Samacheer Kalvi 12th Biology Chapter 8 Exercise Solutions

Restriction enzymes (restriction endonucleases) are bacterial enzymes that cut DNA at specific recognition sequences, which are usually palindromic. They act as "molecular scissors" in recombinant DNA technology. EcoRI is a restriction enzyme isolated from E. coli that recognises the palindromic sequence 5'-GAATTC-3' and cuts between G and A, producing sticky ends with a single-stranded overhang.

Sticky ends are produced when a restriction enzyme cuts DNA asymmetrically, leaving single-stranded overhangs that are complementary and can base-pair with matching ends. This makes ligation of DNA fragments easier and more specific. Blunt ends are produced when the enzyme cuts straight through both strands at the same position, leaving no overhangs. Blunt ends can ligate with any other blunt end but are less specific and less efficient than sticky ends.

PCR (Polymerase Chain Reaction) is an in vitro technique that amplifies a specific DNA segment exponentially through repeated cycles of denaturation, annealing, and extension. Taq polymerase from Thermus aquaticus is used because it is thermostable — it remains active at the high temperatures (94–96°C) required for the denaturation step. Normal DNA polymerase would denature at these temperatures and would need to be added fresh after each cycle, making PCR impractical.

A cloning vector is a DNA molecule that can carry a foreign gene into a host cell and replicate it. Essential features of a good vector are:

• Origin of replication (ori) — ensures the vector can replicate independently in the host
• Selectable marker — gene (usually antibiotic resistance) that allows identification of transformed cells
• Restriction sites (multiple cloning site) — specific sequences where the foreign DNA can be inserted
• Small size — easy to manipulate and transfer into host cells

Gel electrophoresis separates DNA fragments based on their size. DNA fragments are loaded into wells in an agarose gel and an electric current is applied. Since DNA is negatively charged (due to phosphate groups), it migrates toward the positive electrode. Smaller fragments move faster through the gel pores and travel farther, while larger fragments are retarded. The separated fragments are visualised by staining with ethidium bromide and viewing under UV light. A DNA ladder (marker) is run alongside for size comparison.

Competent cells are bacterial cells that have been treated to make them capable of taking up foreign (recombinant) DNA from the surrounding medium. They are prepared by treating bacterial cells (usually E. coli) with cold CaCl₂ solution. The calcium ions create pores in the bacterial cell wall and membrane, allowing the negatively charged DNA to enter. The cells are then subjected to a brief heat shock (42°C for 2 minutes), which further facilitates DNA uptake. This process is called transformation.

Recombinant DNA technology (genetic engineering) involves the following steps:

1. Isolation of the gene of interest:

• The desired gene is cut from the source DNA using a restriction enzyme (e.g., EcoRI)
• The gene can also be synthesised artificially or obtained via PCR amplification
• The enzyme produces sticky ends that are complementary to the vector's cut ends

2. Isolation and preparation of the vector:

• A plasmid (e.g., pBR322) is cut with the same restriction enzyme as the gene of interest
• This ensures that the plasmid has complementary sticky ends to the foreign gene
• The vector must have an origin of replication (ori), selectable marker, and restriction sites

3. Ligation:

• The gene of interest and the cut plasmid are mixed with DNA ligase
• DNA ligase forms phosphodiester bonds, joining the foreign gene into the plasmid
• This creates recombinant DNA (rDNA)
• Sometimes the plasmid re-circularises without the insert — these are non-recombinant

4. Transformation (introduction into host cell):

• The recombinant plasmid is introduced into competent bacterial cells (treated with CaCl₂)
• A brief heat shock (42°C) facilitates DNA uptake
• The host cell is typically E. coli — easy to grow and replicates fast

5. Selection and screening:

• Transformed cells are selected using selectable markers (antibiotic resistance)
• Blue-white screening using lacZ gene can distinguish recombinant (white colonies) from non-recombinant (blue colonies)
• Only cells that have taken up the recombinant plasmid survive on antibiotic plates

6. Cloning and expression:

• Selected transformed cells multiply, producing many copies of the recombinant gene
• The gene is expressed to produce the desired protein (e.g., insulin, growth hormone)

Diagram description: Draw a flow diagram — Source DNA + Plasmid → Cut with restriction enzyme → Mix with DNA ligase → Recombinant DNA → Transform into competent E. coli (CaCl₂ + heat shock) → Select on antibiotic plate → Clone → Express protein.

PCR (Polymerase Chain Reaction) is an in vitro technique that amplifies a specific DNA segment exponentially. It requires the following components:

Components of PCR:

• Template DNA — the DNA containing the target sequence to be amplified
• Primers — short synthetic oligonucleotides (18–25 bases) complementary to sequences flanking the target DNA; one forward primer and one reverse primer
• Taq polymerase — thermostable DNA polymerase from Thermus aquaticus that synthesises new DNA strands at 72°C
• dNTPs — deoxynucleotide triphosphates (dATP, dTTP, dGTP, dCTP) — building blocks for new DNA
• Buffer — provides optimal pH and ionic conditions for the enzyme

Three steps of each PCR cycle:

1. Denaturation (94–96°C):

• The double-stranded template DNA is heated to separate into two single strands
• Hydrogen bonds between base pairs are broken
• Duration: ~30 seconds

2. Annealing (50–65°C):

• Temperature is lowered to allow primers to bind (anneal) to complementary sequences on each single strand
• Primers mark the starting points for DNA synthesis
• Duration: ~30 seconds

3. Extension (72°C):

• Taq polymerase adds dNTPs to the 3' end of each primer, synthesising new DNA strands complementary to the template
• Duration: ~1 minute per kilobase

Amplification calculation:

• Each cycle doubles the DNA: 1 → 2 → 4 → 8 → 16...
• After n cycles: 2ⁿ copies
• 30 cycles → 2³⁰ ≈ 10⁹ copies from a single DNA molecule

Applications of PCR:

• Disease diagnosis — detecting viral/bacterial DNA (HIV, tuberculosis, COVID-19)
• DNA fingerprinting — forensic identification and paternity testing
• Gene cloning — amplifying genes before cloning into vectors
• Mutation detection — identifying genetic disorders
• Sequencing — providing enough DNA for sequencing reactions

Essential features of a cloning vector:

• Origin of replication (ori): Sequence from which DNA replication initiates; ensures the vector replicates independently in the host cell
• Selectable marker: Gene that allows identification of transformed cells — usually antibiotic resistance genes (e.g., ampicillin resistance in pBR322)
• Multiple cloning site (MCS): Region containing several unique restriction enzyme recognition sites where foreign DNA can be inserted
• Small size: Makes the vector easy to manipulate and transfer into host cells
• High copy number: Ensures multiple copies of the recombinant gene are produced

Comparison of cloning vectors:

Example: pBR322 — a well-known plasmid vector with:

• Ampicillin resistance gene (amp⁺)
• Tetracycline resistance gene (tet⁺)
• Origin of replication (ori)
• Several restriction sites (EcoRI, HindIII, BamHI, PstI, PvuI)

Gel electrophoresis is a technique used to separate DNA fragments based on their molecular size.

Principle: DNA molecules carry a negative charge due to phosphate groups in their backbone. When placed in an electric field, they migrate toward the positive electrode (anode). Smaller fragments move faster through the agarose gel matrix, while larger fragments are retarded.

Procedure: 1. Prepare the gel: Agarose powder is dissolved in buffer (e.g., TAE or TBE), boiled, cooled slightly, and poured into a casting tray with a comb to form wells.

2. Load samples: DNA samples are mixed with loading dye (contains glycerol to make them sink) and loaded into wells. A DNA ladder (marker) of known sizes is loaded in one well for reference.

3. Run electrophoresis: Electric current is applied (~50–100V). DNA, being negatively charged, migrates from cathode (negative) to anode (positive).

4. Staining and visualisation: After electrophoresis, the gel is stained with ethidium bromide (or safer alternatives like SYBR Green). Ethidium bromide intercalates between DNA base pairs and fluoresces under UV light, making DNA bands visible.

5. Interpretation: Bands are compared against the DNA ladder to determine fragment sizes. Smaller fragments are found farther from the wells.

How gel electrophoresis confirms rDNA success:

• After restriction enzyme digestion of the recombinant plasmid, the gene insert should separate from the vector
• The gel should show two bands: one for the vector and one for the insert
• If the gene was not inserted (empty vector), only one band corresponding to the vector size will appear
• PCR-amplified products can be run on gel to confirm the correct size of the amplified gene
• In blue-white screening, white colonies (recombinant) when digested should show the insert band

Diagram description: Draw an agarose gel with wells at the top. Show DNA ladder in lane 1 (multiple bands of known sizes). Show undigested plasmid in lane 2 (single band). Show digested recombinant plasmid in lane 3 (two bands — vector + insert). Label cathode (−) at top and anode (+) at bottom.