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Single-Strand Conformation Polymorphism









Figure 1: SSCP Procedure.
 The three equal-length double-stranded DNA fragments are shown with the corresponding single-stranded structures, the red fragment folding into the smallest molecule and the green the largest (Panel A).  The desired polymorphism is selected with PCR primers; primer A is in excess to amplify only a single strand (Panel B).  Both the double-stranded and single-stranded fragments are run through gel electrophoresis (Panel C).  If not for the color labels, there would be no distinction between the double-stranded fragments.  The single-stranded fragments, however, show considerable variation in mobility; the small red molecule migrates more quickly through the gel than either the blue or the large green molecule.  Using this SSCP result, it becomes clear that the different lanes (red, blue, or green) contain strands with different sequences; the more far apart the bands, the less similar the nucleotide sequences.
Source with Permission from Dr. Ulrich Melcher

              1.  A specific pair of PCR primers (forward and reverse) is used to amplify the desired DNA fragments from individuals.

              2.  Single-stranded DNA is produced by asymmetric PCR: the primer on one side of the fragment is greatly in excess over the other primer.  After the            
                         low-concentration primer supply is exhausted, continued PCR produces only the target single strand.

              3.  The mobilities of the single stranded fragments are compared by electrophoresis on a neutral polyacrylamide gel.

              4.  Bands are detected by radioactive labeling or (more often) silver staining, and the pattern is interpreted (Melcher, 2000).


Figure 2: Sample SSCP Gel Result and Interpretation.  DNA was isolated and amplified from sand flies (Lutzomyia longipalpis).  SCCP analysis of the DNA shows multiple haplotypes, or sets of alleles usually inherited as a unit.  Lanes 3 and 4 were identical haplotypes from two individuals.  The difference in band migration in adjacent lanes is associated with the number of nucleotide differences (in parentheses): lanes 2-3 (2),  lanes 3-4 (0),  lanes 4-5 (3),  lanes 5-6 (1),  lanes 6-7 (3),  lanes 7-8 (1),  lanes 8-9 (1),  and lanes 9-10 (4).
Source: Hodgkinson, et al,. 2002  






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