5 Single-Strand Conformation Polymorphism (SSCP) Analysis
83
Fig. I. A Schematic diagram of the SSCP
principle. Thetarget sequence (represented by a black block) of a normal (left
panel) and a homozygous affected (right
panel) individual is amplified by PCR using
primer 1 and primer 2. The singlebasepair
substitution in the affected individual is
represented by an unfllled box in the target
sequence. The PCR product is denatured
prior to running in a nondenaturing
polyacrylamide gel. The single base change
causes altered conformations of the singlestranded DNA molecules. The conformational differences are detected by the
altered migration rate of each singlestranded DNA molecule. B A typical
example of an SSCP pattern. Generally,
each strand of an allele runs as a single
band representing a single conformation
when electrophoresed through a nondenaturing gel (lanes 1, 2). PCR products
amplified from individuals homozygous
for such an allele will run as two bands.
Heterozygous individuals having alleles A
and B can be easily identified by the
presence of four bands. On occasion, one
strand of an allele will have 2 conformations at a given temperature, buffer, or
matrix composition (lane 4, strand C' of
allele C) and migrate as two bands. In this
case, a homozygous individual will have
three bands, and a heterozygous individual
may have five (lane 5). Alternatively, the
strands of an allele may not be distinguished under some running conditions,
and all four strands run together as a single
band (lane 6) . It is important to keep in
mind when interpreting SSCP gels that the
band intensity of complementary strands
often is not equal if a-[ 32 P]-dCTP is added
to the nucleotide mix of the PCR reaction.
This problern can be circumvented by
using 5' end-labeled primers (see Chap. 7)
in the PCR reaction.
A
B
Normal sequence
Disease-causing sequence
with a single base pair change
...
.....
...
Prim er
Prim er 2
Prim er
Prim er 2
PC R
=
~ ....,
-
~
....0
~
~ Strand a
Single·stranded
- : l
Strand B
DNA molecule
- - : l
Strand b
Electropho re sis
~ Nondenatur;n/
Cathode
~
~
Strand a
Strand A
-
~
Strand B
c5jß
Anode
2
3
4
5
6
GENOTYPE: AA
BB
AB
cc AC DD
c
- -
A
- - -
B
D, D'
-
strand A '
-
B'
- -
C'
- -
C'
- -
83
Fig. I. A Schematic diagram of the SSCP
principle. Thetarget sequence (represented by a black block) of a normal (left
panel) and a homozygous affected (right
panel) individual is amplified by PCR using
primer 1 and primer 2. The singlebasepair
substitution in the affected individual is
represented by an unfllled box in the target
sequence. The PCR product is denatured
prior to running in a nondenaturing
polyacrylamide gel. The single base change
causes altered conformations of the singlestranded DNA molecules. The conformational differences are detected by the
altered migration rate of each singlestranded DNA molecule. B A typical
example of an SSCP pattern. Generally,
each strand of an allele runs as a single
band representing a single conformation
when electrophoresed through a nondenaturing gel (lanes 1, 2). PCR products
amplified from individuals homozygous
for such an allele will run as two bands.
Heterozygous individuals having alleles A
and B can be easily identified by the
presence of four bands. On occasion, one
strand of an allele will have 2 conformations at a given temperature, buffer, or
matrix composition (lane 4, strand C' of
allele C) and migrate as two bands. In this
case, a homozygous individual will have
three bands, and a heterozygous individual
may have five (lane 5). Alternatively, the
strands of an allele may not be distinguished under some running conditions,
and all four strands run together as a single
band (lane 6) . It is important to keep in
mind when interpreting SSCP gels that the
band intensity of complementary strands
often is not equal if a-[ 32 P]-dCTP is added
to the nucleotide mix of the PCR reaction.
This problern can be circumvented by
using 5' end-labeled primers (see Chap. 7)
in the PCR reaction.
A
B
Normal sequence
Disease-causing sequence
with a single base pair change
...
.....
...
Prim er
Prim er 2
Prim er
Prim er 2
PC R
=
~ ....,
-
~
....0
~
~ Strand a
Single·stranded
- : l
Strand B
DNA molecule
- - : l
Strand b
Electropho re sis
~ Nondenatur;n/
Cathode
~
~
Strand a
Strand A
-
~
Strand B
c5jß
Anode
2
3
4
5
6
GENOTYPE: AA
BB
AB
cc AC DD
c
- -
A
- - -
B
D, D'
-
strand A '
-
B'
- -
C'
- -
C'
- -