Chapter 23: Biotechnology – Important Short Questions & Answers (Class 12 Biology)
Short Answers (2–3 Marks) – Punjab Board / RTS / HED Exams (Exam-style: Point-wise, examples, tables where needed – full marks guaranteed!)
1) Give ways to get gene of interest
Ways to isolate/obtain the gene of interest:
- From DNA library — Construct genomic or cDNA library and screen using probes or hybridization.
- PCR amplification — Use specific primers to amplify the gene if sequence is known.
- Restriction enzyme digestion — Cut genomic DNA and isolate fragment by size or cloning.
- Chemical synthesis — Synthesize gene artificially if sequence is known.
- From mRNA — Reverse transcribe mRNA to cDNA (for expressed genes).
2) What are restriction enzymes? Give examples.
Restriction enzymes (molecular scissors): Endonucleases from bacteria that cut DNA at specific recognition sites (palindromic sequences), producing sticky or blunt ends. Used in recombinant DNA technology.
Examples:
- EcoRI (from Escherichia coli RY13) — cuts GAATTC.
- HindIII (from Haemophilus influenzae) — cuts AAGCTT.
- BamHI — cuts GGATCC.
3) What are plasmids?
Plasmids: Small, circular, extrachromosomal double-stranded DNA molecules in bacteria (e.g., E. coli). They replicate independently and carry genes for antibiotic resistance.
Used as vectors in recombinant DNA technology to carry foreign genes because:
- Easy to isolate.
- Have origin of replication.
- Multiple cloning sites.
- Selectable markers.
Example: pBR322, pUC18.
4) What is probe? Give its uses.
Probe: Short, single-stranded DNA or RNA fragment (labeled with radioactive/fluorescent tag) complementary to target sequence.
Uses:
- Detect specific DNA/RNA in Southern/Northern blotting (hybridization).
- Screen genomic/cDNA libraries for gene of interest.
- Diagnose genetic disorders (e.g., in FISH).
- DNA fingerprinting and gene mapping.
5) What is genomic library?
Genomic library: Collection of recombinant DNA clones (in vectors like plasmids, cosmids, BACs) representing the entire genome of an organism (all DNA fragments after restriction digestion).
Used to isolate genes, study genome organization, and map genes.
(Contrast: cDNA library contains only expressed genes.)
6) What is Taq polymerase?
Taq polymerase: Thermostable DNA polymerase from bacterium Thermus aquaticus (hot springs).
Used in PCR because:
- Survives high temperatures (94°C denaturation step).
- No need to add fresh enzyme each cycle.
- High processivity.
7) Give uses of DNA fingerprinting
DNA fingerprinting (DNA profiling): Uses VNTRs/STRs to create unique genetic profile.
Uses:
- Forensic science — Identify criminals from crime scene samples.
- Paternity/maternity testing.
- Identify disaster victims.
- Organ transplantation matching.
- Population genetics and biodiversity studies.
8) What are primary goals of Human Genome Project?
Primary goals of Human Genome Project (1990–2003):
- Sequence the entire human genome (3 billion base pairs).
- Map all human genes (identify and locate).
- Develop tools for data analysis (bioinformatics).
- Sequence model organisms (E. coli, yeast, mouse).
- Address ethical, legal, social issues (ELSI).
Achieved: Draft in 2000, complete in 2003.
9) What is gene pharming?
Gene pharming (molecular pharming / biopharming): Genetic engineering of plants or animals to produce pharmaceutical proteins (transgenic organisms).
Proteins expressed in milk (animals like goats/sheep), leaves/seeds (plants like tobacco/maize).
Example: Transgenic sheep producing human clotting factor in milk for haemophilia treatment.
10) Difference between ex vivo and in vivo gene therapy
| Feature | Ex vivo Gene Therapy | In vivo Gene Therapy |
|---|---|---|
| Method | Cells removed from body, modified in lab, reintroduced | Therapeutic gene directly injected/delivered into body |
| Cells targeted | Blood cells, stem cells (easy to extract) | Tissues/organs (e.g., eye, liver, muscle) |
| Advantages | Controlled modification, safer screening | Simpler, no cell extraction |
| Example | CAR-T cell therapy for cancer | Luxturna for retinal dystrophy (eye injection) |
| Risk | Lower immune response | Higher risk of off-target effects |
11) What are totipotent cells in plants?
Totipotent cells in plants: Cells capable of regenerating a complete plant (whole organism) from a single cell due to totipotency.
Found in meristems, explants in tissue culture.
Used in:
- Micropropagation.
- Somatic embryogenesis.
- Callus formation → organogenesis.
Example: Any plant cell (e.g., leaf, root) can form whole plant in vitro.
12) What is cystic fibrosis?
Cystic fibrosis (CF): Autosomal recessive genetic disorder caused by mutations in CFTR gene (chromosome 7).
CFTR protein regulates chloride transport → defective protein causes thick mucus in lungs, pancreas, intestines.
Symptoms:
- Persistent lung infections, breathing difficulty.
- Poor growth, salty sweat.
- Digestive problems (pancreatic insufficiency).
- Infertility in males.
Write a note on genomic library
Genomic Library Long Note for Class 12 Biology (Important 5–8 Marks Question – RTS/HED/Punjab Board Pattern)
Definition
A genomic library is a collection of recombinant DNA clones that together represent the entire genome of an organism. It contains all the DNA fragments (coding + non-coding) of the organism’s genome inserted into suitable vectors.
It is like a “book library” where each “book” is a fragment of the total DNA, and the complete set of books contains the whole genetic information of that organism.
Construction of Genomic Library (Step-by-Step)
- Isolation of Genomic DNA High molecular weight genomic DNA is extracted from the organism.
- Fragmentation of DNA DNA is cut into smaller fragments using restriction enzymes (partial digestion is done so that overlapping fragments are produced).
- Insertion into Vectors
These DNA fragments are ligated into suitable cloning vectors such as:
- Plasmids
- Bacteriophages (λ phage)
- Cosmids
- BACs (Bacterial Artificial Chromosomes)
- YACs (Yeast Artificial Chromosomes)
- Transformation / Transfection Recombinant vectors are introduced into host cells (usually E. coli).
- Selection & Storage Host cells are grown on selective media. Each colony or plaque contains a different fragment of the genome. All these clones together form the genomic library.
Types of Genomic Libraries
| Type | Vector Used | Insert Size | Best For |
|---|---|---|---|
| Plasmid library | Plasmid | 5–10 kb | Small genomes, easy handling |
| Phage library | λ bacteriophage | 15–25 kb | Most commonly used |
| Cosmid library | Cosmid | 35–45 kb | Larger inserts |
| BAC library | Bacterial Artificial Chromosome | Up to 300 kb | Large genomes, human genome project |
| YAC library | Yeast Artificial Chromosome | 100–1000 kb | Very large fragments |
Uses / Applications of Genomic Library (Very Important)
- Isolation of specific gene of interest using probes.
- Genome mapping and physical mapping of genes.
- Study of gene structure, regulatory sequences and introns.
- Comparative genomics between different species.
- Preparation of cDNA library comparison (genomic library has both coding and non-coding DNA).
- Human Genome Project heavily relied on BAC and YAC libraries.
Genomic Library vs cDNA Library (Difference Table – Must Draw)
| Feature | Genomic Library | cDNA Library |
|---|---|---|
| Source | Total genomic DNA | mRNA (expressed genes only) |
| Contains introns? | Yes | No (introns are spliced out) |
| Represents | Entire genome (coding + non-coding) | Only expressed genes |
| Size of insert | Large | Smaller |
| Used for | Gene structure, regulatory sequences | Gene expression studies, protein coding |
Key Lines for Exam (Write these)
- “Genomic library is a complete representation of the genome in the form of cloned fragments.”
- “It is constructed using restriction enzymes and suitable vectors.”
- “It contains both exons and introns while cDNA library contains only exons.”
Explain PCR with diagram.
PCR (Polymerase Chain Reaction) Long Note for Class 12 Biology (Very Important 7–8 Marks Question – RTS/HED/Punjab Board/NCERT)
Definition
Polymerase Chain Reaction (PCR) is a revolutionary technique used to amplify (make millions of copies) a specific segment of DNA in vitro (in a test tube) within a few hours. It was invented by Kary Mullis in 1983 (Nobel Prize 1993).
PCR is based on the natural process of DNA replication but is carried out artificially using thermostable Taq polymerase.
Principle of PCR
PCR is based on three cyclic steps that are repeated 25–35 times:
- Denaturation
- Annealing
- Extension (Polymerisation)
Each cycle doubles the number of DNA molecules, so after n cycles, the number of copies = 2ⁿ.
Requirements / Components of PCR
- Template DNA (the DNA to be amplified)
- Taq polymerase (heat-stable DNA polymerase from Thermus aquaticus)
- Primers (short oligonucleotides, 18–22 nucleotides, complementary to the 3' ends of target DNA)
- dNTPs (dATP, dTTP, dGTP, dCTP)
- Buffer (Mg²⁺ ions)
- Thermal cycler (PCR machine)
Three Steps of PCR (With Diagram Explanation)
1. Denaturation (94–95°C for 30–60 sec) Double-stranded DNA is heated to separate into two single strands. Hydrogen bonds break.
2. Annealing (50–65°C for 30–60 sec) Temperature is lowered so that primers can anneal (bind) to their complementary sequences on the single-stranded DNA.
3. Extension / Polymerisation (72°C for 1–2 min) Taq polymerase extends the primers by adding dNTPs in 5'→3' direction, synthesizing new complementary strands.
These three steps are repeated 25–35 times in a thermal cycler.
Diagrammatic Representation (Summary of One Cycle)
Cycle 1:
dsDNA → (Denaturation) → 2 ssDNA
↓ (Annealing)
Primer binds
↓ (Extension)
2 dsDNA copies
Cycle 2: 4 copies
Cycle 3: 8 copies
...
After 30 cycles ≈ 1 billion copies (2³⁰)Applications of PCR (Very Important for Exams)
- Amplification of specific genes for cloning.
- Diagnosis of genetic disorders (e.g., sickle cell anaemia, cystic fibrosis).
- Forensic science (DNA fingerprinting from small samples).
- Detection of pathogens (COVID-19 RT-PCR).
- Gene expression studies.
- DNA sequencing.
Advantages
- Very sensitive (can amplify DNA from a single cell).
- Extremely fast (few hours).
- Highly specific due to primers.
- No need for living cells.
Key Exam Lines:
- “PCR is an in vitro technique to amplify specific DNA segment using Taq polymerase.”
- “It involves three steps: Denaturation, Annealing and Extension.”
- “One cycle doubles the DNA; after 30 cycles, 2³⁰ copies are produced.”
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