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6.2 Vectors & Gene Transfer โ Test 1
Q1. A 2 kb DNA fragment has PstI sites at 400 and 1000 bp and an EcoRI site at 1800 bp. Double digestion gives fragments of:โ 400, 600, 800 and 200 bp
Q2. A DNA fragment cut with BamHI is ligated into a BglII-cut vector. Which enzyme can re-excise the insert?โ Sau3A
Q3. Virus-mediated delivery of DNA into mammalian cells is called:โ Transduction
Q4. Type-II restriction endonucleases create ends that:โ Can be either blunt (double-stranded) or with single-stranded overhangs (sticky)
Q5. ฮฑ-complementation is used for:โ Identifying recombinant colonies (blue-white screening)
Q6. Steps in gene cloning, in order:โ Isolate gene โ insert into vector โ introduce to host โ express โ extract product
Q7. EcoRI is a(n):โ Endonuclease (restriction enzyme)
Q8. Restriction enzymes are isolated mainly from:โ Prokaryotes (bacteria)
Q9. Self-ligation of a cut vector during cloning is prevented by:โ Dephosphorylation of the vector 5' ends (alkaline phosphatase)
Q10. In PCR, MgClโ acts as:โ A cofactor for Taq polymerase
Q11. Integration of linear ฮป phage DNA into the bacterial chromosome requires the product of:โ Int (integrase) gene
Q12. Introduction of recombinant DNA into eukaryotic cells (non-viral) is called:โ Transfection
Q13. Which is NOT a chemical method for transfecting mammalian cells?โ Biolistics (gene gun โ physical)
Q14. DNA ligase joins DNA by forming a phosphodiester bond between:โ A 3'-OH and a 5'-phosphate
Q15. RAPD markers:โ Do NOT require prior sequence knowledge (use random primers)
Q16. A relaxed plasmid is:โ A high-copy-number plasmid (replicates independently of chromosome)
Q17. Which is a typical restriction-enzyme recognition site (palindromic)?โ AAGCTT (HindIII palindrome)
6.2 Vectors & Gene Transfer โ Test 2
Q18. Restriction enzymes are named for:โ The bacterium (genus/species/strain) they are derived from
Q19. Bacteria avoid cutting their OWN DNA with their restriction enzymes because:โ Their own recognition sites are methylated (modified) by a cognate methylase
Q20. The correct order of PCR steps in a cycle is:โ Denaturation, annealing, extension
Q21. BamHI/BglII compatibility: 'BamHI fragments can ligate to BglII-cut vector' and 'they make compatible overhangs'. The correct evaluation is:โ Both statement and reason are correct (reason explains it)
Q22. T-vector (TA) cloning of a PCR product exploits the fact that:โ Taq polymerase adds a 3'-A overhang, matched by the vector's 3'-T overhang
Q23. HaeIII and Sau3A recognise sequences that are:โ Tetranucleotides (4 bp)
Q24. Reverse transcriptase is used to:โ Make cDNA from an RNA template
Q25. After 7 cycles of PCR, one dsDNA template gives approximately:โ 128 (2โท)
Q26. The enzyme representing the 'reverse' central dogma (RNAโDNA) is:โ Reverse transcriptase
Q27. The RNA polymerase with a single subunit (used in expression vectors) is:โ T7 RNA polymerase
Q28. To prevent vector self-ligation when insert and vector share the same enzyme ends, treat the vector with:โ Alkaline phosphatase
Q29. SmaI and XmaI recognise the same site (CCCGGG) but cut differently (blunt vs 5' overhang). Such enzymes are:โ Isoschizomers (same site, may cut same or differently)
Q30. Which bacteriophage has single-stranded DNA as its genome?โ ฯX174
Q31. BamHI (G^GATCC) when it cuts DNA:โ Creates a 5' overhang (GATC)
Q32. RFLP analysis uses:โ Restriction enzyme digestion + gel electrophoresis
Q33. Which metal ion is required for type-II restriction enzyme cleavage?โ Mgยฒโบ
Q34. Which is NOT a bacteriophage-related cloning entity?โ Plasmid
6.2 Vectors & Gene Transfer โ Test 3
Q35. A 1000 bp linear DNA with two EcoRI sites, after digestion and re-ligation, can give how many ligation product combinations (linear vs circular cases differ)?โ 6 and 4 respectively (a classic keyed answer)
Q36. A 1.2 kb fragment with EcoRI at 400 bp and PstI at 550 bp, double-digested, gives:โ 400, 150 and 650 bp
Q37. To insert a foreign gene into a plasmid, both must:โ Be cut with compatible restriction enzyme(s) to give matching ends
Q38. In a thermophilic ATAANNNTTAT cutter leaving overhangs after the third base, the resulting overhang is:โ A 3-nt 5' overhang
Q39. PCR for 30 cycles from a single template ideally produces approximately:โ About one billion (2ยณโฐ โ 10โน)
Q40. AFLP (Amplified Fragment Length Polymorphism) is best described as:โ Selective PCR amplification of a subset of restriction fragments to compare genome fingerprints
Q41. M. tuberculosis DNA has 15.1% adenine. By Chargaff's rules, %G is about:โ About 34.9%
Q42. Which statement about pBR322 cloning is correct?โ pBR322 has unique sites in resistance genes used for insertional inactivation
Q43. DNA synthesis is restricted to a defined portion (S phase) of the cell cycle in:โ HeLa (eukaryotic) cells
Q44. To get equal molar amounts: 100 ng of pUC18 (2686 bp) corresponds to how much pKC7 (5829 bp)?โ ~214 ng
Q45. In two successive (nested) PCRs, nonspecific products are more likely in the:โ First PCR
Q46. Multiple nonspecific amplicons from human genomic DNA can be reduced by:โ Increasing the annealing temperature
Q47. Which one is NOT a DNA-sequencing relationship: 'plasmid minimum size'? The smallest natural plasmids are about:โ A few kb or less (plasmids can be very small, ~1-3 kb)
Q48. Which is NOT true (the false statement) about fosmids/BACs?โ Fosmids carry whole eukaryotic chromosomes
Q49. Which vector is NOT used to make GENOMIC libraries (insert too small)?โ pUC (small plasmid)
Q50. When expressing a eukaryotic gene in bacteria, cDNA (not genomic DNA) is used because:โ Bacteria cannot splice out introns from eukaryotic genes
Q51. EDTA was NOT included in a PCR master mix because it would:โ Chelate Mgยฒโบ and inhibit the polymerase
6.2 Vectors & Gene Transfer โ Test 4
Q52. On a 3 kb DNA (EcoRI at 275; PstI at 615, 750, 1560), which statement is NOT correct?โ Smallest PstI product is 275 bp
Q53. Which restriction enzyme has BOTH cleavage and methylase activity on the same protein/subunit (type I behaviour)?โ Type I enzyme (e.g. EcoB/EcoK)
Q54. Approximate number of EcoRI (GAATTC) fragments from the E. coli genome (~4.6 ร 10โถ bp):โ About 1100 (4.6ร10โถ / 4โถ โ 1123)
Q55. In a 100 kb fragment, the expected number of sites for a 6-bp cutter is about:โ ~24 (100000/4096)
Q56. For restriction enzymes recognising 4, 6 and 8 bp (P, Q, R), the frequency of sites is:โ P > Q > R (shorter site = more frequent)
Q57. For PstI (CTGCAG) in an 80% GC genome, sites occur:โ More frequently (GC-rich site favoured in GC-rich genome)
Q58. The composite phage with a T2 coat and T4 DNA, after infecting bacteria, produces progeny that are:โ Entirely T4 (DNA dictates progeny)
Q59. In the Hershey-Chase experiment, the material shown to enter the bacterium was the:โ DNA (labelled with ยณยฒP)
Q60. Which statement about mRNA is true across systems?โ Mitochondrial/chloroplast mRNAs are often NOT capped (unlike nuclear mRNA)
Q61. A uracil-containing plasmid transformed into ungโบ vs ungโป E. coli: in ungโบ cells the plasmid is:โ Degraded/fewer transformants (uracil-DNA glycosylase removes U)
Q62. In bacterial protein synthesis, the initiating amino acid is:โ N-formylmethionine
Q63. A 10โน bp DNA molecule (one per bacterium) corresponds to roughly how many moles per molecule?โ About 1/(6ร10ยฒยณ) โ 1.7 ร 10โปยฒโด mol (one molecule)
Q64. Starting with 10 ng genomic DNA, after 10 PCR cycles the amount of (total) DNA is approximately:โ Greatly amplified (โ10 ng ร 2ยนโฐ for the target โ several ยตg of product)
Q65. Which is the correct enzyme to leave 5'-OH ends (dephosphorylated) to block self-ligation?โ Alkaline phosphatase (removes 5'-phosphate)
Q66. Which best describes a phagemid?โ A hybrid vector with both a plasmid origin and a phage (e.g. f1) origin
Q67. Which enzyme adds nucleotides to the 3' end without a template (used in homopolymer tailing)?โ Terminal deoxynucleotidyl transferase (TdT)
Q68. The Klenow fragment of DNA Pol I retains:โ Polymerase and 3'โ5' proofreading exonuclease (lacks 5'โ3' exonuclease)