Tools of Molecular Biology

Overview – Tools of Molecular Biology

Tools of molecular biology underpin the modern study of genes and proteins. From nucleic acid extraction to gene cloning and DNA sequencing, these techniques allow scientists to manipulate, replicate, and analyze genetic material for research, diagnostics, and therapeutics. Whether used for producing recombinant insulin or detecting pathogenic DNA in a patient, mastering these tools is essential for medical students aiming to understand molecular diagnostics and biotechnology.

Nucleic Acid Extraction

mRNA Extraction (for Cloning or PCR)

  • Start with eukaryotic cells expressing the gene of interest.
  • Lyse cells → solution contains DNA, RNA, mRNA, proteins, and membranes.
  • Centrifuge → removes membranes and insolubles.
  • Add phenol → dissolves proteins. Decant protein-rich layer.
  • Add DNase → degrades contaminating DNA.
  • Add poly-T beads → bind to mRNA’s poly-A tail.
  • Magnetise and wash → removes residual RNA.
  • Use hot water to release mRNA → leaves purified mRNA without introns or junk DNA.

Convert mRNA to cDNA

  • Use reverse transcriptase → forms mRNA/cDNA hybrid.
  • Add RNase → removes RNA strand.
  • Add DNA polymerase → synthesises second cDNA strand.
  • Result: double-stranded complementary DNA (cDNA) suitable for cloning or PCR.

DNA Extraction for Sequencing

  • Lyse cells in aqueous buffer with EDTA.
  • Centrifuge to remove debris.
  • Add phenol → removes proteins.
  • Add RNase → removes RNA.
  • Add ethanol (EtOH) → precipitates DNA.
  • Centrifuge again → isolate purified DNA.

Gel Electrophoresis

  • Separates DNA fragments by size using an electric field.
  • DNA is negatively charged → migrates toward positive electrode.
  • Shorter fragments move faster through the gel matrix.
  • DNA is stained with ethidium bromide → fluoresces under UV.
  • DNA can be transferred to nitrocellulose membrane for storage or hybridization.
tools of molecular biology: Gel Electrophoresis
Available from: https://www.researchgate.net/figure/representation-schematique-de-la-separation-de-proteines-par-electrophorese-sur-gel-de_fig33_281657002

DNA Sequencing

What DNA Sequencing Can Reveal:

  • Gene location, structure, and length
  • Start and stop codons
  • Protein-binding sites
  • Splice sites and domains
  • Predict amino acid sequences and protein function

Sequencing Methods

Pyrosequencing

  • Each DNA synthesis step is linked to a light-emitting reaction via ATP and luciferase.
  • Light burst indicates nucleotide incorporation.
  • DNA+dNTP → PPi → ATP → Light emission via luciferase → detected by computer.

Sanger Sequencing (ddNTP method)

  • Use single-stranded DNA template and primers.
  • Mix with normal dNTPs and fluorescently-labelled ddNTPs (which lack 3′-OH group).
  • When a ddNTP is incorporated, synthesis halts.
  • Result: terminated strands of varying lengths.
  • Separate by gel electrophoresis, read with a laser, and digitized by software.
tools of molecular biology: DNA Sequencing
https://www.sigmaaldrich.com/AU/en/technical-documents/protocol/genomics/sequencing/sanger-sequencing

Plasmid Cloning (DNA Amplification)

1. Transformation

  • Some bacteria take up plasmids (small circular DNA).
  • Plasmid requirements:
    • ORI (origin of replication)
    • Selectable marker (e.g. antibiotic resistance gene)
    • Polylinker (MCS: multiple cloning site)
    • Must be transformable and separable from chromosomal DNA.

2. Insertion

  • Gene of interest and plasmid are cut with same restriction enzymes.
  • Sticky ends facilitate binding.
  • DNA ligase seals the recombinant plasmid.

3. Replication

  • Bacteria are grown on agar + antibiotics.
  • Only plasmid-containing bacteria survive and replicate.
  • Plasmid is copied and passed to daughter cells during mitosis.

4. Extraction & Screening

  • Bacteria are lysed → plasmid DNA extracted.
  • DNA fragments cut using restriction enzymes.
  • Separate by gel electrophoresis → screen for mutations.

5. Protein Production

  • If the inserted gene (e.g. insulin) is correct, colonies are scaled up.
  • Used in biopharmaceutical production.
tools of molecular biology: DNA Amplification
https://www.jobilize.com/course/section/genetic-engineering-cloning-and-genetic-engineering-enbio-by-openstax

Polymerase Chain Reaction (PCR)

What’s Needed

  • DNA template (target sequence)
  • Two oligonucleotide primers (for each 3′ end)
  • Taq polymerase (heat-stable)
  • dNTPs (building blocks)
  • MgClâ‚‚-containing buffer

Steps

  1. Denaturation (94°C): DNA strands separate.
  2. Annealing (40–65°C): Primers bind to 3′ ends.
  3. Extension (74°C): Taq polymerase adds nucleotides from primers.

Output

  • 20–30 cycles → over 1 billion copies.
  • Products visualised by agarose gel electrophoresis.

Applications

  • Pathogen detection (e.g. COVID-19, tuberculosis)
  • Paternity & forensic DNA analysis
  • Gene cloning for protein synthesis in bacteria
Polymerase Chain Reaction
Enzoklop, CC BY-SA 4.0 <https://creativecommons.org/licenses/by-sa/4.0>, via Wikimedia Commons

Summary – Tools of Molecular Biology

Tools of molecular biology enable precise manipulation of DNA and RNA for cloning, sequencing, and diagnostics. Techniques like PCR, electrophoresis, and plasmid cloning are foundational to modern medicine and research, from producing therapeutic proteins to identifying pathogens. For a broader context, see our Genetics & Cancer Overview page.

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