module 18

Introduction to Bioinformatics and Christopher Porter

• Guest Speaker Overview: Christopher Porter is a Bioinformatician at the Ottawa Hospital Research Institute (OHRI) and the Ottawa Bioinformatics Core Facility.

• Professional Specialization: Porter specializes in the transformation of large-scale biological data into meaningful insights to drive modern biomedical research.

• Core Competencies:

  • Applying computational approaches for the analysis of genomic and transcriptomic datasets.

  • Supporting researchers in experimental design, data interpretation, and ensuring reproducibility.

  • Developing scalable pipelines for Next-Generation Sequencing (NGS) analysis.

• Major Projects:

  • Development of RNA-seq analysis pipelines for differential gene expression within various disease models.

  • Cancer genomics studies focused on the identification of molecular signatures and specific biomarkers.

  • Single-cell RNA-seq projects to explore cellular heterogeneity within complex tissues.

  • Integration of multi-omics datasets (combining genomics and transcriptomics) to achieve systems-level biological insights.

• Lecture Objective: The presentation demonstrates how bioinformatics converts raw sequencing data into actionable biological knowledge.

Restriction Fragment Length Polymorphism (RFLP)

• Definition: RFLPs are a specific type of length polymorphism found in the genomes of homologous DNA segments across different individuals after the DNA has been digested with a specific restriction enzyme.

• Mechanism:

  • DNA from different sources varies due to polymorphisms in their sequences.

  • Restriction enzymes are used to identify these differences.

  • Digested DNA is resolved using agarose gels.

  • Specific probes are utilized to identify DNA regions exhibiting length polymorphisms.

• Sequence Example:

  • Normal DNA: GCGTGACCGCTACACTTGCCAGCGCCCGAATTCCCTAGCG.

  • Diseased DNA: GCGTGACCGCTACACTTGCCAGCGCCCGAATACCCTAGCG.

  • Note: The diseased sequence differs by a single base at the restriction site location.

• Restriction Enzyme Application:

  • Example enzyme: EcoR1.

  • Specific recognition site: GAATCC.

• Data Interpretation:

  • Sample A: This individual is homozygous for the same allele, resulting in a single visible band on the gel.

  • Sample B: This individual is heterozygous, resulting in the revelation of three distinct bands.

• Applications of RFLP:

  • Mapping genes associated with human diseases.

  • Forensics: Precise identification of DNA samples.

• Reference Citation: Mentioned in "Molecular biomethods handbook second edition," by John Walker and Ralph Rapley, page $33$.

Variable Number Tandem Repeats (VNTRs) and Short Tandem Repeats (STRs)

• Definition (Nakamura et al. 1987; 1988): VNTRs are short DNA sequences repeated multiple times in tandem (one after another) at specific genomic locations.

• Polymorphism Mechanism: Polymorphism occurs when these repeats expand or contract within non-coding regions. The number of repeat units varies significantly between individuals.

• Terminology and Classification:

  • VNTRs: Referred to as minisatellites.

  • STRs: Referred to as microsatellites.

• Genetic Variation Statistics:

  • The DNA of two randomly chosen humans differs by approximately $1$ in $1000$ bases.

  • Overall genetic similarity between humans is approximately $99.9\%$.

• STR Alleles: In standard genetics, alleles are often associated with physical manifestations or variations in RNA and protein synthesis. In STR analysis, alleles are defined simply by the difference in the number of repeats, meaning the DNA length at a specific locus is either longer or shorter.

• Loci Persistence: STRs are observed at the same chromosomal positions (loci) across different population members, but the repeat number ($n$) is the variable factor.

• Molecular Example of STR:

  • DNA Sequence: CGTCAGCACACACACACACACACACACACACATGGCGTG.

  • In this sequence, the dinucleotide CA is repeated $13$ times ($n = 13$).

  • Patterns of repeating nucleotides can be two or more (e.g., $(CA)_n$ or $(ACGT)_n$).

• Genome Scale: Tens of thousands of different microsatellites (STRs) have been identified within the human genome.

DNA Fingerprinting by STR Analysis

• Utility: Individuals are differentiated based on establishing the specific number of repeats at given loci.

• Process Steps:

  1. Sample Collection: Biological samples such as blood or saliva are collected from individuals or crime scenes.

  2. DNA Extraction: Pure DNA is isolated from the collected samples.

  3. PCR Amplification: Specific STR loci are amplified via PCR. This often involves "multiplex PCR" where multiple loci are targeted simultaneously.

  4. Electrophoresis: Amplified STR fragments are separated by size using gel or capillary electrophoresis. This produces peaks that represent specific STR alleles.

  5. STR Profile Analysis: The number of repeats at each locus is determined to generate a unique STR profile.

  6. Comparison: STR profiles are compared between samples (e.g., a suspect versus a crime scene sample, or a parent versus a child).

  7. Statistical Analysis: Calculations are performed to determine the probability that a matching profile could belong to a different, unrelated person.

Forensic Applications and CODIS

• CODIS (Combined DNA Index System):

  • Utilized by the FBI.

  • Consists of a set of $20$ specific loci that are tested together.

  • Used by law enforcement to differentiate individuals based on allele repeat lengths.

• The AMEL Locus: The twentieth locus in the CODIS set is named "AMEL." This specific locus is used to discriminate between male and female biological sex.

• Paternity Testing: STR analysis is a standard tool used for determining biological parentage.