Lecture 9 - Unit 3 II

Question 1

Describe the structure of a tetramer

Answer 1

• 4 x MHC/CD1d molecules
  
  • Presenting peptide / lipid Ag
  • Biotinylated
• Streptavidin binds 4 biotin molecules
  
  • ​Thus binding 4 x MHC/CD1d molecules

Question 2

Describe how the α and β chain sequences of T cells of interest can be determined

Answer 2

1. Isolation of T cells that are specific for the Ag of interest using a tetramer
   
   • ​Eg PA224
   • Tetramer: PA224 presented in MHC I, biotinylated and bound to streptavidin
2. Single cell sort
3. PCR to amplify the α and β chain genes
   
   • ​​Forward and reverse primers for α and β chains
   • External round
   • Internal round:
     
     • Helps focus in on region of interest
4. Sanger sequencing
   
   • ​Fluorescently tagged nucleotides
     
     • ​ddATP, ddGTP, ddCTP, ddTTP
   • Generation of a chromatogram and trace
   • ⇒ sequence of nucleotides
   • ⇒ sequence of amino acids