Lecture 2

• Genotyping


  Determining the genetic makeup of an individual
• Techniques for genotyping in pharmacogenetics/genomics


  • Genotyping for known polymorphisms
  • PCR-RFLP
  • Allele-specific PCR
  • Primer extension methods
• PCR-RFLP


  Digestion of NAT2 with *Ddel*
• Allele-specific PCR


  • Involves using primers + allele-specific oligonucleotides
  • Probe includes fluorescent reporter and quencher
  • Only see fluorescence if probe binds + reporter released by 5'-nuclease of Taq polymerase as it extends
  • Diff colour reporters for each allele
  • Colour detected in RT-PCR
• Taqman system


  1. Target DNA: Isolate the DNA to study

  2. Primers and Probes: Design short DNA pieces that match the target sequence

  3. PCR: Copy the target DNA many times

  4. Probe Cleavage: Probes are labeled with dyes; when cleaved, they release fluorescence

  5. Fluorescence Detection: Measure emitted light with a fluorometer

  6. Signal Measurement: Detect fluorescence to determine DNA quantity

  7. Genotyping: Analyse fluorescence patterns to determine genetic makeup
• Primer extension methods


  1. PCR: Amplify DNA region containing the polymorphism

  2. Primer extension: Add primer near the polymorphism and extend

  3. Minisequencing: Determine base at polymorphism site
• Sequenom


  1. Need to determine which polymorphism needs testing

  2. Create a specific test for this polymorphism

  3. Primer Selection: Use a normal primer matching the DNA sequence except for the polymorphic site, where dGTP is complementary to C. Other primers (A, C, and T) are dideoxynucleotides that can't be extended
• Pyrosequencing


  1. Prepare PCR Product: Label one strand with biotin

  2. Isolate Biotin-labeled Strand: Separate and isolate the biotin-labeled DNA strand

  3. Add Primer: Introduce a primer that attaches near the polymorphism site

  4. DNA Polymerase & Detection System: Combine DNA polymerase with a detection system using luciferin

  5. Add Single DNA Bases: Sequentially add single DNA bases: dATP, dCTP, dGTP, and dTTP

  6. Real-Time Binding: The complementary base to the polymorphism site binds and releases pyrophosphate (PPi) in real-time

  7. Light Production: PPi reacts with the detection system, producing light

  8. Camera Measurement: Measure the emitted light with a camera and send the signal to a detector

  9. Heterozygous Detection: If the individual is heterozygous, there will be decreased signals for two different bases
• Genome-wide association studies (GWAS)


  • Identify novel genes involved in disease or drug response
  • Strong linkage disequilibrium in human genome allows connections with genes some distance away from marker to be detected
• Affymetrix gene chip


  • Oligonucleotides specific to DNA sequences are attached to quartz surface-gene chip
  • Detect hybridisation of fluorescently labelled DNA
• Illumina bead chip


  • Need primer specific to each SNP
  • Can screen 1mil diff variations simultaneously
  • Each bead has specific primers attached
  • Input DNA not labelled
• Typical GWAS result


  • Y-axis = -log of p values. Lower p value = higher number
  • Significance usually set to 0.05 x 10^-6 to correct for multiple testing
  • Red dots are genome wide significant
  • Green dots close to genome wide significant
• Exome sequencing


  • Widely used in clinical genetics
  • Led to new information of genetic disease
• Whole genome sequencing


  • Increasingly possible
  • Challenging to assemble and interpret data
• Sanger sequencing


  1. DNA Breakdown: Use enzymes or heat to break DNA into pieces

  2. PCR: Mix DNA pieces, primers, DNA polymerase, and special nucleotides (ddNTPs)

  3. Gel Electrophoresis: Run PCR products through a gel to separate by size

  4. Visualization: Stain gel, view under UV light to see DNA bands

  5. Sanger Sequencing: Perform sequencing reactions with primers, polymerase, regular nucleotides, and ddNTPs

  6. Capillary Electrophoresis: Separate sequencing reaction products by size using capillary tubes and electric field

  7. Detection: Measure fluorescence or absorbance to determine DNA sequence
• Illumina sequencing


  1. Library Prep: Break DNA, add adapters

  2. Cluster Gen: Attach fragments to surface, amplify into clusters

  3. Seq-by-Synthesis: Add fluorescent nucleotides, detect incorporation

  4. Imaging: Scan surface, capture fluorescent signals

  5. Base Calling: Analyze signals to determine sequence

  6. Data Analysis: Align and analyze sequences for final results