1.
Molecular Biotechnology Basics
● Cells contain nucleic acids (DNA, RNA) and proteins.
● DNA can exist as the genome (chromosomal DNA) or as plasmids (extra-chromosomal
DNA).
● Proteins are expressed from nucleic acids through transcription (DNA → RNA) and
translation (RNA → proteins).
(Ref: Diagram 1 – nucleic acids and proteins overview)
2. Important Molecular Biology Procedures
Key methods used in molecular biology:
● PCR (Polymerase Chain Reaction) → amplification of DNA
● Sequencing (skipped as per instructions)
● Microarray → gene expression studies
● Gel electrophoresis → separation of DNA/RNA by size
(Ref: Diagram 2 – overview of procedures)
3. Polymerase Chain Reaction (PCR)
Principle
PCR is a technique to amplify a single piece of DNA, generating millions of copies. It was
invented by Kary Mullis in 1987 (Nobel Prize 1993). PCR is applied in cloning, diagnostics, and
molecular identification .
Requirements
To perform PCR, we need:
1. Thermal cycler – to change temperatures rapidly.
2. Template DNA – the sequence we want to amplify.
3. Primers – short synthetic DNA fragments flanking the target sequence.
4. DNA polymerase – usually Taq polymerase, heat-stable.
5. dNTPs – nucleotides (dATP, dTTP, dGTP, dCTP).
6. Buffer solution – maintains optimal conditions.
7. MgCl₂ – cofactor for the enzyme .
(Ref: Diagram 3 – PCR overview components)
4. PCR Cycle
Each cycle consists of three steps:
1. Denaturation (≈95 °C): Double-stranded DNA → single-stranded.
2. Annealing (50–60 °C): Primers bind to complementary target sequences.
3. Extension (≈72 °C): Taq polymerase synthesizes new DNA strands.
● Typically, 25–30 cycles are performed.
● The process is exponential: after 30 cycles, billions of DNA copies can be produced .
(Ref: Diagrams 4 & 5 – PCR cycle with temperature profile)
Primers and Melting Temperature (Tm)
● Primers are 18–25 nucleotides long.
● Tm is calculated:
Tm = 4(G + C) + 2(A + T)
● Annealing temperature is usually 5 °C below Tm .
(Ref: Diagrams 6 & 7 – Tm calculation with example)
5. Examination of PCR Products
Gel Electrophoresis
● PCR products are analyzed using agarose gel electrophoresis.
● DNA fragments migrate according to size (small fragments move faster).
● A DNA ladder/marker of known sizes is run in parallel for comparison.
● DNA is stained with ethidium bromide (EtBr) and visualized under UV light .
(Ref: Diagrams 8–10 – Gel electrophoresis and visualization)
6. Types of PCR
1. Conventional (single-plex) PCR – one target amplified, single band.
2. Multiplex PCR – multiple genes amplified simultaneously, multiple bands.
3. RT-PCR (Reverse Transcriptase PCR) – RNA is converted into cDNA before
amplification.
4. qRT-PCR (Quantitative Real-Time PCR) – DNA/RNA quantified during amplification .
(Ref: Diagram 11 – types of PCR)
7. Quantitative Real-Time PCR (qRT-PCR)
● Monitors DNA amplification in real time using fluorescent dyes or probes.
● A fluorescent signal increases proportionally with DNA copies.
● Key concept: Ct (threshold cycle) = the cycle number when fluorescence exceeds
background.
● Lower Ct = higher starting DNA concentration .
Detection Methods
1. SYBR Green dye – binds to double-stranded DNA, fluoresces upon binding.
2. Fluorescent probes (TaqMan) – release fluorescence when DNA polymerase cleaves
them.
(Ref: Diagrams 12–15 – qRT-PCR workflow, fluorescence, Ct curves)
8. Applications of PCR
● Gene cloning: amplification for further genetic engineering.
● Diagnostics: detection of pathogens and genetic disorders.
● Molecular identification: identifying organisms using DNA.
● Quantification: measuring gene expression (qPCR).
(Ref: Diagrams 16–20 – practical applications with gel images and Ct graph)
Final Notes
● PCR is a powerful molecular biology tool for amplifying and analyzing DNA.
● Mastery of PCR involves understanding both the theoretical basis (enzymes, primers,
cycles) and the practical analysis (gel electrophoresis, qPCR detection).
● The diagrams included serve as visual memory aids alongside the explanations.
End of Study Paper
