Lecture Notes on DNA Sequencing Technology and Genetic Analysis

Syllabus Update

  • Minor update to the syllabus due to issues with electronic devices.
  • Requirement to remove wearable electronic devices (smartwatches, headphones, etc.) during quizzes and exams to prevent flagging due to notifications.

DNA Sequencing Technology

  • Focus on DNA sequencing technologies and DNA-based technologies that aid forensic science.
  • Importance of DNA collection in criminal cases; implication on committing felonies due to high DNA identification accuracy.

Background on PCR (Polymerase Chain Reaction)

  • Previous discussion on PCR and its variant, real-time PCR.
  • Real-time PCR used for making numerous copies of messenger RNA.
Reverse Transcriptase Enzyme
  • Reverse transcriptase synthesizes DNA from messenger RNA, creating complementary DNA (cDNA).
  • This process is reverse to natural transcription.
Regular PCR vs. Real-Time PCR
  • Regular PCR: Ingredients mixed, thermocycler used for 2.5 to 3 hours to amplify target DNA. Requires gel electrophoresis and UV light for visualization.
  • Real-Time PCR: Incorporation of Cyber Green fluorescent dye detects DNA in real-time; fluorescence increases with more DNA copies, allowing observation through the thermocycler.
  • Real-Time PCR is typically more expensive but more efficient.

DNA Sequencing Basics

  • Frederick Sanger's Sequencing Method: Developed in the 1970s, widely used for DNA sequencing.
  • Involves dideoxynucleotides (ddNTPs) which halt DNA replication when incorporated.
  • Each nucleotide has a phosphodiester bond formation; ddNTP lacks a hydroxyl group at the 3\' carbon, preventing further elongation.

Mechanism of Sanger Sequencing

  • The process produces varying lengths of DNA fragments, each terminating at a random ddNTP.
  • As DNA copy is made, regular nucleotides (dNTPs) and ddNTPs are used, creating a mix of fragment sizes for electrophoresis.
  • Fluorescently labeled ddNTPs enable detection via sequencing machines.
Reading the Sequence
  • Smaller fragments pass through the electrophoresis gel faster, leading to a sequence readout based on fluorescence of colored nucleotides.
  • Peaks in chromatograms represent nucleotide sequences, including detection of heterozygotes by colored signals.

Next-Generation Sequencing (NGS)

  • NGS has many improvements, such as Pyrosequencing:
    • Template strand attached to a bead; nucleotides added one at a time.
    • Light emitted indicates successful incorporation of a complementary nucleotide.

Site-Specific Mutagenesis

  • Technique utilized to study mutations in a gene relevant to diseases such as diabetes.
  • Amplification of gene fragments with primers, introducing mutations at a specific point, allows exploration of gene function.
  • Techniques like CRISPR potentially enhance this method significantly.

Gene Knockout Technology

  • Involves replacing one gene in target cells (like mouse embryos) with a mutated version to study effects on phenotype (e.g., diabetes incidence).
  • Aims to explore how mutations impact health and facilitates understanding of gene function in disease.

Polymorphism and DNA Variability

  • Defined as the ability to assume different forms within a species, exemplified by genetic variation in reed frogs.
  • DNA Polymorphism: Variability in alleles at a chromosomal locus due to mutations.
    • Can involve tandem repeats, often found in non-coding regions (junk DNA).
    • Approx. 2.5% of the human genome consists of genes, while the rest is considered non-coding.

Forensic Applications

  • Forensic analysis can utilize DNA polymorphism for identification via STR analysis and SNP profiling.
    • Probability of matching DNA profiles in criminal cases are significantly high.
    • CODIS (Combined DNA Index System) is a database for DNA profiles aiding law enforcement.
Historical Cases of DNA in Forensics
  • Notable cases where DNA profiling has exonerated suspects or identified criminals through forensic evidence.
  • Example: Narborough, England, where the first use of DNA profiling helped resolve a murder case using VNTR analysis.

Technologies in Genetic Testing and Gene Therapy

  • Gene Therapy: Insert normal genes into patients' cells to address genetic disorders. Includes mutational fixes and potential for treating cancers.
  • Genetic testing for prospective parents to identify carrier status for diseases, aiding in planning for family.
    • Emphasizes importance of genetic variation and its implications for health.

Summary of Modern DNA Techniques

  • Overview of older technologies (Southern blot, RFLP) compared to newer methods (NGS, microarrays).
  • Transcriptomics as a means to study RNA expression dynamics in different conditions.

Final Thoughts

  • Future prospects with CRISPR and other gene editing technologies hold promise for revolutionizing genetic treatments.
  • Ethical considerations in genetic testing and genetic modification remain crucial as technology advances.