+5
14. Describe the principle of Clark’s oxygen electrode.    8
15. What is bioinformatics? Mention the scope of bioinformatics in clinical diagnosis.   2+5
16. Enlist the scope of biotechnology.     8

Write a short note    5

• Ion selective electrode
• Hybridization
• DNA sequencing  
• Column chromatography
• Capacity factor in chromatography
• Fluorescence polarization immunoassay
• Electrochemiluminescence

BCM BT513-II Biochemistry Model Question.

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Model Questions for Biochemistry (M.Sc. Biotechnology, I-Semester, set II)

Long Answer Question: (7.5 × 1 = 7.5) (any one)

1. Write the structure and function of polysaccharides in the living organisms.
2. Classify the lipids and describe the structure of saturated and unsaturated fatty acids.
3. Explain the major enzyme-catalyzed reactions with examples.
4. What is PUFA? Describe the structure and functions of steroids.
5. Write the structure and function of mono-saccharides.
6. Define Michaelis-Menten expression for single substrate single enzyme catalyzed reaction. What is the significance of Michaelis constant?

Short Answer Question: (2.5 × 3 = 7.5) (any three)

1. Write a short note on competitive inhibitor.
2. What are the biological roles of nucleic acids?
3. Describe the structure and biological role of glycoprotein.
4. Explain the two major hypotheses for mode of enzyme action.
5. Define enzyme. Describe Six-major types of enzyme-catalysed reaction.
6. Suppose we have a mixture of an enzyme that contains total number of enzyme 0.1M. At this concentration (when all active site are filled), the maximal rate of reaction is 60,000M/S. Calculate the turnover number of that enzyme and time it takes to convert one substrate into a product molecule.
7. Draw the structure of lactose.
8. Draw the structure of sucrose.
9. Write a short note on starch.

Very Short Answer Question: (1 × 5 = 5) (any five)

1. Write the Michaelis-Menten equation.
2. Write the Henderson and Hasselbach equation.
3. What do you mean by Turnover Number of an enzyme?
4. Define cofactor and coenzyme.
5. What is an isoenzyme?
6. What do you mean by steady state assumption?
7. Draw the structure of alpha-D-glucose.
8. Draw the structure of alpha-D-fructose.
9. Draw the structure of dGTP.
10. What is glycogenin?
11. Write a short note on sphingolipid.

 

Molecular and Cell Biology; and Genetics

Long Answer Question: (8)

1. Describe the initiation of the replication process.
2. Describe the structure and type of gene.
3. What is a nucleosome? Describe the formation of a solenoid (condensed fiber) of DNA.
4. What are the functions of RNA? Describe mRNA.
5. Describe transcription briefly (in Prokaryotes).
6. What are the Post-transcriptional Modifications? Describe in brief.
7. Describe the initiation of the translation process in Prokaryotes.
8. What are the Post-translational Modifications? Describe in brief.
9. Describe the type of mutation and results due to base substitution. 
10. Describe in  brief about the chemical mutagens.
11. Describe the lac operon system.
12. How is gene regulation promotor work in prokaryotes? illustrate with diagram.
13. What is DNA cloning? Describe restriction enzymes (type II endonuclease).
14. Illustrate with diagram of pUC19 and pBR322 as cloning vector.
15. What are the applications of genetic engineering in biomedical sciences?
16. Describe phenol-chloroform extraction method of DNA isolation.
17. Describe the southern blot hybridization.
18. Describe the steps in PCR.
19. Highlight some gene editing techniques.
    
    

Short Answer Question: (5)

1. Enlist the enzymes involved in the replication of DNA.
2. Describe the proof-reading process of DNA polymerase.
3. Describe tRNA.
4. Draw the structure of adenine and cytosine. 
5. Write a very short note on the human genome. 
6. List the RNA polymerases and their product in Eukaryotes.
7. Differentiate between RNA polymerase and DNA polymerase.
8. Describe briefly inhibitors of transcription and their mode of action in Prokaryotes.
9. What are the genetic codes and their characters?
10. Describe translocation process during protein synthesis.
11. Shine-Dalgarno sequence. 
12. What is base excision repair.
13. TATA binding protein.
14. Inhibitors of DNases.
    
    

BCM 513-I List of Practicals

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List of Practicals (M.Sc. Biotechnology, I-Semester)

1. Preparation of normal, molar, Parts per million (PPM), and percent solutions.
2. Preparation of different buffer solutions and measure their pH.
3. Estimation of given protein concentration by Bradford, Lowry (Folin) protein assay and by Biuret test.
4. Determination of the isoelectric point of casein (of milk).
5. Titration of amino acids and find the physical constants.
6. Qualitative detection of glucose in urine (the Trommer reaction).
7. Quantitative determination of glucose in blood by a glucose-oxidase method.
8. Determination of total cholesterol concentration in serum.
9. Determination of urea concentration in serum and urine.
10. Determination of saponification value of the fat.
11. Qualitative tests for carbohydrates(Iodine test, Benedict’s test, Barfoed’s  test, Seliwanoff’s test, Bial’s test)
12. Separation of amino acids by chromatographic techniques and detection by nin-hydrin test.
13. Separation of lipids by TLC, and detection.

BCM 513-I Biochemistry Model Question

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Model Questions for Biochemistry (M.Sc. Biotechnology, I-Semester)

Long Answer Question: (7.5 × 1 = 7.5) (any one)
1) Explain the biological functions of proteins with examples.
2) Explain the essential amino acids.
3) Explain the Ramachadran plot.

Short Answer Question: (2.5 × 3 = 7.5) (any three)
1) Describe how peptide bond is formed.
2) Draw the structures of non-polar and non-charged aliphatic amino acids.
3) Mention the the enzymes and co-factors involved in hydroxylation of proline and lysine in collagen synthesis.
4) Differentiate between fibrous and globular proteins.
4) Describe the structure of collagen.

Very Short Answer Question: (1 × 5 = 5) (any five)
1) Define denaturation of proteins.
2) Which bonds are flexible for rotation in a dipeptide?
3) Mention the replaced amino acids that leads to sickle cell anaemia.
4) Define the quaternary structure of proteins.
5) What are the major amino acids in collagen?
6) Describe the supersecondary structure of protein.
7) Describe in brief about the structure of haemoglobin.
8) What are chaperones.

Model Questions for Biochemistry-IV

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for M.Sc. Biotechnology
Long Answer Questions        (@7)

1. Describe the regulation of cholesterol synthesis.
2. Describe the terpenoids biosynthesis.
3. Describe the reaction that involves pyridoxal-5-phosphate and explain the mechanism of any one of them.
4. Describe the beta-oxidation of odd-chain fatty acids.
5. Describe the gluconeogenesis pathway and its regulation.
6. Describe the degradation of purine.
7. Describe the biosynthesis of flavonoids.
8. Describe the urea cycle.

Short Answer Questions      (@2)

1. Write a brief note on fate of shikimate in metabolism.
2. Why alkaloids are imporatant?
3. Describe the metabolic role of niacin.
4. Describe the link between TCA and urea cycle.
5. Explain the mechanism of digestion of carbohydrate in brief.
6. What are steroid? Mention their role in metabolism.
7. What are the pharmacological role of coumarin?
8. Write a short note on monoterpenes.
9. Describe allelopathy.
10. Biosynthesis of ribose sugar.

Very Short Answer Questions                             (@1)

1. Write a short note on carbamoyl phosphate synthetase?
2. Enlist some organic reaction mechanisms.
3. Function of biotin.
4. What are saponin?
5. Write a short note on flavanone.
6. Glucocorticosteroid
7. Role of carnitine.
8. PUFA
9. Ascorbic acid
10. Cholecalciferol

Model Questions for Pharmacology

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Model Questions for Pharmaceutical Microbiology (Food Microbiology)

1. What are the sources of impurities in pharmaceutical products? 
2. Explain the “bio-assay” linking with it to the drug discovery process. 
3. Highlight about the spoilage of pharmaceutical raw materials. 
4. What is pharmacopoeia and monograph development? Highlight about it in the context of Nepal.
5. Mention some quality assurance and management practices for pharmaceutical products. 
6. Describe preservation methods of pharmaceutical products. 
7. What are the factors affecting microbial spoilage of pharmaceutical products? How the humidity is crucial for it? 
8. What are the biological indicators of sterilization? Explain any two of them. 
9. Explain some methods of testing for the presence of antibiotics in meat. 
10. Explain some in vitro assays that are useful in drug discovery. 


11. What are the routes of drug administration?
12. What is pharmacodynamics? Explain with paracetamol (acetaminophen).
13. What are the "potency and efficacy" of drug? Explain with examples.
14. What is therapeutic index? Explain and draw a graph for warfarin.
15. How can we select a molecule to be taken as a medicine? Explain some theoretical aspects.


16. What are the components of augmentin? Explain the mode of action of augmentin. 
17. What are the criteria to select the antibiotics as the drug of choice? 
18. Enlist some biological sources and the antibiotics extracted/isolated from them. 
19. What are super-bugs? “Overuse of antibiotics may create super-bugs.” Explain the statement.
20. Describe the MIC and MBC. Explain some methods of searching new antibacterials.

Active Nepalese Blogger and Future of Blogging

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There were hundreds of Nepalese blogger writing in Nepali and English from Nepal and abroad. Despite many hurdles, political imbalances, difficulty in internet and electricity access; many of them were frequently feeding us. However, the majority of them disappear from blogverse (Universe of blog), nowadays. Mainly due to Facebook and Twitter, which are proving platforms for mini and micro-blogging, several online newspapers, and may be due to chaos in the country.

Country is suffering from unannounced blockade making distasteful festivals and life. We are struggling to get minimum living things such as gas for cooking, vehicles, even medicines, just a few to mention here. We have to spend majority of time to get such minimum living standard impeding for other creativity. However, still some blogger are paying more attention to the aesthetic value of blog for Nepalese and promulgating voluntarily. According to contribution since past month, the following are active blogs for the Nepalese by Nepalese.  News blog, copy-paste blog, the blog which are filled only with personal bio-data, and malfunctioned or virus infected blogs are removed from the list. Please, suggest me if remains to be included.

1. साहित्य संग्रहालय 2. मेरो संसार 3. केही आफ्नै कथा, ब्यथा अनि भावनाहरु 4. समय र परिवेश 5. नेपालप्लस 6. सापेक्ष बुझाई 7. रामकुमार परियार 8. Sarad Kumar Gauchan's DIARY 9. Nepalvidhya 10. Tales of Sun and Rain (घामपानीका कहानी) 11. hataru/हटारु 12. Apawad 13. अनलाइन कम्प्युटर इनिस्टीच्युट 14. अभिनव.कम  15. गाउँले मन 16. मेरो चौतारी डट कम 17. DAUTARI :: दौंतरी

18. शब्द (Shabda) ... 19. SCIENCE TO SOCIETY 20. Aakar Post 21. Bikash Koirala | What I Feel 22. मेरो खेस्रा whatever i feel  23. Palpali Milan 24. मेरो आफ्नै सेरोफेरो 25. शब्द चित्र डट कम 26. शहरबाट शब्दहरु 27. साहित्यः जीवनको निम्ति 28. मेरो ब्लग 29. समकालीन साहित्य 30. Rabindra Neupane 31. यात्रीको ब्लग 32. merokalam.com 33. प्रगतिशील लेखक सङ्घ, नेपाल 34. Kapilvastu Day Blog

Notable blogs, which were active in the past, are here. Hopefully, these will be reactivated again.

मेरो विचार र अभिव्यक्तिको साझा चौतारी
क्यामज्जा
☽कृष्णपक्ष☆
दूर्जेय चेतना
साइबर चौतारी
Because of gradual loss of enthusiasm in blogging and many hurdles in Nepal, blogverse may shrink in future, however, it gives immense self-satisfaction.

Model Questions for Immunology

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Model Questions for Immunology (MSc Microbiology, Faculty of Science, TU, Nepal)
Long Answer Questions (10)
   1. Explain in brief about type III hypersensitivity. Differentiate between immediate and delayed type hypersensitivity.

Short Answer Questions (5 marks for each)
   2. What is autoimmunity? Explain with some abnormal physiology in human body.
   3. Explain the entry of HIV in CD4 cell.
   4. Write in brief about immunotolerance.
   5. Explain the mechanism of tissue injury in type I hypersensitivity.

Very Short Answer Questions (2 marks for each)
   6. Describe the neonatal jaundice in brief.
   7. What is myasthenia gravis?
   8. What is arthus reaction?
   9. Describe in brief about chronic granulomatous disease.
   10. Explain the manifestation of mutations occur in the Bruton's tyrosine kinase (Btk) gene.
   11. Enlist some diseases due to congenital immunodeficiency.

MCQs (1 mark for each)
1. Type I hypersensitivity can be blocked using:
      A  Histamine.
      B  An IgA myeloma.
      C  A myeloma protein of mixed antibody class.
      D  Interleukin-5.
      E  Sodium cromoglycate.

2. Mycoplasma pneumoniae infection can lead to:
      A  Hashimoto's thyroiditis.
      B  Cold hemagglutinin disease.
      C  Agranulocytosis.
      D  Thrombocytopenic purpura.
      E  Neonatal alloimmune thrombocytopenia.

3. The term reactive lysis usually refers to a sequence of events involving:
      A Complement.
      B Natural killer (NK) cells.
      C Cytotoxic T-lymphocytes (CTL).
      D Antibody-dependent cellular cytotoxicity (ADCC).
      E Th1 cells.

4. Which type of hypersensitivity cannot be transferred with serum antibody?:
      A  Type I.
      B  Type II.
      C  Type III.
      D  Type IV.
      E  Type V.

5. Anaphylaxis can be triggerred by cross-linking of IgE receptors on:
      A  Monocytes.
      B  Mast cells.
      C  B-cells.
      D  Eosinophils.
      E  Neutrophils.
         Source: Roitt's Essential Immunology, 12^th Edition.

Structure, properties and classification of amino acids

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Some key-words are:
Proteins
What is the structure ?
What is the role of Proteins?

1. The function of a protein is intrinsically dependent on its structure.
2. Since its structure is maintained by combinations of chemical interactions which are individually weak and flexible, the environment in which a protein finds itself can affect its structure.
3. Heat and pH affect protein structure.
4. The cell uses factors such as phosphorylation or ligand-binding to affect the structure, and therefore the function, of many proteins

Amino Acid

1. The first discovered Amino Acid was asparagine, in 1806.
2. The last, threonine, 1938.
3. All the amino acids have trivial or common names, in some cases derived from the source from which they were first isolated.
4. Asparagine was first found in asparagus,
5. Glutamate in wheat gluten;
6. Tyrosine was first isolated from cheese (its name is derived from the Greek tyros, “cheese”); and glycine (Greek glykos, “sweet”) was so named because of its sweet taste.

Essential Amino acids: They are not synthesized by the body and must be supplied from the diet. These are; Isoleucine, Leucine, Lysine, Methionine, Phenylanine, Threonine, Tryptophan, Valine, Arginine*, and Histidine*
PVTtim H*A*LL (private team  hall)
*Semi essential amino acid
Rest of the materials are found in pdf file. Please, wait in case of slow internet.

Model Questions for Biochemistry-III

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 Model Questions for Biochemistry (Microbiology) 
1. Explain in brief about lipolysis.
2. Explain the urea cycle.
3. What is the salvage pathway, enlist the steps?
4. Why TCA is said to be amphibolic?
5. What are the ketone bodies? Explain their metabolism.

6. What are enzymes, explain with the classification?
7. Give some examples of the special molecules of the body whose precursor are amino acids.
8. Enlist the enzymes involved in TCA cycle and estimate the number of ATP generation.
9. Describe the stoichiometry for palmitic acid oxidation.
10. What are the differences between fatty acid biosynthesis and breakdown.

11. Derive Michaelis-Menten equation.
12. What are coenzymes and isozymes? Explain in brief with examples.
13. Explain pyruvate dehydrogenase complex.
14. What is the glyoxylate cycle? Show its relationship to the citric acid cycle.
15. Explain the De novo synthesis of Pyrimidine (steps only).