Bioinformatics: DNA Sequencing

Nottingham Trent University: Bioinformatics - DNA Sequencing

Module Overview

  • Module Code: FORE20007 Biological Techniques in Forensic Science

Topics to Cover

  1. DNA Sequencing
  2. Bioinformatics
  3. Bioinformatics Workshop

Module Learning Objectives

  • MLO2: Explain the range of biological techniques that are available to the forensic scientist.
  • Session Objectives:
    • Compare sequencing techniques.
    • Consider the forensic applications.

DNA Sequencing: The Basics

  • Definition: DNA sequencing determines the order of nucleotides in a DNA molecule.
  • Human Genome: Comprises approximately 3 billion base pairs that encode the instructions for forming a human being.
  • Application: Can be analyzed using bioinformatics.

Sanger Sequencing

Overview

  • Description: A highly accurate method for determining DNA nucleotide sequences in small targeted regions.
  • Development: First developed in 1977; modern methods are largely automated.
  • Techniques Used: Includes Polymerase Chain Reaction (PCR) and Capillary Electrophoresis (CE).

Steps in Sanger Sequencing

  1. Chain Termination PCR:
    • Conduct normal PCR on a small section of DNA.
    • Nucleotides added contain a small portion lacking the –OH group necessary for bond formation, resulting in millions of copies of the target DNA sequence, terminated at random lengths.
  2. Electrophoresis:
    • Produces fragment lengths that end with different nucleotides (A, C, T, or G).
    • Example: Fragment ending in A has only A terminal nucleotides added to the mix.
    • Separation by Gel Electrophoresis/Ce (GE/CE) allows for determining the terminal nucleotide of each fragment.

Graphical Representation

  • Visual representation of DNA fragments and their respective terminal nucleotides during sequencing.

Human Genome Project (1990–2003)

  • Achievement: The first full sequence of the human genome, completed in 2003, approximated over 90% accuracy based on advanced technologies.
  • Contribution: Provided a reference genome essential for forensic DNA analysis, including Short Tandem Repeats (STRs).
  • Current Use: Enables development of Single Nucleotide Polymorphism (SNP) panels useful for ancestry and phenotype prediction.

Next Generation Sequencing (NGS)

  • Transition: Evolved from Sanger sequencing to include more efficient techniques.

NGS Process:

  1. Fragmentation: The DNA fragments are processed and adaptors are added.
  2. Attachment: Adaptors bind to a flow cell where sequencing occurs.
  3. Cluster Formation: During PCR amplification, fragments cluster at specific locations on the flow cell.
  4. Signal Scanning: Detection of sequences through signal interpretation during the sequencing phase.

Advantages and Disadvantages of Sequencing Techniques

Sanger Sequencing

Advantages:
  • Incredibly accurate; regarded as the gold standard.
  • Cost-effective for urgent, unbatched sample processing.
  • Less dependent on computational tools compared to NGS.
Disadvantages:
  • Limited to sequencing up to approximately 1000 base pairs.
  • Requires large initial DNA amounts.
  • Not ideal for sequencing multiple genes simultaneously.

Next-Generation Sequencing

Advantages:
  • Significantly faster and capable of high throughput.
  • Can analyze many sequences at once (e.g., ForenSeq™ analyzes 231 loci).
  • Suitable for degraded samples with smaller sequences.
Disadvantages:
  • Higher complexity; reduced accuracy.
  • Increased likelihood of technical challenges.

Forensic Applications

Body Fluid Identification

  • Importance of sequencing techniques in accurate identification of body fluids in forensic contexts.

Phenotyping Applications

  • Usage of sequencing in predicting phenotypic traits (e.g., hair, eye color).
  • Statistical probabilities involved in phenotypic determination based on DNA sequences.

Case Studies

  1. Golden State Killer: Illustrates the use of NGS in cold case resolutions.
  2. Russell Marubbio (1987): DNA analysis led to the identification and resolution of a cold case rape incident.

Medical Applications

Disease Screening

  • BRCA1 Gene: Located on chromosome 17, plays a role in tumor suppression and can be involved in disease predisposition.

Sickle Cell Anemia

  • Illustration of how mutations in DNA sequences affect protein coding, resulting in conditions such as sickle cell disease, where a change in amino acid sequence leads to abnormal hemoglobin structure.
    • Normal sequence: CAC GTG GAC results in normal red blood cells.
    • Mutant sequence: GTG CAC CTG causes sickling under low oxygen levels leading to complications.

Conclusion

  • The comprehensive understanding of DNA sequencing technologies is crucial for advancements in forensic science and medical applications, as evidenced by historical projects like the Human Genome Project, ongoing advancements in sequencing technologies, and specific case applications.