Using SSCP to Screen for Chicken B Histocompatibility Haplotypes
Why does anyone care about chicken B histocompatibility haplotypes?
Marek’s disease is highly contagious and fatal. Chickens with the B21 haplotype are resistant to
Marek’s disease. It would be economically safer to ranchers to have
B21+ chickens.
What is SSCP?
SSCP = single strand conformational polymorphism
Steps in SSCP
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
What are the applications of SSCP?
To screen for the presence of small sequence differences without sequencing
Examples: Normal polymorphisms (histocompatibility alleles) Mutations (e.g., of genes associated with cancer)
prenatal screening, pre- and post-surgical decisions Note: there are many other methods for mutation
detection• Heteroduplex analysis, denaturing gradient gel electrophoresis,
cleavage of mismatched duplexes, allele specific PCR, others
Detection of p53 exon 8 sequence variants by SSCP
LANES1 – WT control2-5 – samplesResults in 2-4 indicate sequence variants
What makes SSCP attractive?
Technically simple Need only 5-10 pg DNA as template for PCR Rapid Can be done without radioactivity Highly sensitive to sequence variations;
sequencing not necessary Can detect 5-10% variation within a sample
Example: 10% tumor cells among normal cells
Basic SSCP facts
Single-stranded DNA undergoes intra-strand sequence-dependent base-pairing.
Intra-strand base-pairing a sequence dependent shape.
Different sequences different shapes. Shapes can be discriminated by non-denaturing
polyacrylamide gel electrophoresis.
3 SSCP Steps
PCR To amplify region(s) of interest
Denature To separate strands
Analyze by non-denaturing PAGE To resolve strands on the basis of shape
Critical Parameters of SSCP
Specificity of the PCR reaction Concentration of DNA PCR products in PAGE
sample (fg-pg/ul) Success of the denaturation preceding PAGE
Heat, formamide Successful resolution of folded fragments
What factors affect SSCP resolution? Remember: resolution specificity
Total quantity of PCR products processed by SSCP Too much leads to interstrand reannealing and creates
artifactual bands Strand length
Optimum = 100-300 bp with 6% gels <100 bp resolves poorly
Acrylamide - trial and error to develop a new SSCP assay Percentage
Gradient vs. non-gradient acrylamide Formulation
What factors affect SSCP resolution? Remember: resolution specificity Temperature
too warm denaturation of sequence-specific folding Voltage Buffer components +/- 10% glycerol
glycerol causes strands to migrate more compactly tighter bands make smaller differences in mobility more
apparent
Chicken Histocompatibility B SSCP Essential assay components Sample
Chicken red blood cell DNA Detection scheme/Specificity
Comparison to standards Clearly defined standards
Requires Minimization of reannealing Maximization of electrophoretic band separation
Visualization Silver stain
Sensitivity Silver stain detects small amounts of sample. Background minimization Amplification of sample is NOT a major contributor to sensitivity.
Essential Assay Components (cont’d)
Sensitivity of the technique to the mutated region can depend on the
type of base substitution length of the fragment local base sequence G/C content position of the mutation relative to the ends of the fragment
some labs routinely run a PCR fragment with possible but unknown mutations under many different electrophoresis conditions to minimize chances of missing a mutation
Essential Assay Components (cont’d)
Sensitivity (cont’d)
The visualization procedure must be sufficiently sensitive to reveal small quantities of DNA. Why?
The use of supra-optimal quantities of DNA risk of reannealing during PAGE interpretation difficulties.
Chicken Histocompatibility B SSCP Essential assay components
What is the CONTROL included for each of the following? For purity of PCR For success of PCR For technically successful electrophoresis For reannealing of B21+ samples Any others?
Why multiple bands in chick B Haplotype SSCP? Biological explanations
Conserved priming sites for 2-3 different regions up to 6 single strand bands
Technical explanations > 1 metastable conformer/strand incomplete denaturation