Molecular lab techniques

Molecular Laboratory Techniques Overview

• Focus on basics of immunology, DNA, and RNA

Nucleic Acids Overview

• Two main types:

  • DNA (deoxyribonucleic acid)

  • RNA (ribonucleic acid)

Differences Between DNA and RNA

• Structure:

  • DNA: Double helix

  • RNA: Single-stranded molecule

• Nitrogenous Bases:

  • DNA: A (adenine) with T (thymine), and G (guanine) with C (cytosine)

  • RNA: A (adenine) with U (uracil) and C (cytosine) with G (guanine)

• Purines (A, G) have two-ring structures

• Pyrimidines (T, U, C) have one-ring structures

Base Pairing Rules

• DNA Base Pairing:

  • Adenine (A) pairs with Thymine (T)

  • Cytosine (C) pairs with Guanine (G)

  • Hydrogen Bonds:

  • A and T have 2 hydrogen bonds

  • C and G have 3 hydrogen bonds

• RNA Base Pairing:

  • Adenine (A) pairs with Uracil (U)

  • Cytosine (C) pairs with Guanine (G)

DNA and RNA Directionality

• DNA and RNA synthesis occurs in a 5' to 3' direction

  • 5' Carbon: The fifth carbon in the sugar molecule in nucleotides

  • 3' Carbon: The third carbon in the sugar molecule, where nucleotides are added

Central Dogma of Molecular Biology

• Flow of Genetic Information:

  • DNA → RNA → Proteins

  • Only 1.2% to 1.5% of the human genome is translated into proteins

  • Not all DNA is transcribed into RNA, nor all RNA into proteins

Gene Mutations and Expression

• Mutations in DNA can result in variations in protein expression

  • The appearance (phenotype) may differ from genetic makeup (genotype) due to mutations

Structure of DNA

• Configuration: Double Helix

  • Two strands running in 5' to 3' direction

  • Complementary base pairing: C ↔ G & A ↔ T

  • Variation in hydrogen bonds:

  • C and G have 3 hydrogen bonds

  • A and T have 2 hydrogen bonds

DNA Transcription to mRNA

1. Process:

   • The DNA double helix is split, creating a replication fork

   • Leading Strand: mRNA is synthesized continually in 5' to 3' direction

   • Lagging Strand: mRNA synthesized in sections, also in 5' to 3', but in opposite direction to the DNA strand

2. mRNA Processing:

   • Exons (coding regions) are retained; introns (non-coding regions) are removed

   • Addition of:

     • Poly A tail for stability and regulation

     • 5' cap for recognition by translation machinery

PCR (Polymerase Chain Reaction)

• Purpose: DNA amplification technique

• Steps in PCR:

  1. Denaturation: Heating DNA to become single-stranded

  2. Annealing: Primers attach to complementary sequences on denatured DNA

  3. Extension: DNA polymerase replicates the target DNA

• Cycle Doubling:

  • After n cycles: Number of strands = $2^n$

  • E.g., 1 strand becomes 2 after cycle 1, 4 after cycle 2, 8 after cycle 3, etc.

Applications of PCR

• Detection of:

  • Genetic mutations

  • Cancers

  • Viruses and bacterial infections

• Advantages:

  • High sensitivity and specificity

  • Small sample volume needed

  • Rapid results compared to culture methods

qPCR (Quantitative PCR)

• Difference from Traditional PCR:

  • Measures the quantity of nucleic acid in the sample

  • Uses fluorescent probes to visualize and quantify the amplification process

• Real-Time PCR (rtPCR) and qPCR are interchangeable terms

Next Generation Sequencing (NGS)

• Overview:

  • Allows random amplification of nucleic acids without prior knowledge of the target sequence

  • Can identify new diseases, viruses, and other genetic material

• Procedure Duration:

  • Template preparation: Approximately 1 day

  • Sequencing on analyzer: Approximately 18 hours

  • Data analysis: Often performed online using databases like NCBI or BLAST

Advantages and Disadvantages of NGS

• Advantages:

  • Simultaneous sequencing of multiple samples (up to 96 or more)

• Disadvantages:

  • High expenses

  • Requires specialized expertise for data analysis

Conclusion

• Molecular techniques are crucial in clinical and research settings for advancing knowledge in genetics, diseases, and diagnostics.