Protocol

Detection of Mutations by Single-strand Conformational Polymorphism

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

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INTRODUCTION

Single-strand conformational polymorphism (SSCP), one of several methods used to scan segments of DNA for mutations, exploits the electrophoretic differences in mobilities between single-stranded mutant and wild-type DNAs.

MATERIALS

10x Amplification buffer

Include 0.01% (w/v) gelatin in the buffer.

10x TBE electrophoresis buffer

Use TBE at a working strength of 1x (89 mM Tris-borate, 2 mM EDTA) for polyacrylamide gel electrophoresis.

6x Gel-loading buffer I

Formamide loading buffer

Human genomic DNA to be screened for point mutations

Dissolve the DNA at 10 µg/ml in TE (pH 7.6).

Oligonucleotide primers, forward and reverse (35 µM each) in TE (pH 7.6)

Restriction enzymes . . . [Full Text of this Article]

METHOD