DNA and RNA Analysis Techniques

Nucleic Acid Extraction & QC - Getting the Goods

• Goal: Isolate pure, intact DNA/RNA.
• Core Steps: Lysis → Separation → Precipitation → Wash → Elute.

• Lysis: Mechanical (homogenization) or Chemical (detergents, Proteinase K).
• Separation: Organic (Phenol-Chloroform) or Solid-phase (silica columns).
• Quality Control (QC):
  • Quantity: Spectrophotometry (OD at 260 nm).
    • DNA: $1 \text{ OD} = 50 \text{ µg/mL}$
    • RNA: $1 \text{ OD} = 40 \text{ µg/mL}$
  • Purity: A260/A280 (protein), A260/A230 (organics).
  • Integrity: Gel electrophoresis (DNA: high MW band; RNA: 28S/18S rRNA, RIN).

⭐ A260/A280 ratio for pure DNA is ~1.8, for pure RNA is ~2.0. <1.7 suggests protein. A260/A230 ideally 2.0-2.2.

PCR Techniques - Copy Cat Genetics

• Principle: Exponential in-vitro amplification of specific DNA.
• Key Reagents:
  • Template DNA
  • Primers (Forward & Reverse)
  • Taq Polymerase (heat-stable)
  • dNTPs (deoxynucleotide triphosphates)
  • Buffer with Mg²⁺
• Thermal Cycling Steps: 📌 "Dad Ate Apples" (Denaturation, Annealing, Extension)
  • Denaturation: ~95°C (dsDNA → ssDNA).
  • Annealing: ~50-65°C (Primers bind to template).
  • Extension: ~72°C (Taq polymerase synthesizes new DNA).
• Outcome: $2^n$ fold amplification of target DNA (where n = number of cycles).

⭐ Taq polymerase, isolated from the bacterium Thermus aquaticus, is a heat-stable enzyme crucial for PCR due to its ability to withstand repeated high-temperature denaturation steps.

Hybridization Techniques - Finding Your Match

• Core Principle: Labeled nucleic acid probe binds to complementary target sequence.
• 📌 SNoW DRoP Mnemonic:
  • Southern = DNA
  • Northern = RNA
  • Western = Protein (Note: Western blot uses antibodies, not nucleic acid probes, but often taught together).

⭐ FISH can detect chromosomal abnormalities like translocations (e.g., BCR-ABL in CML) and aneuploidies directly on slides, even in interphase nuclei.

• Dot/Slot Blot: Simpler; detects presence/absence of sequence, no size info.
• ASO Probes: Allele-Specific Oligonucleotides for single nucleotide polymorphism (SNP) detection (e.g., Cystic Fibrosis).

Sequencing & Typing - Reading Blueprints

⭐ Sanger sequencing uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis, allowing for sequence determination.

• Sanger Sequencing (Chain Termination):
  • Principle: Incorporates chain-terminating ddNTPs during in-vitro DNA replication. Fragments are size-separated.
  • Uses: Gold standard for targeted sequencing, validating single gene mutations, sequencing PCR products.
• Next-Generation Sequencing (NGS):
  • Principle: Massively parallel sequencing of millions of DNA fragments simultaneously. High throughput.
  • Uses: Whole Exome/Genome Sequencing (WES/WGS), RNA-Seq, cancer genomics, identifying novel/rare variants.
• DNA Typing Methods:
  • RFLP (Restriction Fragment Length Polymorphism): Detects variations in DNA fragment lengths after restriction enzyme digestion. Used for genetic mapping.
  • VNTR/STR (Variable Number Tandem Repeats/Short Tandem Repeats): Analyzes length variations in tandemly repeated DNA sequences. Key for DNA fingerprinting, forensics.

High‑Yield Points - ⚡ Biggest Takeaways

• PCR amplifies DNA; RT-PCR for RNA (via cDNA).
• Sanger sequencing for nucleotide sequence; NGS for high-throughput analysis.
• FISH localizes DNA sequences on chromosomes; CISH uses bright-field.
• Southern blotting detects DNA fragments; Northern blotting for RNA.
• Microarrays analyze gene expression of many genes simultaneously.
• RFLP identifies DNA variations via restriction enzyme cleavage.
• AS-PCR detects single nucleotide polymorphisms (SNPs).