Materi SSCP

Single-strand conformational polymorphism (SSCP) analysis is a simple and sensitive technique for
mutation detection and genotyping. The principle of SSCP analysis is based on the fact that single-
stranded DNA has a defined conformation. Altered conformation due to a single base change in the
sequence can cause single-stranded DNA to migrate differently under nondenaturing electrophoresis
conditions. Therefore wild-type and mutant DNA samples display different band patterns.

SSCP analysis involves the following four steps:

(1) polymerase chain reaction (PCR) amplification of DNA sequence of interest

(2) denaturation of double-stranded PCR products dengan formamida dan NaOH

(3) cooling of the denatured DNA (single-stranded) to maximize self-annealing

(4) detection of mobility difference of the single-stranded DNAs by electrophoresis under non-
denaturing conditions.

Several methods have been developed to visualize the SSCP mobility shifts. These include

a. incorporation of radioisotope labeling

b. silver staining: simple, rapid, and cost-effective, and can be routinely performed in clinical
laboratories
c. fluorescent dye-labeled PCR primers
d. capillary-based electrophoresis.

The use of SSCP analysis to discover and genotype single-nucleotide polymorphisms (SNPs) has
been widely applied to the genetics of hypertension, including both monogenic (e.g., Liddle's
syndrome) and polygenic disorders (e.g., essential hypertension).
What is SSCP?
SSCP Analysis: Single-Strand Conformation Polymorphism Analysis

SSCP is the simplest and most used method of mutation detection. PCR is
used to amplify the region of interest and the resultant DNA is separated as
single-stranded molecules by electrophoresis in a non-denaturing
polyacrylamide gel (Orita et al,1989). A strand of single-stranded DNA folds
differently from another if it differs by a single base, and it is believed that
mutation-induced changes of tertiary structure of the DNA results in
different mobilities for the two strands. These mutations are detected as the
appearance of new bands on autoradiograms (radioactive detection), by
silver staining of bands or the use of fluorescent PCR primers which are
subsequently detected by an automated DNA sequencer (non-radioactive
detection).

The tertiary structure of single stranded DNA changes under different

physical conditions e.g. temperature and ionic environment. Hence the
sensitivity of SSCP depends on these (and many other) conditions (see
below). Whilst some empirical rules have emerged for the choice of
separation conditions for sequence variants in particular sequence
contexts, it is not possible to predict whether a certain mutation can be
detected under given conditions, especially when the mutation is in a new
sequence context. Mutation detection for PCR-SSCP is generally high,
>80% in a single run for fragments shorter than 300bp (Hayashi and
Yandell, 1993). As sensitivity is not 100%, the absence of a new band does
not prove that there is no mutation in the analysed molecule.

The sensitivity of PCR-SSCP decreases with increasing fragment length,

<300bp being the optimum. For mutation detection in longer fragments
(exons >300bp and whole cDNAs) overlapping short primer sets can be
used, or long PCR products digested with appropriate restriction enzymes
prior to SSCP (however, reamplification of individual new bands with the
original primer set is now no longer possible).

Advantages

SSCP screening has two primary advantages as a mutation-screening

technique:

You can screen for mutations in a specified DNA region by choosing

PCR primers that span that region.
 You can screen a large number of samples because the technique is
simple and fast.

The only step necessary after PCR amplification is a heat denaturation in

formamide and NaOH.

Limitations

SSCP screening only tells you that a mutation exists. You must perform
subsequent DNA sequencing to determine the nature of the mutation that
caused an electrophoretic mobility shift in a given sample.

Moreover, not all point mutations in a given sequence will cause a

detectable change in electrophoretic mobility. However, by optimizing PCR
reactions and run conditions before attempting a large-scale screening you
can increase the sensitivity.