1. Assay I HLA-DQ Alpha (A1) Haplotype

2. Purpose To determine which one of several known alleles is present at the HLA DQ α locus on each of an individual’s two copies of Chromosome 6.

3. Why? n People with different HLA types have different likelihoods of developing full immunodeficiency. n Selection of organ donors. n Testing after transplant for best way to treat. n Certain HLA genes are linked to disease u Susceptibility to infections. u Autoimmune disease F Rheumatoid arthritis F Multiple sclerosis F Diabetes mellitus Type I F Systemic lupus erythematosus

4. Steps of the HLA-DQ alpha assay Acquire DNA Sample ↓ Amplify HLA-DQ α region by PCR (Check for success of PCR) ↓ Isolate PCR fragment from primers ↓ Detection Scheme Sequence the PCR fragment Fluorescence visualization ↓ Analysis Compare sequence of unknown to known allele sequences

5. HLA-DQ α alleles Allele Number

6. Sample n Start with human buccal cells u Can use any human cells containing nuclear DNA PCR the HLA DQ α region of the nuclear genome u PCR is an enzymatic technique used to increase the amount of sample

8. Ignore the blue highlighting.

9. PCR for HLA DQ-alpha  Primers amplify a region of ~300 bp.  Sequence of primers is common to all individuals.  Sequence between primers is polymorphic – may differ between an individual’s two chromosomes and among individuals.  PCR picks out the 300 bp HLA DQα region from 3.3 X 10 9 bp of DNA present in 23 chromosomes. 11.06.00 11

10. The human male karyotype: 22 homologous chromosomes + X and Y

11.  Products of PCR reaction  >10 6 double strand fragments ~ 300 bp in length.  1/2 = DQ-α allele on one copy of your Chromosome 6; 1/2 = DQ-α allele on the other copy. PCR for HLA DQ-alpha

12. 100bp 300bp Gel Electrophoresis: 2% SB Gel at 90Volts

13. Sequence with internal primer common to all individuals Analyze by using BLAST to align your sequence to known sequence of polymorphic region Compare results to sequences of known polymorphic alleles

14. ~250 bp Chromosome 6 } Polymorphic region of HLA-DQα PCR primers Sequencing primer: one for each direction

15. Detection Scheme n Capillary electrophoresis separates sequencing products that differ from each other by only one nucleotide in length n Each product terminates with one of the four bases A,G,C,T n Each terminal base is labeled with a fluorescent dye of a different color, to allow for visualization and discrimination n Each product, from shortest to longest is detected by a scanner for fluorescence

16. Fluorescence automated sequencing system Gel electrophoresis..

17. Fluorescence automated sequencing system capillary electrophoresis

18. Computerized visualization from a single capillary run of an automated sequencer. Method uses fluorescent labeling.

19. Determine your alleles by BLASTing 2 sequences Query = your sequence Subject = reference sequence – Haplotype 1.1

20. HLA-DQalpha alleles Allele Number

21. Contributions to Specificity u Exact sequence from sequence reaction and known standards of comparison on the preceding chart u Precise hybridization conditions limit pairing of PCR primers to exact complements in genome – HotStart PCR F Hybridization = complementary base pairing of two single DNA or RNA strands to each other u Internal sequencing primer limits sequencing to only those PCR products that represent the HLA-DQα region F Just in case the PCR primer hybridization is not entirely specific to the HLA-DQα region so that multiple PCR products are obtained, the sequencing primer adds additional specificity.

22. Contributions to sensitivity n Sample amplification by PCR n Signal amplification by cycle sequencing u (more later) n Background reduction u Separation of sequencing products from unincorporated nucleotides

23. Controls n PCR reaction u Positive control template – controls for failure of PCR reagents u No template control – controls for presence of contaminant DNA u Agarose gel electrophoresis – checks for PCR yield, expected and unexpected products n Sequencing u Control PCR product F Checks for known haplotype (starting next year?) u Control sequencing template (one volunteer team) F Checks for integrity of sequencing kit reagents

24. Ask yourself n How has each of the essential assay components been incorporated into this assay?