Using SSCP to Screen for Chicken B Histocompatibility Haplotypes.

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