GATE Biotechnology Recombinant DNA technology and Other Tools in Biotechnology Syllabus

Recombinant DNA technology and Other Tools in Biotechnology syllabus — full chapter and topic list

Expand any chapter to see its topics and sub-topics. This is the whole examinable outline for Recombinant DNA technology and Other Tools in Biotechnology in GATE Biotechnology, not a summary of it.

1. 1

   Recombinant DNA technology

   7 topics

   • Restriction and modification enzymes
   • Vectors
     • Plasmids
     • Bacteriophage and other viral vectors
     • Cosmids
     • Ti plasmid
     • Bacterial and yeast artificial chromosomes
   • Expression vectors
   • cDNA and genomic DNA library
   • Gene isolation and cloning
   • Strategies for production of recombinant proteins
   • Transposons and gene targeting
2. 2

   Molecular tools

   8 topics

   • Polymerase chain reaction
   • DNA/RNA labelling and sequencing
   • Southern and northern blotting
   • In-situ hybridization
   • DNA fingerprinting
     • RAPD
     • RFLP
   • Site-directed mutagenesis
   • Gene transfer technologies
     • CRISPR-Cas
   • Biosensing and biosensors
3. 3

   Analytical tools

   8 topics

   • Principles of microscopy
     • Light
     • Electron
     • Fluorescent
     • Confocal
   • Principles of spectroscopy
     • UV
     • Visible
     • CD
     • IR
     • Fluorescence
     • FT-IR
     • MS
     • NMR
   • Electrophoresis
   • Micro-arrays
   • Enzymatic assays
   • Immunoassays
     • ELISA
     • RIA
     • Immunohistochemistry
     • Immunoblotting
   • Flow cytometry
   • Whole genome and ChIP sequencing
4. 4

   Computational tools

   10 topics

   • Bioinformatics resources and search tools
   • Sequence and structure databases
   • Sequence analysis
     • Sequence file formats
     • Scoring matrices
     • Alignment
     • Phylogeny
   • Genomics, proteomics, metabolomics
   • Gene prediction
   • Functional annotation
   • Secondary structure and 3D structure prediction
   • Knowledge discovery in biochemical databases
   • Metagenomics
   • Metabolic engineering and systems biology

Recombinant DNA technology and Other Tools in Biotechnology flashcards for GATE Biotechnology

24 of 51 cards from the Recombinant DNA technology and Other Tools in Biotechnology deck — real questions with worked answers.

1. What are restriction endonucleases and what type of bond do they cleave?

   Restriction endonucleases are bacterial enzymes that recognize specific short (usually 4-8 bp) palindromic DNA sequences and cleave the phosphodiester backbone of double-stranded DNA, producing either blunt or sticky (cohesive) ends. They are part of the bacterial restriction-modification defense system against foreign (phage) DNA.

2. Compare the three types (I, II, III) of restriction enzymes.

   Type I: single multifunctional enzyme with both restriction and methylation activity; cleaves DNA at random sites far from the recognition sequence; requires ATP, $\text{Mg}^{2+}$ and SAM. Type II: separate restriction and methylation enzymes; cleaves at or very near the specific recognition site; requires only $\text{Mg}^{2+}$ (most useful in cloning). Type III: cleaves DNA ~24-26 bp downstream of recognition site; requires ATP and $\text{Mg}^{2+}$.

3. What is the role of modification (methylase) enzymes in the restriction-modification system?

   Modification methylases add a methyl group (from SAM) to specific bases within the host's own recognition sequences (e.g., on adenine to form N6-methyladenine or cytosine to form 5-methylcytosine). This methylation protects the host's own DNA from being cleaved by its cognate restriction enzyme, while unmethylated foreign DNA is degraded.

4. What is a palindromic recognition sequence? Give the example for EcoRI.

   A palindrome reads the same $5' \to 3'$ on both complementary strands. EcoRI recognizes $5'\text{-GAATTC-}3'$ / $3'\text{-CTTAAG-}5'$ and cuts between G and A, leaving $5'$ AATT sticky overhangs.

5. Distinguish isoschizomers, neoschizomers, and isocaudomers.

   Isoschizomers: different enzymes recognizing the same sequence and cutting at the same position. Neoschizomers: recognize the same sequence but cut at different positions. Isocaudomers: recognize different sequences but generate identical (compatible) cohesive ends.

6. What essential properties must a cloning vector possess?

   (1) An origin of replication (ori) for autonomous replication in the host; (2) a selectable marker (e.g., antibiotic resistance) to identify transformed cells; (3) unique restriction sites in a multiple cloning site (MCS/polylinker) for inserting foreign DNA; (4) small size and high copy number; (5) a means to distinguish recombinants (e.g., insertional inactivation / blue-white screening).

7. What is a plasmid and what is meant by its copy number?

   A plasmid is a small, circular, double-stranded extrachromosomal DNA molecule that replicates independently of the host chromosome. Copy number is the number of plasmid copies maintained per host cell, controlled by the ori; relaxed plasmids (e.g., pUC) are high-copy, while stringent plasmids are low-copy.

8. How does blue-white screening identify recombinant clones?

   The vector carries the lacZ' gene encoding the $\alpha$-fragment of $\beta$-galactosidase, with the MCS inside it. Insertion of foreign DNA disrupts lacZ' (insertional inactivation), so no functional enzyme forms via $\alpha$-complementation. On X-gal/IPTG plates, recombinant (insert-containing) colonies are white, while non-recombinant colonies are blue.

9. What is plasmid incompatibility?

   Two plasmids are incompatible if they cannot be stably co-maintained in the same cell line, because they share the same replication control system (same incompatibility group / ori) and compete for the same regulatory machinery. Plasmids of different incompatibility groups can coexist.

10. What is the maximum insert size that can be cloned in a typical plasmid vector?

    Plasmid vectors efficiently accommodate inserts up to about 10-15 kb (typically <10 kb); larger inserts reduce transformation efficiency and plasmid stability.

11. Describe the bacteriophage $\lambda$ as a cloning vector, including insertion vs. replacement vectors.

    Bacteriophage $\lambda$ has a ~48.5 kb genome with a central ~15-20 kb dispensable region (non-essential for lytic growth). Insertion vectors have a single cloning site and accept small inserts (up to ~10 kb). Replacement (substitution) vectors have a removable central 'stuffer' fragment replaced by foreign DNA, accepting inserts of ~9-23 kb. Packaging constraints require the recombinant genome to be ~75-105% of wild-type length.

12. What is the role of cos sites and in vitro packaging in $\lambda$ vectors?

    cos (cohesive end) sites are the 12-nucleotide single-stranded complementary ends of $\lambda$ DNA. During in vitro packaging, the terminase enzyme recognizes cos sites and cuts concatemeric DNA into unit-length genomes that are inserted into phage heads. Only DNA between ~38-52 kb (two cos sites at correct spacing) is efficiently packaged into infectious particles.

13. What is M13 phage used for in molecular biology?

    M13 is a filamentous bacteriophage with a single-stranded circular DNA genome. It is used to produce single-stranded recombinant DNA, which is valuable for Sanger dideoxy sequencing and site-directed mutagenesis. It does not lyse the host but is continuously extruded.

14. What is a cosmid and what insert sizes can it carry?

    A cosmid is a hybrid vector combining plasmid features (ori, selectable marker, MCS) with the $\lambda$ cos sequence. This lets it be packaged into $\lambda$ phage particles for efficient delivery, then replicate as a plasmid. Cosmids carry large inserts of about 35-45 kb.

15. What is a phagemid?

    A phagemid is a hybrid vector containing both a plasmid origin of replication and a filamentous phage (f1/M13) origin. It can replicate as a double-stranded plasmid, but upon superinfection with a helper phage it produces single-stranded DNA packaged into phage particles.

16. Describe the Ti plasmid and its role in plant genetic engineering.

    The Ti (Tumour-inducing) plasmid is a large (~200 kb) plasmid of Agrobacterium tumefaciens that causes crown gall disease. It contains the T-DNA region (transferred to and integrated into the plant genome) and the vir (virulence) genes that mediate transfer. For engineering, the tumour-causing (onc) genes in T-DNA are removed (disarmed) and replaced with the gene of interest, making it a natural vector for plant transformation.

17. What are the T-DNA borders and vir genes of the Ti plasmid?

    T-DNA is flanked by 25 bp imperfect direct repeats called the left and right border sequences; these define the region transferred into the plant genome (the right border is essential). The vir genes (virA, virB, virC, virD, virE, virG), located outside T-DNA on the Ti plasmid, encode the machinery that processes and transfers the T-DNA into the plant cell.

18. What is a binary vector system in Agrobacterium-mediated transformation?

    A binary system splits the functions onto two plasmids: a small binary vector containing the engineered T-DNA (with the gene of interest, between the borders) that replicates in both E. coli and Agrobacterium, and a separate disarmed helper Ti plasmid supplying the vir genes in trans. This avoids manipulating the very large Ti plasmid directly.

19. Compare BAC and YAC vectors (host, basis, insert capacity).

    BAC (Bacterial Artificial Chromosome): based on the F-plasmid (single-copy), propagated in E. coli, carries inserts ~100-300 kb. YAC (Yeast Artificial Chromosome): based on yeast chromosomal elements, propagated in Saccharomyces cerevisiae, carries the largest inserts ~100 kb up to ~1000 kb (1 Mb). BACs are more stable and easier to handle; YACs hold larger inserts but show more chimerism and instability.

20. What three functional elements must a YAC contain to behave like a chromosome?

    (1) A centromere (CEN) for proper segregation during mitosis; (2) two telomeres (TEL) at the ends to protect and replicate chromosome termini; (3) an autonomously replicating sequence (ARS, the yeast origin of replication). It also includes selectable markers (e.g., TRP1, URA3).

21. What is an expression vector and what features distinguish it from a cloning vector?

    An expression vector is designed to produce protein from a cloned gene. In addition to cloning-vector features, it contains expression signals: a strong promoter, a ribosome binding site (Shine-Dalgarno sequence in prokaryotes / Kozak in eukaryotes), a start codon, a transcription terminator, and often an inducible regulatory element and an affinity tag for purification.

22. Name common strong/inducible promoters used in bacterial expression vectors.

    lac/lacUV5, tac and trc (hybrid trp-lac, IPTG-inducible), trp (tryptophan-regulated), $\lambda$ $p_L$/$p_R$ (temperature-regulated via cI857), araBAD (arabinose-inducible, BAD promoter), and the T7 promoter (requires T7 RNA polymerase, as in the pET system).

23. How does the T7/pET expression system work?

    The gene of interest is placed under a T7 promoter on a pET vector. Expression requires T7 RNA polymerase, supplied by a host strain (e.g., E. coli BL21(DE3)) carrying the T7 polymerase gene under control of the IPTG-inducible lacUV5 promoter. Adding IPTG induces T7 polymerase, which then drives high-level, selective transcription of the target gene.

24. What is a cDNA library and how is it constructed?

    A cDNA library is a collection of clones derived from the mRNA of a cell/tissue. mRNA is isolated, reverse transcribed into first-strand cDNA using reverse transcriptase (primed by oligo-dT), the second strand is synthesized, and the double-stranded cDNA is ligated into a vector. It represents only the expressed (exon-only) genes of that cell type and contains no introns.

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