Chapter 5
Single-Strand Conformation Polymorphism (SSCP)
Analysis
FENG QIAN AND GREGORY G. GERMINO
lntroduction
The detection ofDNA sequence variation is important for the identification
of disease-causing mutations, as well as for the detection of clinically silent
DNA polymorphisms. Among the techniques capable of detecting a single
base change, the single-strand conformation polymorphism (SSCP) analysis is most commonly used due to its simplicity and effectiveness ( Orita et al.
1989a, 1989b; Sheffield et al. 1993). SSCP analysis permits the detection of
mutations in very small amounts of genomic DNA or cDNA. This technique
is rapid and inexpensive. It is based on the principle that a single-stranded
DNA molecule adopts a sequence-specific conformation. A change in conformation resultung from a single base Substitution can be detected as an
alteration of its migration rate in a nondenaturing polyacrylamide gel (Figure 1).
The detection of conformation differences depends on a number of parameters. The optimal condition varies markedly from fragment to fragment and must be determined empirically for each DNA segment.
Procedure
Generating SSCP template by PCR reactions
The desired SSCP template is amplified using PCR. A total PCR reaction
volume of 10 1-11 is sufficient. Add 0.1 flCi of 32 P-dCTP to each reaction.
Feng Qian, The Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA, Correspondence to Gregory G.
Germino, The Johns Hopkins University, School of Medicine, Division of Nephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA (phone +01-410-614-1650;/ax
+01-410-614-5129; e-mail ggermino@welch.jhu.edu)
Single-Strand Conformation Polymorphism (SSCP)
Analysis
FENG QIAN AND GREGORY G. GERMINO
lntroduction
The detection ofDNA sequence variation is important for the identification
of disease-causing mutations, as well as for the detection of clinically silent
DNA polymorphisms. Among the techniques capable of detecting a single
base change, the single-strand conformation polymorphism (SSCP) analysis is most commonly used due to its simplicity and effectiveness ( Orita et al.
1989a, 1989b; Sheffield et al. 1993). SSCP analysis permits the detection of
mutations in very small amounts of genomic DNA or cDNA. This technique
is rapid and inexpensive. It is based on the principle that a single-stranded
DNA molecule adopts a sequence-specific conformation. A change in conformation resultung from a single base Substitution can be detected as an
alteration of its migration rate in a nondenaturing polyacrylamide gel (Figure 1).
The detection of conformation differences depends on a number of parameters. The optimal condition varies markedly from fragment to fragment and must be determined empirically for each DNA segment.
Procedure
Generating SSCP template by PCR reactions
The desired SSCP template is amplified using PCR. A total PCR reaction
volume of 10 1-11 is sufficient. Add 0.1 flCi of 32 P-dCTP to each reaction.
Feng Qian, The Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA, Correspondence to Gregory G.
Germino, The Johns Hopkins University, School of Medicine, Division of Nephrology,
720 Rutland Avenue, Ross 958, Baltimore, Maryland, USA (phone +01-410-614-1650;/ax
+01-410-614-5129; e-mail ggermino@welch.jhu.edu)