1. Using SSCP to Screen for Chicken B Histocompatibility Haplotypes

2. Why does anyone care about chicken B histocompatibility haplotypes? n Marek’s disease is highly contagious and fatal. n Chickens with the B21 haplotype are resistant to Marek’s disease. n It would be economically safer to ranchers to have B21+ chickens.

3. What is SSCP? n SSCP = single strand conformational polymorphism

4. Steps in SSCP

5. SSCP for Chicken B-F Haplotype Silver-stained PAGE LANES M –  X174 x HinfI (not denatured) 1,11 – B23 2,3,4,7 – B2 5,6,10 – B21 8 – B8 9 – B19 12 – B24

6. What are the applications of SSCP? n To screen for the presence of small sequence differences without sequencing n Examples: u Normal polymorphisms (histocompatibility alleles) u Mutations (e.g., of genes associated with cancer) F prenatal screening, pre- and post-surgical decisions F Note: there are many other methods for mutation detection Heteroduplex analysis, denaturing gradient gel electrophoresis, cleavage of mismatched duplexes, allele specific PCR, others

7. Detection of p53 exon 8 sequence variants by SSCP LANES 1 – WT control 2-5 – samples Results in 2-4 indicate sequence variants

8. What makes SSCP attractive? n Technically simple n Need only 5-10 pg DNA as template for PCR n Rapid n Can be done without radioactivity n Highly sensitive to sequence variations; sequencing not necessary n Can detect 5-10% variation within a sample u Example: 10% tumor cells among normal cells

9. Basic SSCP facts n Single-stranded DNA undergoes intra-strand sequence-dependent base-pairing. n Intra-strand base-pairing  a sequence dependent shape. n Different sequences  different shapes. n Shapes can be discriminated by non-denaturing polyacrylamide gel electrophoresis.

10. 3 SSCP Steps n PCR u To amplify region(s) of interest n Denature u To separate strands n Analyze by non-denaturing PAGE u To resolve strands on the basis of shape

12. Critical Parameters of SSCP n Specificity of the PCR reaction n Concentration of DNA PCR products in PAGE sample (fg-pg/ul) n Success of the denaturation preceding PAGE u Heat, formamide n Successful resolution of folded fragments

13. What factors affect SSCP resolution? Remember:  resolution   specificity n Total quantity of PCR products processed by SSCP u Too much leads to interstrand reannealing and creates artifactual bands n Strand length u Optimum = 100-300 bp with 6% gels u <100 bp resolves poorly n Acrylamide - trial and error to develop a new SSCP assay u Percentage F Gradient vs. non-gradient acrylamide u Formulation

14. What factors affect SSCP resolution? Remember:  resolution   specificity n Temperature u too warm  denaturation of sequence-specific folding n Voltage n Buffer components n +/- 10% glycerol u glycerol causes strands to migrate more compactly F tighter bands make smaller differences in mobility more apparent

15. Chicken Histocompatibility B SSCP Essential assay components n Sample u Chicken red blood cell DNA n Detection scheme/Specificity u Comparison to standards F Clearly defined standards u Requires F Minimization of reannealing F Maximization of electrophoretic band separation n Visualization u Silver stain n Sensitivity u Silver stain detects small amounts of sample. u Background minimization u Amplification of sample is NOT a major contributor to sensitivity.

16. Essential Assay Components (cont’d) n Sensitivity u of the technique to the mutated region can depend on the F type of base substitution F length of the fragment F local base sequence F G/C content F position of the mutation relative to the ends of the fragment u some labs routinely run a PCR fragment with possible but unknown mutations under many different electrophoresis conditions to minimize chances of missing a mutation

17. Essential Assay Components (cont’d) n Sensitivity (cont’d) u The visualization procedure must be sufficiently sensitive to reveal small quantities of DNA. Why? u The use of supra-optimal quantities of DNA  risk of reannealing during PAGE  interpretation difficulties.

18. Chicken Histocompatibility B SSCP Essential assay components n What is the CONTROL included for each of the following? u For purity of PCR u For success of PCR u For technically successful electrophoresis u For reannealing of B21+ samples u Any others?

19. Why multiple bands in chick B Haplotype SSCP? n Biological explanations u Conserved priming sites for 2-3 different regions  up to 6 single strand bands n Technical explanations u > 1 metastable conformer/strand u incomplete denaturation