Single Strand Conformation Polymorphism Analysis (1).pdf

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1. Single Strand
Conformation
Polymorphism
Analysis
Rida Bashir
Final year MLT

2. Content
●Introduction
●DNA Polymorphism
●Principle
●Process
●Steps
●Features
●Sensitivity
●Advantages and Disadvantages

3. SSCP
Single-strand conformational polymorphism (SSCP)
analysis is a simple and sensitive technique for mutation
detection and genotyping.
It is single strand chain polymorphism, is defined as
conformational difference of single stranded nucleotide
sequence of identical length as induced by difference in the
sequence under experimental condition.

4. ●It uses to distinguish the sequence by means of gel
electrophoresis with separate different conformation.

●Like RFLP, SSCPs are allelic variant of inherited,genetic
trait that can be used as genetic markers.

5. Features of SSCPs

●Efficient screen for DNA polymorphism

●Monomorphic markers are often times polymorphic with this assay

●Mutant bands separated from wild-type can be isolated for analysis

●Co-dominant or dominant

●Locus-specific

●Less applicable to DNA with unknown sequence

6. DNA polymorphism
●It is formed by differential folding (intramolecular interaction) of single stranded DNA harboring
mutation
●Heat-denatures PCR amplified Dna is detected by Assay while non-denaturing DNA on
sequencing gels.

7. Principle of SSCP
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 non denaturing electrophoresis conditions.
Therefore wild-type and mutant DNA samples display
different band patterns.

8. Principle of SSCP
The 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.

13. PROCEDURE

The procedure used during the development of SSCP was as follows:

1.Digestion of genomic DNA with restriction endonucleases.

2.Denaturation in an alkaline (basic) solution.

3.electrophoresis on a neutral polyacrylamide gel.

4.transfer to a nylon membrane.

5.hybridization with either DNA fragments or more clearly with

6.RNA copies synthesized on each strands as probes.

14. PROCEDURE

7. 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).

8. The desired polymorphism is selected with PCR primers; primer A is in excess to amplify
only a single strand (Panel B).

9. 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.

10. The single-stranded fragments, however, show considerable Nariation in mobility; the
small red molecule migrates more quickly through the gel than either the blue or the large
green molecule.

11. 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.

21. Thank you!!