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Single-Strand Conformation Polymorphism
- SSCP is the electrophoretic separation of
single-stranded nucleic acids based on subtle differences in sequence
(often a single base pair) which results in a different secondary
structure and a measurable difference in mobility through a gel.
mobility of double-stranded DNA in gel electrophoresis is dependent on
strand size and length but is relatively independent of the particular
nucleotide sequence. The mobility of single strands, however, is
noticeably affected by very small changes in sequence, possibly one
changed nucleotide out of several hundred. Small changes are
noticeable because of the relatively unstable nature of single-stranded
DNA; in the absence of a complementary strand, the single strand may experience
intrastrand base pairing, resulting in loops and folds that give the
single strand a unique 3D structure, regardless of its length. A
single nucleotide change could dramatically affect the strand's mobility
through a gel by altering the intrastrand base pairing and its resulting
3D conformation (Melcher, 2000).
Single-strand conformation polymorphism analysis takes advantage of this
quality of single-stranded DNA. First announced in 1989 as a new
means of detecting DNA polymorphisms, or sequence variations, SSCP
analysis offers an inexpensive, convenient, and sensitive method for
determining genetic variation (Sunnucks et al., 2000).
restriction fragment length polymorphisms (RFLPs), SSCPs are allelic
variants of inherited, genetic traits that can be used as genetic markers.
Unlike RFLP analysis, however, SSCP analysis can detect DNA
polymorphisms and mutations at multiple places in DNA fragments (Orita et
al., 1989). As a mutation scanning technique, though, SSCP is more
often used to analyze the polymorphisms at single loci, especially when
used for medical diagnoses (Sunnucks et al., 2000).
- The procedure
used during the development of SSCP was as follows:
- digestion of genomic
DNA with restriction endonucleases
- denaturation in an
alkaline (basic) solution
- electrophoresis on a
neutral polyacrylamide gel
- transfer to a nylon
- hybridization with
either DNA fragments or more clearly with RNA copies synthesized on each
strand as probes (Orita et al., 1989).
then, more convenient procedures have been developed, taking into account
other molecular techniques, although sometimes it is simpler to amplify
the double strand and then denature it into single strands instead of
trying to find suitable primers for the below PCR method if the targeted
sequence is unknown.
experiments involving SSCP are designed to evaluate polymorphisms at
single loci and compare the results from different individuals.
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
Procedure as illustrated in Figure 1:
1. A specific pair of PCR primers (forward and
reverse) is used to amplify the desired DNA fragments from individuals.
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.
The mobilities of the single stranded fragments are compared by
electrophoresis on a neutral polyacrylamide gel.
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).
et al,. 2002
SSCP LIMITATIONS AND CONSIDERATIONS
DNA mobilities are dependent on temperature. For best results, gel
electrophoresis must be run in a constant temperature.
Sensitivity of SSCP is affected by pH. Double-stranded DNA fragments
are usually denatured by exposure to basic conditions: a high pH. Kukita
et al. found that adding glycerol to the polyacrylamide gel lowers the pH
of the electrophoresis buffer--more specifically, the Tris-borate
buffer--and the result is increased SSCP sensitivity and clearer data
length also affects SSCP analysis. For optimal results, DNA fragment
size should fall within the range of 150 to 300 bp, although SSCP analysis
of RNA allows for a larger fragment size (Wagner, 2002). Tthe
presence of glycerol in the gel may also allow a larger DNA fragment size
at acceptable sensitivity (Kukita et al., 1997).
- Under optimal conditions,
approximately 80 to 90% of the potential base exchanges are detectable by
SSCP (Wagner, 2002).
- If the specific nucleotide
responsible for the mobility difference is known, a similar technique
called Single Nucleotide Polymorphism (SNP) may be
- Kukita, Y., et al.
SSCP Analysis of Long DNA Fragments in Low pH Gel. Human
Mutation; 1997, (10): 400-7.
- Orita, M., et al.
Detection of Polymorphisms of Human DNA by Gel Electrophoresis as
SSCPs. Proceedings of the National Academy of Sciences of the
United States of America; 1989, (86): 2766-70.
- Sunnucks, P., et al.
SSCP Is Not So Difficult: The Application and Utility of
Single-Stranded Conformation Polymorphism in Evolutionary Biology and
Molecular Ecology. Molecular Ecology; 2000, (9): 1699-710.