MLAB 2378 Fundamentals of Molecular Diagnostics Links

MLAB 2378 Fundamentals of Molecular Diagnostics

Unit 11 Notes - Gene Mutations

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SSCP analysis is based on the principle that under non-denaturing conditions, single-stranded DNA molecules assume unique conformations that vary depending on their nucleotide sequence. The change of only one base may cause a conformational change in the DNA molecule that is sufficient to be detected on a non-denaturing polymer, e.g., electrophoretic mobility differences compared with wild-type (wt) sequences.

The SSCP technique includes three processes:

• PCR amplification using primers that flank the DNA region of interest;

• Denaturation of the resulting double-stranded PCR product, followed by rapid chilling to prevent re-annealing of the strands;

• Electrophoretic separation of the single-stranded DNA on a nondenaturing sieving medium, such as a flowable polymer.

Reference: Biocompare