BI August ’23 Viva

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Table Of Contents
  1. Chemistry of Nucleic acid
  2. Structure of DNA, RNA & Cell Cycle
  3. Metabolism of Nucleic acid
  4. DNA Replication
  5. DNA Repair & Mutation
  6. Transcription
  7. Blotting Techniques
  8. Gene Therapy
  9. Translation
  10. Gene Expression
  11. PCR & RFLP
  12. Recombinant DNA Technology

Chemistry of Nucleic acid

  1. Name the purines. Adenine, Guanine
  2. Name the pyrimidines. Thymine, Cytosine, Uracil
  3. Difference between nucleoside and nucleotide. Nitrogenous Base + Sugar = Nucleoside. Nucleoside + Phosphate = Nucleotide
  4. Name the Synthetic nucleotides and their uses. Cytarabine for chemotherapy, Azidothymidine for AIDS, Azathioprine as immunosuppressant, 5-iododeoxyuridine as antivirus.
  5. Functions of Cyclic AMP. Second messenger for hormones like epinephrine, glucagon, etc. Stimulates lipolysis and glycogenolysis.
  6. Give an example and function of biologically important nucleotide? ATP is the energy currency. AMP is a part of many essential coenzymes. cAMP is a secondary messenger, GTP is an energy source for protein synthesis.
  7. Define Chargaff’s rule.  Purines = Pyrimidine, Purine: Pyrimidine = 1:1, A = T, G = C,
  8. Name the bond which holds the two strands of DNA together. Hydrogen Bond
  9. Types of RNA. mRNA, tRNA, rRNA.

Structure of DNA, RNA & Cell Cycle

  1. Name the Nucleotides in DNA. ATGC
  2. Name the Nucleotides in RNA. AUGC
  3. Structure of DNA. Double stranded helical, antiparallel, complementary pairing
  4. Structure of tRNA. Single stranded, unbranched linear polymer of ribonucleotides joined by 3`-5` Phosphodiester Bonds

Metabolism of Nucleic acid

  1. What is the difference in the structure of uracil and thymine? Thymine has an additional Methyl group on its 5th carbon.
  2. Name the sources of carbon and nitrogen atoms in Purine ring. Glutamine (N3, N9), Glycine (C4, C5, N7), Aspartate(N1), N-10-Formyl THF(C8), CO2(C6)
  3. Name the sources of carbon and nitrogen atoms in the Pyrimidine ring. Aspartate (N1, C4, C5, C6)
  4. What is the rate limiting enzyme of purine synthesis? Formation of 5-Phosphoribosylamine from PRPP with the help of enzymes PRPP Glutamyl Amido Transferase and the amide group Glutamine.
  5. Where does salvage pathway of purine synthesis take place? Brain, Erythrocytes, Polymorphonuclear Leukocytes, and Bone Marrow
  6. What is uric acid? End product of Purine Catabolism
  7. What is normal serum uric acid levels in males and females? 3-7 g/dl (males). 2-5 g/dl(females)
  8. What is gout? A disorder related to defects in Uric Acid metabolism characterized by elevated level of uric acid in the serum either due to increased production or decreased excretion of uric acid.
  9. What are Tophi? Tophi are depositions of Monosodium Urate crystals deposited in case of Gout in the cooler areas of the body. The 1st metatarsophalangeal joint in the great toe is principally involved (Condition called Podagra Gout).Other areas of deposition are knee joint, ankle joint, etc.
  10. Name the enzyme defective in Lesch-Nyhan syndrome. Complete deficiency of HGPRT- Hypoxanthine Guanyl Phosphoribosyl Transferase
  11. What is the cause of SCID? Decreased levels of Adenosine Deaminase (ADA) leading to T-cell and B-cell defect and thus decreased uric acid levels.
  12. What is the mechanism of action of anti-cancer drugs 5-flurouracil & 5- lodouracil? They are pyrimidine analogues which are competitive inhibitors of Thymidylate synthase and thus inhibit DNA replication.
  13. Name the enzyme deficient in orotic aciduria. UMP Synthase (OPRTase + OMP decarboxylase). OPRTase = Orotic Phosphoribosyl Transferase. OMP = Orotine Monophosphate.
  14. List the characteristic findings in orotic aciduria. Urine is positive for Orotate Crystals. Deficiency of UMP Synthase, Failure to thrive, Megaloblastic Anemia, Remission with Uridine.

DNA Replication

  1. What is replication? Formation of Daughter DNA molecule from parent.
  2. Name the enzymes involved in replication and their functions. Primase (Attached RNA primer at 3`end), DNA Polymerase III and II (5`3` Polymerase activity + 3`5`Exonuclease activity with proofreading), DNA polymerase I (5`3` Polymerase + 5`3`Exonuclease activity with proofreading). DNA polymerase II also has repair activity.
  3. What are the differences in replication between prokaryotes and eukaryotes? Prokaryotic replication has 3 DNA polymerase (Eukaryotic- 5) and single ORI site (eukaryotic- multiple) with faster replication and longer Okazaki fragments.
  4. In which direction does DNA synthesis occur? 5`-3`
  5. What is leading strand? The strand on which a single RNA primer attaches at the 3`end and replication takes place continuously.
  6. What is lagging strand? The strand running in 5`-3` direction on which DNA polymerase III acts in discontinuous manner thus producing many Okazaki fragments.
  7. What are Okazaki fragments? On the lagging strand, polymerization takes place in a discontinuous manner requiring multiple RNA primers. As fork moves, the primers are extended by DNA polymerase III into short pieces of DNA called Okazaki fragments.
  8. What is proof reading of DNA? 3’-5’ exonuclease activity of DNA polymerase I, II and III and 5`-3` exonuclease activity of DNA polymerase I is called proofreading as it keeps the error in replication to a very low level. DNA polymerase I and II excise the mismatched nucleotide.
  9. Name inhibitors of replication.  DNA gyrase inhibitors (Ciprofloxacin, Norfloxacin), Topoisomerase I Inhibitor (Topotecan). Topoisomerase II inhibitor (Doxorubicin, Daunonibicin)
  10. What is mitochondrial DNA? DNA present in Mitochondria. It is maternally inherited. Synthesized by Prokaryotic DNA polymerase II and Eukaryotic DNA polymerase ɣ.
  11. What are the phases of Cell cycle. G1, S, G2 and M.
  12. Where are the check points in cell cycle? G1 to S, G2 to Mitotic Phase (Maturation Promoting Factor-MPF). Metaphase to Anaphase (Anaphase Promoting Factor-APF)

DNA Repair & Mutation

  1. Causes of DNA damage. Copying errors where 1-5 bases are unpaired, chemical damage to a single base or a segment, attack by free radicals and radiation.
  2. Types of DNA repair. Mismatch repair, base excision repair, nucleotide excision repair, double strand break respectively.
  3. Diseases associated with DNA repair. Xeroderma Pigmentosum due to defect in  NER= Nucleotide Excision Repair. Autosomal Recessive. Patients are photosensitive and susceptible to skin cancer.
  4. Define mutagens. Any agent which will increase DNA damage or cell proliferation cause increased rate of mutations are called mutagens. X-ray, gamma-ray, UV ray, acridine orange, etc. are well-known mutagens.
  5. Types of mutagens. Physical (X-rays, gamma rays, UV rays, Alpha particles), Chemical (Arsenic, Acridine Orange, Benzene) and Biological (Transposons, Viruses)
  6. Define mutation. Gross or subtle but abrupt and spontaneous change in nucleotide sequence of DNA.
  7. Types of mutation.  2 types- Point and Frame Shift
  8. Types of Point mutation. Silent, Missense, Nonsense (Thalassemia) 
  9. Examples of Transition and Transversion. Transition (Same Type: A⥐G, C⥐T). Transversion (Different Type: A⥐T, A⥐C, G⥐T, G⥐C)
  10. What is Frame shift mutation?  Due to insertion or deletion, from that point onwards, the reading frame shifts and changes all the triplet codons thereafter.
  11. Effects of mutation. Lethal (intrauterine death of embryo since alpha chain not produced due to mutation). Silent (no metabolic effect). Carcinogenic, Beneficial (Basis of Mutation, Tryptophan rich Maize)

Transcription

  1. Define transcription.  Transfer of information from DNA to RNA.
  2. Name the promoters of transcription in prokaryotes. Pribnow Box (TATAAT, -10bp).
  3. Name the promoters of transcription in eukaryotes. Hogness Box (TATA, -25bp)
  4. Name the enzyme required for transcription in eukaryotes. RNAP = RNA Polymerase (I, II and III), Helicase,
  5. What are the post-Transcriptional modifications of mRNA? 5` Capping, 3` Poly-A-Tail, Splicing, Alternate Splicing, mRNA editing.
  6. What is reverse transcription? Formation of DNA from RNA.
  7. Give another name of reverse transcriptase. RNA dependent DNA Polymerase.
  8. Give the importance of capping and tailing. Capping functions in protein biosynthesis and tailing enhances translational efficiency. Both provide stability to mRNA.
  9. 10. Give examples of inhibitors of transcription. Alpha-amanitin, Rifampicin (TB Drug), Dactinomycin(Cancer Drug)
  10. How is transcription terminated in prokaryotes? RHO Independent/Intrinsic ( Due to Poly “U” part and Due to Hairpin loop formation at high GC region), RHO dependent (ATP dependent RNA stimulated helicase activity of Rho factor causes degradation of RNA)

Blotting Techniques

  1. Define probes. A single strand of RNA or DNA molecule (15-30 bp) tagged with a radioactive or non-radioactive molecule used to detect complimentary nucleotide sequence.
  2. Types of probes. cDNA probe, genomic DNA probe, antibody probe, chemically synthesized oligonucleotides, chemically synthesized RNA, Radioactive, Non-Radioactive, Fluorescent.  
  3. Types of Blotting techniques. Northern, Southern, Western.
  4. Steps in Southern blotting. Extraction of DNA, Digestion by Restriction Enzyme, Separation by Agarose or Polyacrylamide Gel electrophoresis, Denaturation by Alkali, Transfer to Nitrocellulose paper, Addition of Probe, Autoradiograph Obtained showing specific bands corresponding to DNA fragment.
  5. Steps in Northern blotting. Extraction of RNA, Separation by Agarose or Polyacrylamide Gel electrophoresis, Transfer to Nitrocellulose paper, Addition of Probe, Autoradiograph Obtained showing specific bands corresponding to DNA fragment. [NB: Alkali not used since it causes denaturation of RNA]
  6. Steps in Western blotting. Electrophoresis, Transfer to Nitrocellulose paper, Probing with labelled antibodies.
  7. Applications of Southern blotting.  Determining number of copies of a gene present in a given tissue and mutations in the gene. E.g., Detection of HbS (Sickle Cell Anemia), DMD (Duchenne’s Muscular Dystrophy), PKU (Phenylketonuria), HBV (Hepatitis B Virus), etc.  
  8. Applications of Northern blotting. To size and quantitate specific RNA molecules.
  9. Applications of Western blotting. To size and quantitate specific protein molecules. Test for AIDS and Ebola virus.

Gene Therapy

  1. What are Restriction Endonucleases (RE)? Endonuclease enzymes which cut DNA of any source in a sequence specific manner.
  2. Steps in Recombinant DNA technology. Isolation of DNA,  Fragmentation of DNA by RE, Isolation of specific human DNA, Insertion of isolated human DNA into vector to form chimeric or hybrid DNA molecule, Joining of 2 different cut DNA fragments by DNA ligase.
  3. Define vectors. DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called as Vector.
  4. Examples of vectors. Plasmids, Bacteriophage, Cosmid, BAC ( Bacterial Artificial Chromosome), YAC (Yeast AC), MAC (Mammalian AC),  
  5. How are genes introduced (transfected)? Chemically (Liposome mediated gene transfer, Calcium & Phosphorous mediated gene transfer, Ethylene Glycol mediated gene transfer). Physical (Electroporation, Gene Gun, Particle bombardment), Biological (Transduction, Transformation, Conjugation)
  6. Diseases cured by Gene therapy. ADA Deficiency, SCID = Severe Combined Immunodeficiency Syndrome, Sickle Cell Disease, Thalassemia.
  7. Diseases cured by RDNA technology- Diabetes Mellitus (Humulin), Hepatitis B Vaccine, Anemia (Erythropoietin), Neutropenia (Filgrastim), Viral Infections (Interferons), RCC=Renal Cell Carcinoma (Interleukins), Burns (EGF = Epidermal growth Factors).

Translation

  1. What is codon?  Codons are a group of three adjacent bases that specify the amino acids of a protein.
  2. What are the salient features of genetic code? Triplet in Nature, Universal(Same codon codes same Amino acid in all living organisms), Unambiguous(Each codon codes for only one amino acid- OR- No two amino acids have same codon), Degenerate(more than one codon can code for the same amino acid), Non-Overlapping(A base in sequence is part of one codon only), Comma less(each codon is immediately followed by the next), Start Codon, Stop Codon.
  3. Name the start codon. AUG (Methionine in Eukaryotes and N-formyl methionine in prokaryotes), GUG (Sometimes -normally codes for valine, bit when start- it codes for methionine)
  4. List the stop codons. Ochre (UAA), Amber (UAG), Opal (UGA)
  5. What is the wobbling phenomenon?  A change in nitrogen base at the 3rd position does not normally cause any change in expression of codon because sometimes the anticodon recognizes more than 1 codon because of which the codon is mostly read by the first two nitrogen bases. The 3rd nitrogen base does not affect the reading of the codon and this position is known as “Wobble” Position. Concept given by “Crick”.
  6. What is translation? The pathway of protein biosynthesis  
  7. What are the basic requirements of the translation process? mRNA, tRNA, Ribosomes, ATP, GTP, Enzymes, Specific Protein Factors (IF- Initiation Factor, EF- Elongation Factor)
  8. List the stages of translation. Activation of Amino Acid, Initiation, Elongation, Termination.
  9. Name the steps in initiation of translational process. Ribosomal dissociation, formation of 43S pre-initiation complex, formation of 48S initiation complex, formation of 80S initiation complex.
  10. List the post-translational modifications of translation. Proteolysis and Covalent Modification, Amino Terminal Modification, Loss of Signal Sequence.
  11. What is proteolytic processing in post-translational modification? Removal of Initiator Methionine, Removal of Signal Peptide, and Activation of Latent Protein/Proprotein (E.g., Pepsinogen→Pepsin, Procarboxypeptidase→Carboxypeptidase)
  12. List the inhibitors of translation. Both Eukaryotic & Prokaryotic (Puromycin), Only Prokaryotic (Streptomycin, Erythromycin, Neomycin, Chloramphenicol, Tetracycline & Doxycycline), Only Eukaryotic (Cycloheximide, Ricin, Diphtheria Toxin)
  13. Mechanism of action of streptomycin. Binds to 30S ribosomal subunit blocking initiation and elongation, Causes mRNA misreading.
  14. Mechanism of action of erythromycin. Binds to 50S ribosomal subunit and blocks translocation through eEF-2(Eukaryotic Elongation Factor-2)
  15. List the toxins that inhibit eukaryotic translational process. Cycloheximide, Ricin, Diphtheria Toxin

Gene Expression

  1. What are constitutive genes? Genes which are always expressed, and which are not regulated. Also called housekeeping genes.
  2. What is a cistron? Basic unit of gene expression which encodes one polypeptide chain. Equivalent to “gene” in classical genetics.
  3. Name the set of genes in Lac Operon. Lac Z, Lac Y, Lac A.
  4. Which are the positive modulators of Lac Operon?  Lactose, Isopropyl Thiogalactoside (Gratuitous Inducer)
  5. What is epigenetic regulation? Changes in characteristics of a cell or an organism that are not due to changes in the nucleotide sequence of DNA but rather through modulation of chromatin structure via histone modification or DNA methylation. These modifications are also inheritable.
  6. What is gene amplification? Mechanism by which gene expression is increased several times. E.g., Resistance to Methotrexate (Inhibitor of dihydrofolate reductase) is developed by cancerous cells by gene amplification in cancer patients who have been taking this drug for a long time.
  7. What is m RNA editing?  0.01% of mRNAs undergo editing leading to changes in coding information. Apo-48 (Present in Chylomicrons) and Apo-100 (Present in VLDL, LDL) both are derived from the same gene Apo-B. But in the Intestines, the Apo-B mRNA undergoes editing. CAA→UAA thus causing premature termination of translation leading to form a shorter polypeptide. Whereas in liver no such modification takes place and so full polypeptide Apo-100 is produced.

PCR & RFLP

  1. What is PCR? PCR stands for Polymerase Chain Reaction. It is a cell free technique (In-vivo) of DNA or gene amplification that proceeds in a continuous and exponential manner.  
  2. What are the steps of PCR?  Denaturation, Annealing (=Renaturation), Synthesis (=Extension).
  3. Which enzyme is used for PCR and why?  Taq DNA polymerase enzyme is used because it can withstand an extremely high temperature of 90°-95°C.
  4. What are the applications of PCR?  Viral load of Covid-19 and HIV, HIV detection in latency period, TB diagnosis, Diagnosis of prenatal disease like Sickle Cell and Thalassemia, DNA fingerprinting, Gene library.  
  5. What is DNA fingerprinting?  Also known as DNA profiling, it is analysis of DNA base sequence in the DNA of an individual.
  6. Name the DNA Markers. RFLP, VNTR, STR, SNP. (Random Fragment Length Polymorphism-Earlier Used), (Variable Number of Tandem Repeats), (Simple Tandem Repeats OR Microsatellites), (Single Nucleotide Polymorphism – Nowadays used)
  7. What is Restriction Fragment length Polymorphism (RFLP)? It represents a stretch of DNA which exists in a fragment of different length (polymorphism) derived by the action of restriction enzymes. Using restriction enzymes and gel electrophoresis, DNA of different individuals can be analyzed and compared.

Recombinant DNA Technology

  1. What is recombinant DNA technology? A technique of gene amplification where modification of nucleic acid is done to achieve a desired goal in a pre-determined way.
  2. What are molecular scissors? Restriction Endonuclease.
  3. Name some restriction endonucleases. EcoR-I, Hpa-I, BamH-I, Hind-III, Mst-II, Pst-I. (Escherichia coli RY 13, Hemophilus parainfluenza I, Bacillus amylolique faciens H, Hemophilus Influenza Rd, Microcoleus Strain, Providencia stuart ii 164 respectively)
  4. What is chimeric DNA? Recombinant DNA is also known as Chimeric or hybrid DNA.
  5. Name the vectors used in rDNA technology? Plasmids, Bacteriophage, Cosmid, BAC (Bacterial Artificial Chromosome), YAC (Yeast AC), MAC (Mammalian AC).
  6. What are plasmids? name any two. Plasmids are extra-chromosomal, self-replicatory nucleic acids which are circular, covalently closed, double stranded DNA (ccc ds DNA). E.g., pBR322 plasmid, PUC18.
  7. What is restriction map? It is a map of known restriction sites within a DNA sequence used to determine the location and relative distance of restriction sites on a DNA molecule.
  8. Applications of rDNA technology? Diabetes Mellitus (Humulin), Hepatitis B Vaccine, Anemia (Erythropoietin), Neutropenia (Filgrastim), Viral Infections (Interferons), RCC=Renal Cell Carcinoma (Interleukins), Burns (EGF = Epidermal growth Factors).
  9. Why do bacteria produce restriction endonuclease (RE)?  REs doesn’t damage the cells’ own DNA because the sequences recognized by its own REs are methylated, thereby protected from the action of REs.
  10. What is a vector? DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called Vector.